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Cell size and morphological properties of yeast Saccharomyces cerevisiae in relation to growth temperature

Abstract (Expand)

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast … Saccharomyces cerevisiae CEN.PK 113–7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (μmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 μm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 μm. The size of the bud linearly depends on μmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on μmax. There are two temperature regions (5–31°C vs. 33–40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33–40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the μmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

Authors: Maksim Zakhartsev, Matthias Reuss

Date Published: 26th Apr 2018

Publication Type: Not specified

Phosphoglycerate kinase acts as a futile cycle at high temperature.

Abstract (Expand)

In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate … to glyceraldehyde-3-phosphate at 30 degrees C and at 70 degrees C. At 30 degrees C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 degrees C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 degrees C and at 70 degrees C, however, at 70 degrees C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.

Authors: T. Kouril, J. J. Eicher, B. Siebers, J. L. Snoep

Date Published: 7th Oct 2017

Publication Type: Not specified

Systems biology of the modified branched Entner-Doudoroff pathway in Sulfolobus solfataricus.

Abstract (Expand)

Sulfolobus solfataricus is a thermoacidophilic Archaeon that thrives in terrestrial hot springs (solfatares) with optimal growth at 80 degrees C and pH 2-4. It catabolizes specific carbon sources, such … as D-glucose, to pyruvate via the modified Entner-Doudoroff (ED) pathway. This pathway has two parallel branches, the semi-phosphorylative and the non-phosphorylative. However, the strategy of S.solfataricus to endure in such an extreme environment in terms of robustness and adaptation is not yet completely understood. Here, we present the first dynamic mathematical model of the ED pathway parameterized with quantitative experimental data. These data consist of enzyme activities of the branched pathway at 70 degrees C and 80 degrees C and of metabolomics data at the same temperatures for the wild type and for a metabolic engineered knockout of the semi-phosphorylative branch. We use the validated model to address two questions: 1. Is this system more robust to perturbations at its optimal growth temperature? 2. Is the ED robust to deletion and perturbations? We employed a systems biology approach to answer these questions and to gain further knowledge on the emergent properties of this biological system. Specifically, we applied deterministic and stochastic approaches to study the sensitivity and robustness of the system, respectively. The mathematical model we present here, shows that: 1. Steady state metabolite concentrations of the ED pathway are consistently more robust to stochastic internal perturbations at 80 degrees C than at 70 degrees C; 2. These metabolite concentrations are highly robust when faced with the knockout of either branch. Connected with this observation, these two branches show different properties at the level of metabolite production and flux control. These new results reveal how enzyme kinetics and metabolomics synergizes with mathematical modelling to unveil new systemic properties of the ED pathway in S.solfataricus in terms of its adaptation and robustness.

Authors: A. S. Figueiredo, T. Kouril, D. Esser, P. Haferkamp, P. Wieloch, D. Schomburg, P. Ruoff, B. Siebers, J. Schaber

Date Published: 12th Jul 2017

Publication Type: Not specified

Metabolic efficiency in yeast Saccharomyces cerevisiae in relation to temperature dependent growth and biomass yield

Abstract (Expand)

Canonized view on temperature effects on growth rate of microorganisms is based on assumption of protein denaturation, which is not confirmed experimentally so far. We develop an alternative concept, … which is based on view that limits of thermal tolerance are based on imbalance of cellular energy allocation. Therefore, we investigated growth suppression of yeast Saccharomyces cerevisiae in the supraoptimal temperature range (30–40 °C), i.e. above optimal temperature (Topt). The maximal specific growth rate (μmax) of biomass, its concentration and yield on glucose (Yx/glc) were measured across the whole thermal window (5–40 °C) of the yeast in batch anaerobic growth on glucose. Specific rate of glucose consumption, specific rate of glucose consumption for maintenance (mglc), true biomass yield on glucose (View the MathML source), fractional conservation of substrate carbon in product and ATP yield on glucose (Yatp/glc) were estimated from the experimental data. There was a negative linear relationship between ATP, ADP and AMP concentrations and specific growth rate at any growth conditions, whilst the energy charge was always high (~0.83). There were two temperature regions where mglc differed 12-fold, which points to the existence of a ‘low’ (within 5–31 °C) and a ‘high’ (within 33–40 °C) metabolic mode regarding maintenance requirements. The rise from the low to high mode occurred at 31–32 °C in step-wise manner and it was accompanied with onset of suppression of μmax. High mglc at supraoptimal temperatures indicates a significant reduction of scope for growth, due to high maintenance cost. Analysis of temperature dependencies of product formation efficiency and Yatp/glc revealed that the efficiency of energy metabolism approaches its lower limit at 26–31 °C. This limit is reflected in the predetermined combination of View the MathML source, elemental biomass composition and degree of reduction of the growth substrate. Approaching the limit implies a reduction of the safety margin of metabolic efficiency. We hypothesize that a temperature increase above Topt (e.g. >31 °C) triggers both an increment in mglc and suppression of μmax, which together contribute to an upshift of Yatp/glc from the lower limit and thus compensate for the loss of the safety margin. This trade-off allows adding 10 more degrees to Topt and extends the thermal window up to 40 °C, sustaining survival and reproduction in supraoptimal temperatures. Deeper understanding of the limits of thermal tolerance can be practically exploited in biotechnological applications.

Authors: Maksim Zakhartsev, Xuelian Yang, Matthias Reuss, Hans Otto Pörtner

Date Published: 1st Aug 2015

Publication Type: Not specified

Fast sampling for quantitative microbial metabolomics: new aspects on cold methanol quenching: metabolite co-precipitation

Abstract (Expand)

The intra- and extracellular concentrations of 16 metabolites were measured in chemostat (D = 0.1 h−1) anaerobic cultures of the yeast Saccharomyces cerevisiae CEN.PK-113-7D growing on minimal medium. … Two independent sampling workflows were employed: (i) conventional cold methanol quenching and (ii) a differential approach. Metabolites were quantified in different sample fractions (total, extracellular, quenching supernatant, methanol/water extract and pellet) in order to derive their mass balance. The differential method in combination with absolute metabolite quantification by gas-chromatography with isotope dilution mass spectrometry (GC–IDMS) was used as a benchmark to assess quality of the cold methanol quenching procedure. Quantitative comparison of metabolite concentrations in all fractions collected by different quenching techniques indicates asystematic loss of the total mass of various metabolites in course of the cold methanol quenching. Pellet resulting from the cold methanol quenching besides biomass contains considerable amounts of precipitated inorganic salts from the fermentation media. Quantitative analysis has revealed significant co-precipitation of polar extracellular metabolites together with these salts. This phenomenon is especially significant for metabolites with large extracellular mass-fraction. We report that the co-precipitation is a hitherto neglected phenomenon and concluded that its degree strongly linked to culturing conditions (i.e. media composition) and chemical properties of the particular metabolite. Thus, intracellular metabolite levels measured from samples collected by cold methanol quenching might be uncertain and variably biased due to corruption by described phenomena.

Authors: Maksim Zakhartsev, Oliver Vielhauer, Thomas Horn, Xuelian Yang, Matthias Reuss

Date Published: 1st Apr 2015

Publication Type: Not specified

Using a Genome-Scale Metabolic Model of Enterococcus faecalis V583 To Assess Amino Acid Uptake and Its Impact on Central Metabolism

Abstract (Expand)

Increasing antibiotic resistance in pathogenic bacteria necessitates the development of new medication strategies. Interfering with the metabolic network of the pathogen can provide novel drug targets … but simultaneously requires a deeper and more detailed organism-specific understanding of the metabolism, which is often surprisingly sparse. In light of this, we reconstructed a genome-scale metabolic model of the pathogen Enterococcus faecalis V583. The manually curated metabolic network comprises 642 metabolites and 706 reactions. We experimentally determined metabolic profiles of E. faecalis grown in chemically defined medium in an anaerobic chemostat setup at different dilution rates and calculated the net uptake and product fluxes to constrain the model. We computed growth-associated energy and maintenance parameters and studied flux distributions through the metabolic network. Amino acid auxotrophies were identified experimentally for model validation and revealed seven essential amino acids. In addition, the important metabolic hub of glutamine/glutamate was altered by constructing a glutamine synthetase knockout mutant. The metabolic profile showed a slight shift in the fermentation pattern toward ethanol production and increased uptake rates of multiple amino acids, especially l-glutamine and l-glutamate. The model was used to understand the altered flux distributions in the mutant and provided an explanation for the experimentally observed redirection of the metabolic flux. We further highlighted the importance of gene-regulatory effects on the redirection of the metabolic fluxes upon perturbation. The genome-scale metabolic model presented here includes gene-protein-reaction associations, allowing a further use for biotechnological applications, for studying essential genes, proteins, or reactions, and the search for novel drug targets.

Authors: N. Veith, M. Solheim, K. W. van Grinsven, B. G. Olivier, J. Levering, R. Grosseholz, J. Hugenholtz, H. Holo, I. Nes, B. Teusink, U. Kummer

Date Published: 19th Dec 2014

Publication Type: Not specified

Evolution, dynamics and specialized functions of glycosomes in metabolism and development of trypanosomatids

Abstract (Expand)

Kinetoplastea such as trypanosomatid parasites contain specialized peroxisomes that uniquely contain enzymes of the glycolytic pathway and other parts of intermediary metabolism and hence are called … glycosomes. Their specific enzyme content can vary strongly, quantitatively and qualitatively, between different species and during the parasites’ life cycle. The correct sequestering of enzymes has great importance for the regulation of the trypanosomatids’ metabolism and can, dependent on environmental conditions, even be essential. Glycosomes also play a pivotal role in life-cycle regulation of Trypanosoma brucei, as the translocation of a protein phosphatase from the cytosol forms part of a crucial developmental control switch. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative for unique forms of activity regulation, whereas many kinetic activity regulation mechanisms common for glycolytic enzymes are absent in these organisms. Glycosome turnover occurs by autophagic degradation of redundant organelles and assembly of new ones. This may provide the trypanosomatids with a manner to rapidly and efficiently adapt their metabolism to the sudden, major nutritional changes often encountered during the life cycle. This could also have helped facilitating successful adaptation of kinetoplastids, at multiple occasions during evolution, to their parasitic life style.

Authors: Balázs Szöör, , Melisa Gualdrón-López, Paul AM Michels

Date Published: 1st Dec 2014

Publication Type: Not specified

Basic Regulatory Principles of Escherichia coli's Electron Transport Chain for Varying Oxygen Conditions

Abstract (Expand)

For adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known … that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system. In the following a mathematical model of the E. coli ETC linked to basic modules for substrate uptake, fermentation product excretion and biomass formation is introduced. The kinetic modelling focusses on regulatory principles of the ETC for varying oxygen conditions in glucose-limited continuous cultures. The model is based on the balance of electron donation (glucose) and acceptance (oxygen or other acceptors). Also, it is able to account for different chemostat conditions due to changed substrate concentrations and dilution rates. The parameter identification process is divided into an estimation and a validation step based on previously published and new experimental data. The model shows that experimentally observed, qualitatively different behaviour of the ubiquinone redox state and the ArcA activity profile in the micro-aerobic range for different experimental conditions can emerge from a single network structure. The network structure features a strong feed-forward effect from the FNR regulatory system to the ArcBA regulatory system via a common control of the dehydrogenases of the ETC. The model supports the hypothesis that ubiquinone but not ubiquinol plays a key role in determining the activity of ArcBA in a glucose-limited chemostat at micro-aerobic conditions.

Editor:

Date Published: 30th Sep 2014

Publication Type: Not specified

What makes the lac-pathway switch: identifying the fluctuations that trigger phenotype switching in gene regulatory systems

Abstract (Expand)

Multistable gene regulatory systems sustain different levels of gene expression under identical external conditions. Such multistability is used to encode phenotypic states in processes including … nutrient uptake and persistence in bacteria, fate selection in viral infection, cell-cycle control and development. Stochastic switching between different phenotypes can occur as the result of random fluctuations in molecular copy numbers of mRNA and proteins arising in transcription, translation, transport and binding. However, which component of a pathway triggers such a transition is generally not known. By linking single-cell experiments on the lactose-uptake pathway in E. coli to molecular simulations, we devise a general method to pinpoint the particular fluctuation driving phenotype switching and apply this method to the transition between the uninduced and induced states of the lac-genes. We find that the transition to the induced state is not caused only by the single event of lac-repressor unbinding, but depends crucially on the time period over which the repressor remains unbound from the lac-operon. We confirm this notion in strains with a high expression level of the lac-repressor (leading to shorter periods over which the lac-operon remains unbound), which show a reduced switching rate. Our techniques apply to multistable gene regulatory systems in general and allow to identify the molecular mechanisms behind stochastic transitions in gene regulatory circuits.

Editor:

Date Published: 24th Sep 2014

Publication Type: Not specified

Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae

Abstract (Expand)

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such … pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in 'transcription traffic jams' on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

Authors: , , M. Herber, L. Attaiech, , , S. Klumpp, ,

Date Published: 6th Sep 2014

Publication Type: Not specified

Transcriptome-wide analysis of trypanosome mRNA decay reveals complex degradation kinetics and suggests a role for co-transcriptional degradation in determining mRNA levels

Abstract (Expand)

African trypanosomes are an excellent system for quantitative modelling of post-transcriptional mRNA control. Transcription is constitutive and polycistronic; individual mRNAs are excised by trans … splicing and polyadenylation. We here measure mRNA decay kinetics in two life cycle stages, bloodstream and procyclic forms, by transcription inhibition and RNASeq. Messenger RNAs with short half-lives tend to show initial fast degradation, followed by a slower phase; they are often stabilized by depletion of the 5'-3' exoribonuclease XRNA. Many longer-lived mRNAs show initial slow degradation followed by rapid destruction: we suggest that the slow phase reflects gradual deadenylation. Developmentally regulated mRNAs often show regulated decay, and switch their decay pattern. Rates of mRNA decay are good predictors of steady state levels for short mRNAs, but mRNAs longer than 3 kb show unexpectedly low abundances. Modelling shows that variations in splicing and polyadenylation rates can contribute to steady-state mRNA levels, but this is completely dependent on competition between processing and co-transcriptional mRNA precursor destruction.

Authors: , M. Ryten, D. Droll, , V. Farber, , C. Merce, , ,

Date Published: 26th Aug 2014

Publication Type: Not specified

The silicon trypanosome: a test case of iterative model extension in systems biology

Abstract (Expand)

The African trypanosome, Trypanosoma brucei, is a unicellular parasite causing African Trypanosomiasis (sleeping sickness in humans and nagana in animals). Due to some of its unique properties, it has … emerged as a popular model organism in systems biology. A predictive quantitative model of glycolysis in the bloodstream form of the parasite has been constructed and updated several times. The Silicon Trypanosome is a project that brings together modellers and experimentalists to improve and extend this core model with new pathways and additional levels of regulation. These new extensions and analyses use computational methods that explicitly take different levels of uncertainty into account. During this project, numerous tools and techniques have been developed for this purpose, which can now be used for a wide range of different studies in systems biology.

Authors: , , , , , , T. Papamarkou, , , , , , , ,

Date Published: 7th May 2014

Publication Type: Not specified

Towards a Systems Level Understanding of the Oxygen Response of Escherichia coli

Abstract (Expand)

Escherichia coli is a facultatively anaerobic bacterium. With glucose if no external electron acceptors are available, ATP is produced by substrate level phosphorylation. The intracellular redox balance … is maintained by mixed-acid fermentation, that is, the production and excretion of several organic acids. When oxygen is available, E. coli switches to aerobic respiration to achieve redox balance and optimal energy conservation by proton translocation linked to electron transfer. The switch between fermentative and aerobic respiratory growth is driven by extensive changes in gene expression and protein synthesis, resulting in global changes in metabolic fluxes and metabolite concentrations. This oxygen response is determined by the interaction of global and local genetic regulatory mechanisms, as well as by enzymatic regulation. The response is affected by basic physical constraints such as diffusion, thermodynamics and the requirement for a balance of carbon, electrons and energy (predominantly the proton motive force and the ATP pool). A comprehensive systems level understanding of the oxygen response of E. coli requires the integrated interpretation of experimental data that are pertinent to the multiple levels of organization that mediate the response. In the pan-European venture, Systems Biology of Microorganisms (SysMO) and specifically within the project Systems Understanding of Microbial Oxygen Metabolism (SUMO), regulator activities, gene expression, metabolite levels and metabolic flux datasets were obtained using a standardized and reproducible chemostat-based experimental system. These different types and qualities of data were integrated using mathematical models. The approach described here has revealed a much more detailed picture of the aerobic-anaerobic response, especially for the environmentally critical microaerobic range that is located between unlimited oxygen availability and anaerobiosis.

Authors: , , , , , , S. Kunz, , , , , ,

Date Published: 7th May 2014

Publication Type: Not specified

Agent-based modeling of oxygen-responsive transcription factors in Escherichia coli

Abstract (Expand)

In the presence of oxygen (O2) the model bacterium Escherichia coli is able to conserve energy by aerobic respiration. Two major terminal oxidases are involved in this process - Cyo has a relatively … low affinity for O2 but is able to pump protons and hence is energetically efficient; Cyd has a high affinity for O2 but does not pump protons. When E. coli encounters environments with different O2 availabilities, the expression of the genes encoding the alternative terminal oxidases, the cydAB and cyoABCDE operons, are regulated by two O2-responsive transcription factors, ArcA (an indirect O2 sensor) and FNR (a direct O2 sensor). It has been suggested that O2-consumption by the terminal oxidases located at the cytoplasmic membrane significantly affects the activities of ArcA and FNR in the bacterial nucleoid. In this study, an agent-based modeling approach has been taken to spatially simulate the uptake and consumption of O2 by E. coli and the consequent modulation of ArcA and FNR activities based on experimental data obtained from highly controlled chemostat cultures. The molecules of O2, transcription factors and terminal oxidases are treated as individual agents and their behaviors and interactions are imitated in a simulated 3-D E. coli cell. The model implies that there are two barriers that dampen the response of FNR to O2, i.e. consumption of O2 at the membrane by the terminal oxidases and reaction of O2 with cytoplasmic FNR. Analysis of FNR variants suggested that the monomer-dimer transition is the key step in FNR-mediated repression of gene expression.

Authors: , , , S. Coakley, , ,

Date Published: 24th Apr 2014

Publication Type: Not specified

A mathematical model of metabolism and regulation provides a systems-level view of how Escherichia coli responds to oxygen

Abstract (Expand)

The efficient redesign of bacteria for biotechnological purposes, such as biofuel production, waste disposal or specific biocatalytic functions, requires a quantitative systems-level understanding of … energy supply, carbon, and redox metabolism. The measurement of transcript levels, metabolite concentrations and metabolic fluxes per se gives an incomplete picture. An appreciation of the interdependencies between the different measurement values is essential for systems-level understanding. Mathematical modeling has the potential to provide a coherent and quantitative description of the interplay between gene expression, metabolite concentrations, and metabolic fluxes. Escherichia coli undergoes major adaptations in central metabolism when the availability of oxygen changes. Thus, an integrated description of the oxygen response provides a benchmark of our understanding of carbon, energy, and redox metabolism. We present the first comprehensive model of the central metabolism of E. coli that describes steady-state metabolism at different levels of oxygen availability. Variables of the model are metabolite concentrations, gene expression levels, transcription factor activities, metabolic fluxes, and biomass concentration. We analyze the model with respect to the production capabilities of central metabolism of E. coli. In particular, we predict how precursor and biomass concentration are affected by product formation.

Editor:

Date Published: 27th Mar 2014

Publication Type: Not specified

Meeting report from the fourth meeting of the Computational Modeling in Biology Network (COMBINE)

Abstract (Expand)

The Computational Modeling in Biology Network (COMBINE) is an initiative to coordinate the development of community standards and formats in computational systems biology and related fields. This report … summarizes the topics and activities of the fourth edition of the annual COMBINE meeting, held in Paris during September 16-20 2013, and attended by a total of 96 people. This edition pioneered a first day devoted to modeling approaches in biology, which attracted a broad audience of scientists thanks to a panel of renowned speakers. During subsequent days, discussions were held on many subjects including the introduction of new features in the various COMBINE standards, new software tools that use the standards, and outreach efforts. Significant emphasis went into work on extensions of the SBML format, and also into community-building. This year’s edition once again demonstrated that the COMBINE community is thriving, and still manages to help coordinate activities between different standards in computational systems biology.

Authors: Dagmar Waltemath, Frank T. Bergmann, Claudine Chaouiya, Tobias Czauderna, Padraig Gleeson, , Martin Golebiewski, Michael Hucka, Nick Juty, , Nicolas Le Novère, Huaiyu Mi, Ion I. Moraru, Chris J. Myers, David Nickerson, Brett G. Olivier, Nicolas Rodriguez, Falk Schreiber, Lucian Smith, Fengkai Zhang, Eric Bonnet

Date Published: 15th Mar 2014

Publication Type: Journal

Analysis of Escherichia coli mutants with a linear respiratory chain

Abstract (Expand)

The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I … and II, as well as of three terminal oxidases. They differ with respect to energetic efficiency (proton translocation) and their affinity to the different quinone/quinol species and oxygen. In order to analyze the advantages of the branched electron transport chain over a linear one and to assess how usage of the different terminal oxidases determines growth behavior at varying oxygen concentrations, a set of isogenic mutant strains was created, which lack NADH dehydrogenase I as well as two of the terminal oxidases, resulting in strains with a linear respiratory chain. These strains were analyzed in glucose-limited chemostat experiments with defined oxygen supply, adjusting aerobic, anaerobic and different microaerobic conditions. In contrast to the wild-type strain MG1655, the mutant strains produced acetate even under aerobic conditions. Strain TBE032, lacking NADH dehydrogenase I and expressing cytochrome bd-II as sole terminal oxidase, showed the highest acetate formation rate under aerobic conditions. This supports the idea that cytochrome bd-II terminal oxidase is not able to catalyze the efficient oxidation of the quinol pool at higher oxygen conditions, but is functioning mainly under limiting oxygen conditions. Phosphorylation of ArcA, the regulator of the two-component system ArcBA, besides Fnr the main transcription factor for the response towards different oxygen concentrations, was studied. Its phosphorylation pattern was changed in the mutant strains. Dephosphorylation and therefore inactivation of ArcA started at lower aerobiosis levels than in the wild-type strain. Notably, not only the micro- and aerobic metabolism was affected by the mutations, but also the anaerobic metabolism, where the respiratory chain should not be important.

Editor:

Date Published: 27th Jan 2014

Publication Type: Not specified

Systems Biology of Monovalent Cation Homeostasis in Yeast

Abstract (Expand)

Maintenance of monovalent cation homeostasis (mainly K(+) and Na(+)) is vital for cell survival, and cation toxicity is at the basis of a myriad of relevant phenomena, such as salt stress in crops and …d diverse human diseases. Full understanding of the importance of monovalent cations in the biology of the cell can only be achieved from a systemic perspective. Translucent is a multinational project developed within the context of the SysMO (System Biology of Microorganisms) initiative and focussed in the study of cation homeostasis using the well-known yeast Saccharomyces cerevisiae as a model. The present review summarize how the combination of biochemical, genetic, genomic and computational approaches has boosted our knowledge in this field, providing the basis for a more comprehensive and coherent vision of the role of monovalent cations in the biology of the cell.

Authors: , Ebru Aydar, Samuel Drulhe, Daniel Ganser, , , , , Lynne Yenush, Olga Zimmermannová, G. Paul H. van Heusden, , , Chris Palmer, ,

Date Published: 2014

Publication Type: Not specified

Handling uncertainty in dynamic models: the pentose phosphate pathway in Trypanosoma brucei

Abstract (Expand)

Dynamic models of metabolism can be useful in identifying potential drug targets, especially in unicellular organisms. A model of glycolysis in the causative agent of human African trypanosomiasis, … Trypanosoma brucei, has already shown the utility of this approach. Here we add the pentose phosphate pathway (PPP) of T. brucei to the glycolytic model. The PPP is localized to both the cytosol and the glycosome and adding it to the glycolytic model without further adjustments leads to a draining of the essential bound-phosphate moiety within the glycosome. This phosphate "leak" must be resolved for the model to be a reasonable representation of parasite physiology. Two main types of theoretical solution to the problem could be identified: (i) including additional enzymatic reactions in the glycosome, or (ii) adding a mechanism to transfer bound phosphates between cytosol and glycosome. One example of the first type of solution would be the presence of a glycosomal ribokinase to regenerate ATP from ribose 5-phosphate and ADP. Experimental characterization of ribokinase in T. brucei showed that very low enzyme levels are sufficient for parasite survival, indicating that other mechanisms are required in controlling the phosphate leak. Examples of the second type would involve the presence of an ATP:ADP exchanger or recently described permeability pores in the glycosomal membrane, although the current absence of identified genes encoding such molecules impedes experimental testing by genetic manipulation. Confronted with this uncertainty, we present a modeling strategy that identifies robust predictions in the context of incomplete system characterization. We illustrate this strategy by exploring the mechanism underlying the essential function of one of the PPP enzymes, and validate it by confirming the model predictions experimentally.

Authors: , , V. P. Alibu, R. J. Burchmore, I. H. Gilbert, M. Trybilo, N. N. Driessen, D. Gilbert, , ,

Date Published: 5th Dec 2013

Publication Type: Not specified

Kinase activity of ArcB from Escherichia coli is subject to regulation by both ubiquinone and demethylmenaquinone

Abstract (Expand)

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is … controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.

Authors: P. Sharma, S. Stagge, M. Bekker, K. Bettenbrock, K. J. Hellingwerf

Date Published: 7th Oct 2013

Publication Type: Not specified

Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging

Abstract (Expand)

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell … biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two "superfolder" GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

Authors: W. Overkamp, K. Beilharz, R. Detert Oude Weme, A. Solopova, H. Karsens, A. Kovacs, J. Kok, ,

Date Published: 16th Aug 2013

Publication Type: Not specified

Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

Abstract

Not specified

Editor:

Date Published: 25th Jul 2013

Publication Type: Not specified

Intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in Sulfolobus solfataricus

Abstract (Expand)

Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature … instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/ phosphatase maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1 hour period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the systems uxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3 bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency. This article is protected by copyright. All rights reserved.

Authors: , Dominik Esser, Julia Kort, , ,

Date Published: 20th Jul 2013

Publication Type: Not specified

Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modelling

Abstract (Expand)

In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione [bis(glutathionyl)spermidine, T(SH)2]. Here we present a … thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays Km-values for GSH, ATP, spermidine and Gsp of 34, 18, 687, and 32 μM, respectively, as well as Ki-values for GSH and T(SH)2 of 1 mM and 360 μM, respectively. As Gsp hydrolysis has a Km-value of 5.6 mM, the in vivo amidase activity is probably negligible. To obtain a deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This systems biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.

Editor:

Date Published: 3rd Jul 2013

Publication Type: Not specified

Kinetic characterization of arginine deiminase and carbamate kinase from Streptococcus pyogenes M49

Abstract (Expand)

Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen causing mild superficial infections of skin and mucous membranes, but also life-threatening systemic diseases. S. … pyogenes and other prokaryotic organisms use the arginine deiminase system (ADS) for survival in acidic environments. In this study, the arginine deiminase (AD), and carbamate kinase (CK) from S. pyogenes M49 strain 591 were heterologously expressed in E. coli DH5α, purified, and kinetically characterized. AD and CK from S. pyogenes M49 share high amino acid sequence similarity with the respective enzymes from Lactococcus lactis subsp. lactis IL1403 (45.6% and 53.5% identical amino acids) and Enterococcus faecalis V583 (66.8% and 66.8% identical amino acids). We found that the arginine deiminase of S. pyogenes is not allosterically regulated by the intermediates and products of the arginine degradation (E. g, ATP, citrulline, carbamoyl phosphate). The Km and Vmax values for arginine were 1.13±0.12 mM (mean ± SD) and 1.51±0.07 μmol/min/mg protein. The carbamate kinase is inhibited by ATP but unaffected by arginine and citrulline. The Km and Vmax values for ADP were 0.72±0.08 mM and 1.10±0.10 μmol/min/mg protein and the Km for carbamoyl phosphate was 0.65±0.07 mM. The optimum pH and temperature for both enzymes were 6.5 and 37°C, respectively.

Editor:

Date Published: 1st Jul 2013

Publication Type: Not specified

Regulation of the Activity of Lactate Dehydrogenases from Four Lactic Acid Bacteria

Abstract

Not specified

Authors: , , H. Messiha, , , , , , ,

Date Published: 17th May 2013

Publication Type: Not specified

A shift in the dominant phenotype governs the pH-induced metabolic switch of Clostridium acetobutylicumin phosphate-limited continuous cultures

Abstract (Expand)

In response to changing extracellular pH levels, phosphate-limited continuous cultures of Clostridium acetobutylicum reversibly switches its metabolism from the dominant formation of acids to the … prevalent production of solvents. Previous experimental and theoretical studies have revealed that this pH-induced metabolic switch involves a rearrangement of the intracellular transcriptomic, proteomic and metabolomic composition of the clostridial cells. However, the influence of the population dynamics on the observations reported has so far been neglected. Here, we present a method for linking the pH shift, clostridial growth and the acetone-butanol-ethanol fermentation metabolic network systematically into a model which combines the dynamics of the external pH and optical density with a metabolic model. Furthermore, the recently found antagonistic expression pattern of the aldehyde/alcohol dehydrogenases AdhE1/2 and pH-dependent enzyme activities have been included into this combined model. Our model predictions reveal that the pH-induced metabolic shift under these experimental conditions is governed by a phenotypic switch of predominantly acidogenic subpopulation towards a predominantly solventogenic subpopulation. This model-driven explanation of the pH-induced shift from acidogenesis to solventogenesis by population dynamics casts an entirely new light on the clostridial response to changing pH levels. Moreover, the results presented here underline that pH-dependent growth and pH-dependent specific enzymatic activity play a crucial role in this adaptation. In particular, the behaviour of AdhE1 and AdhE2 seems to be the key factor for the product formation of the two phenotypes, their pH-dependent growth, and thus, the pH-induced metabolic switch in C. acetobutylicum.

Editor:

Date Published: 3rd May 2013

Publication Type: Not specified

Stealthy annotation of experimental biology by spreadsheets

Abstract (Expand)

The increase in volume and complexity of biological data has led to increased requirements to reuse that data. Consistent and accurate metadata is essential for this task, creating new challenges in … semantic data annotation and in the constriction of terminologies and ontologies used for annotation. The BioSharing community are developing standards and terminologies for annotation, which have been adopted across bioinformatics, but the real challenge is to make these standards accessible to laboratory scientists. Widespread adoption requires the provision of tools to assist scientists whilst reducing the complexities of working with semantics. This paper describes unobtrusive ‘stealthy’ methods for collecting standards compliant, semantically annotated data and for contributing to ontologies used for those annotations. Spreadsheets are ubiquitous in laboratory data management. Our spreadsheet-based RightField tool enables scientists to structure information and select ontology terms for annotation within spreadsheets, producing high quality, consistent data without changing common working practices. Furthermore, our Populous spreadsheet tool proves effective for gathering domain knowledge in the form of Web Ontology Language (OWL) ontologies. Such a corpus of structured and semantically enriched knowledge can be extracted in Resource Description Framework (RDF), providing further means for searching across the content and contributing to Open Linked Data (http://linkeddata.org/)

Authors: , , Matthew Horridge, Simon Jupp, , , , , Robert Stevens,

Date Published: 1st Feb 2013

Publication Type: Journal

Integrative modelling of pH-dependent enzyme activity and transcriptomic regulation of the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum in continuous culture

Abstract (Expand)

In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady-state conditions between pH 5.7 and pH 4.5 … we show that the switch from acidogenesis to solventogenesis occurs between pH 5.3 and pH 5.0 with an intermediate state at pH 5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone–butanol–ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n-butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH-dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C. acetobutylicum.

Editor:

Date Published: 1st Feb 2013

Publication Type: Not specified

Naphthoquinone derivatives exert their antitrypanosomal activity via a multi-target mechanism

Abstract (Expand)

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, … 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Authors: S. Pieretti, , M. Mazet, R. Perozzo, C. Bergamini, F. Prati, R. Fato, G. Lenaz, G. Capranico, R. Brun, , P. A. Michels, L. Scapozza, M. L. Bolognesi, A. Cavalli

Date Published: 17th Jan 2013

Publication Type: Not specified

Simulations of stressosome activation emphasize allosteric interactions between RsbR and RsbT

Abstract (Expand)

Background The stressosome is a bacterial signalling complex that responds to environmental changes by initiating a protein partner switching cascade, which leads to the release of the alternative sigma … factor, sigmaB. Stress perception increases the phosphorylation of the stressosome sensor protein, RsbR, and the scaffold protein, RsbS, by the protein kinase RsbT. Subsequent dissociation of RsbT from the stressosome activates the sigmaB cascade. However, the sequence of physical events that occur in the stressosome during signal transduction is insufficiently understood. Results Here, we use computational modelling to correlate the structure of the stressosome with the efficiency of the phosphorylation reactions that occur upon activation by stress. In our model, the phosphorylation of any stressosome protein is dependent upon its nearest neighbours and their phosphorylation status. We compare different hypotheses about stressosome activation and find that only the model representing the allosteric activation of the kinase RsbT, by phosphorylated RsbR, qualitatively reproduces the experimental data. Conclusions Our simulations and the associated analysis of published data support the following hypotheses: (i) a simple Boolean model is capable of reproducing stressosome dynamics, (ii) different stressors induce identical stressosome activation patterns, and we also confirm that (i) phosphorylated RsbR activates RsbT, and (ii) the main purpose of RsbX is to dephosphorylate RsbS-P.

Authors: , , Jon Marles-Wright, ,

Date Published: 2013

Publication Type: Not specified

Semantic Data and Models Sharing in Systems Biology: The Just Enough Results Model and the SEEK Platform

Abstract (Expand)

Research in Systems Biology involves integrating data and knowledge about the dynamic processes in biological systems in order to understand and model them. Semantic web technologies should be ideal … for exploring the complex networks of genes, proteins and metabolites that interact, but much of this data is not natively available to the semantic web. Data is typically collected and stored with free-text annotations in spreadsheets, many of which do not conform to existing metadata standards and are often not publically released. Along with initiatives to promote more data sharing, one of the main challenges is therefore to semantically annotate and extract this data so that it is available to the research community. Data annotation and curation are expensive and undervalued tasks that have enormous benefits to the discipline as a whole, but fewer benefits to the individual data producers. By embedding semantic annotation into spreadsheets, however, and automatically extracting this data into RDF at the time of repository submission, the process of producing standards-compliant data, that is available for semantic web querying, can be achieved without adding additional overheads to laboratory data management. This paper describes these strategies in the context of semantic data management in the SEEK. The SEEK is a web-based resource for sharing and exchanging Systems Biology data and models that is underpinned by the JERM ontology (Just Enough Results Model), which describes the relationships between data, models, protocols and experiments. The SEEK was originally developed for SysMO, a large European Systems Biology consortium studying micro-organisms, but it has since had widespread adoption across European Systems Biology.

Editor: David Hutchison and Takeo Kanade and Josef Kittler and Jon M. Kleinberg and Friedemann Mattern and John C. Mitchell and Moni Naor and Oscar Nierstrasz and C. Pandu Rangan and Bernhard Steffen and Madhu Sudan and Demetri Terzopoulos and Doug Tygar and Moshe Y. Vardi and Gerhard Weikum and Camille Salinesi and Moira C. Norrie and Óscar Pastor

Date Published: 2013

Publication Type: Journal

Osmoprotection of Bacillus subtilis through Import and Proteolysis of Proline-Containing Peptides

Abstract

Not specified

Authors: , J. Brill, M. Thuring, G. Wunsche, M. Heun, H. Barzantny, ,

Date Published: 28th Dec 2012

Publication Type: Not specified

Novel twin-arginine translocation pathway-dependent phenotypes of Bacillus subtilis unveiled by quantitative proteomics

Abstract (Expand)

The Twin-arginine Translocation (Tat) pathway is known to translocate fully folded proteins across bacterial, archaeal and organellar membranes. To date, the mechanisms involved in processing, proofreading … and quality control of Tat substrates have remained largely elusive. Bacillus subtilis is an industrially relevant Gram-positive model bacterium. The Tat pathway in B. subtilis differs from that of other well-studied organisms in that it is composed of two complexes operating in parallel. To obtain a better understanding of this pathway in B. subtilis and to identify Tat-associated proteins, the B. subtilis 'Tat proteome' was investigated by quantitative proteomics. Metabolically labeled proteins from cytoplasmic, membrane and extracellular fractions were analyzed by LC-MS/MS. Changes in the amounts of identified peptides allowed for quantitative comparisons of their abundance in tat mutant strains. The observed differences were suggestive of indirect or direct protein-protein relationships. The rich data set generated was then approached in hypothesis-driving and hypothesis-driven manners. The hypothesis-driving approach led to the identification of a novel delayed biofilm phenotype of certain tat mutant strains, whereas the hypothesis-driven approach identified the membrane protein QcrA as a new Tat substrate of B. subtilis. Thus, our quantitative proteomics analyses have unveiled novel Tat pathway-dependent phenotypes in Bacillus.

Authors: Vivianne J Goosens, Andreas Otto, Corinna Glasner, Carmine G Monteferrante, René van der Ploeg, , Dörte Becher,

Date Published: 22nd Dec 2012

Publication Type: Not specified

The DEAD-box RNA helicases in Bacillus subtilis have multiple functions and act independent from each other

Abstract (Expand)

DEAD-box RNA helicases play important roles in remodeling RNA molecules and in facilitating a variety of RNA-protein interactions that are key to many essential cellular processes. In spite of the … importance of RNA, our knowledge about RNA helicases is only limited. In this study we investigated the role of the four DEAD-box RNA helicases in Gram positive model-organism Bacillus subtilis. A strain deleted of all RNA helicases is able to grow at 37°C but not at lower temperatures. Especially the deletion of cshA, cshB or yfmL lead to cold-sensitive phenotypes. Moreover, these mutant strains exhibit unique defects in ribosome biogenesis suggesting distinct functions for the individual enzymes in this process. Based on protein accumulation, severity of the cold-sensitive phenotype and the interaction with components of the RNA degradosome, CshA is the major RNA helicase of B. subtilis. To unravel the functions of CshA in addition to ribosome biogenesis we conducted microarray analysis and identified the ysbAB and frlBONMD mRNAs as targets that are strongly affected by the deletion of the cshA gene. Our findings suggest that the different helicases make distinct contributions to the physiology of B. subtilis. Ribosome biogenesis and RNA degradation are two of their major tasks in B. subtilis.

Authors: Martin Lehnik-Habrink, Leonie Rempeters, Akos T Kovács, Christoph Wrede, Claudia Baierlein, Heike Krebber, ,

Date Published: 24th Nov 2012

Publication Type: Not specified

Osmotic control of opuA expression in Bacillus subtilis and its modulation in response to intracellular glycine betaine and proline pools

Abstract (Expand)

Glycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the build-up of large pools, whose size was sensitively determined by the degree … of the imposed osmotic stress. The amassing of glycine betaine caused a repression in the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was up-regulated in response to both sudden and sustained increases in the external osmolarity. Non-ionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. Expression of opuA was down regulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless whether this osmoprotectant was imported or newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was up-regulated in a B. subtilis mutant unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.

Authors: , Annette Wensing, Margot Brosius, , ,

Date Published: 24th Nov 2012

Publication Type: Not specified

GraphAlignment: Bayesian pairwise alignment of biological networks

Abstract (Expand)

ABSTRACT: BACKGROUND: With increased experimental availability and accuracy of bio-molecular networks, tools for their comparative and evolutionary analysis are needed. A key component for such studies … is the alignment of networks. RESULTS: We introduce the Bioconductor package GraphAlignment for pairwise alignment of bio-molecular networks. The alignment incorporates information both from network vertices and network edges and is based on an explicit evolutionary model, allowing inference of all scoring parameters directly from empirical data. We compare the performance of our algorithm to an alternative algorithm, Graemlin 2.0.On simulated data, GraphAlignment outperforms Graemlin 2.0 in several benchmarks except for computational complexity. When there is little or no noise in the data, GraphAlignment is slower than Graemlin 2.0. It is faster than Graemlin 2.0 when processing noisy data containing spurious vertex associations. Its typical case complexity grows approximately as O(N^2.6). On empirical bacterial protein-protein interaction networks (PIN) and gene co-expression networks, GraphAlignment outperforms Graemlin 2.0 with respect to coverage and specificity, albeit by a small margin. On large eukaryotic PIN, Graemlin 2.0 outperforms GraphAlignment. CONCLUSIONS: The GraphAlignment algorithm is robust to spurious vertex associations, correctly resolves paralogs, and shows very good performance in identification of homologous vertices defined by high vertex and/or interaction similarity.

Authors: Michal Kolar, Jörn Meier, Ville Mustonen, Michael Lässig,

Date Published: 21st Nov 2012

Publication Type: Not specified

Malate metabolism in Bacillus subtilis: distinct roles for three classes of malate-oxidizing enzymes

Abstract (Expand)

The Gram-positive soil bacterium Bacillus subtilis uses glucose and malate as the preferred carbon sources. In the presence of either glucose or malate, the expression of genes and operons for the … utilization of secondary carbon sources is subject to carbon catabolite repression. While glucose is a preferred substrate in many organisms from bacteria to man, the factors that contribute to the preference for malate have so far remained elusive. In this work, we have studied the contribution of the different malate-metabolizing enzymes in B. subtilis, and we have elucidated their distinct functions. The malate dehydrogenase and the phosphoenolpyruvate carboxykinase are both essential for malate utilization; they introduce malate into gluconeogenesis. The NADPH-generating malic enzyme YtsJ is important to establish the cellular pools of NADPH for anabolic reactions. Finally, the NADH-generating malic enzymes MaeA, MalS, and MleA are involved in keeping the ATP levels high. Together, this unique array of distinct activities makes malate a preferred carbon source for B. subtilis.

Authors: Frederik M Meyer,

Date Published: 10th Nov 2012

Publication Type: Not specified

Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems

Abstract (Expand)

In Escherichia coli several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase … system (glucose-PTS), but also the mannose-PTS, as well as the galactose and maltose transporters can translocate glucose. Mutant strains which lack the EIIBC protein of the glucose-PTS have been previously investigated because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit has not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as in glucose limited continuous cultivations. Deletion of EIIBC, disturbs glucose transport severely. cAMP-CRP levels rise, induction of the mgl-operon occurs. Nevertheless mgl transcription is not essential, as deletion of this transporter did not affect growth rate; the activities of the remaining transporters seems to be sufficient by induction of the galactose and maltose transporters. Despite the strong up-regulation of mgl under glucose limitations, deletion of this transport-system did not lead to further changes.

Editor:

Date Published: 8th Oct 2012

Publication Type: Not specified

Single cell analysis of gene expression patterns during carbon starvation in Bacillus subtilis reveals large phenotypic variation

Abstract (Expand)

How cells dynamically respond to fluctuating environmental conditions depends on the architecture and noise of the underlying genetic circuits. Most work characterizing stress pathways in the model … bacterium Bacillus subtilis has been performed on bulk cultures using ensemble assays. However, investigating the single cell response to stress is important since noise might generate significant phenotypic heterogeneity. Here, we study the stress response to carbon source starvation and compare both population and single cell data. Using a top-down approach, we investigate the transcriptional dynamics of various stress-related genes of B. subtilis in response to carbon source starvation and to increased cell density. Our data reveal that most of the tested gene-regulatory networks respond highly heterogeneously to starvation and cells show a large degree of variation in gene expression. The level of highly dynamic diversification within B. subtilis populations under changing environments reflects the necessity to study cells at the single cell level.

Editor:

Date Published: 4th Oct 2012

Publication Type: Not specified

‘Domino’ systems biology and the ‘A’ of ATP

Abstract (Expand)

We develop a strategic ‘domino’ approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to … explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in ATP upon glucose addition, (ii) the lack of increase in ADP when ATP is hydrolyzed, and (iii) the rapid disappearance of the ‘A’ (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of AMP explains. Cycling of the ‘A’ moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the ‘A’ component of ATP.

Editor:

Date Published: 1st Sep 2012

Publication Type: Not specified

On the function of the various quinone species in Escherichia coli

Abstract (Expand)

The respiratory chain of Escherichia coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which … has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E. coli accept electrons from this naphtoquinone derivative.

Authors: , , Klaas J. Hellingwerf,

Date Published: 1st Sep 2012

Publication Type: Not specified

High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis

Abstract (Expand)

In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA … (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity.

Authors: , Monika Pabijaniak, Anne de Jong, Robert Dűhring, , ,

Date Published: 17th Aug 2012

Publication Type: Not specified

Systems analysis of transcription factor activities in environments with stable and dynamic oxygen concentrations

Abstract (Expand)

Understanding gene regulation requires knowledge of changes in transcription factor (TF) activities. Simultaneous direct measurement of numerous TF activities is currently impossible. Nevertheless, … statistical approaches to infer TF activities have yielded non-trivial and verifiable predictions for individual TFs. Here, global statistical modelling identifies changes in TF activities from transcript profiles of Escherichia coli growing in stable (fixed oxygen availabilities) and dynamic (changing oxygen availability) environments. A core oxygen-responsive TF network, supplemented by additional TFs acting under specific conditions, was identified. The activities of the cytoplasmic oxygen-responsive TF, FNR, and the membrane-bound terminal oxidases implied that, even on the scale of the bacterial cell, spatial effects significantly influence oxygen-sensing. Several transcripts exhibited asymmetrical patterns of abundance in aerobic to anaerobic and anaerobic to aerobic transitions. One of these transcripts, ndh, encodes a major component of the aerobic respiratory chain and is regulated by oxygen-responsive TFs ArcA and FNR. Kinetic modelling indicated that ArcA and FNR behaviour could not explain the ndh transcript profile, leading to the identification of another TF, PdhR, as the source of the asymmetry. Thus, this approach illustrates how systematic examination of regulatory responses in stable and dynamic environments yields new mechanistic insights into adaptive processes.

Authors: , Andrea Ocone, Melanie R Stapleton, Simon Hall, Eleanor W Trotter, , ,

Date Published: 8th Aug 2012

Publication Type: Not specified

Uncoupling of substrate-level phosphorylation in Escherichia coli during glucose-limited growth

Abstract (Expand)

The respiratory chain of Escherichia coli contains three different cytochrome oxidases. Whereas the cytochrome bo oxidase and the cytochrome bd-I oxidase are well characterized and have been shown to … contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochrome bd-II oxidase. Recently, however, this view was challenged by an in vitro biochemical analysis that showed that the activity of cytochrome bd-II oxidase does contribute to proton translocation with an H(+)/e(-) stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growing E. coli strains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochrome bd-II oxidase, compatible with an H(+)/e(-) ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation in E. coli under energy-limited growth conditions.

Authors: , Klaas J Hellingwerf, Maarten J Teixeira de Mattos,

Date Published: 27th Jul 2012

Publication Type: Not specified

Multiple active centers of multi-subunit RNA polymerases

Abstract (Expand)

The active center of multi-subunit RNA polymerase consists of two modules, the Mg(2+) module, holding the catalytic Mg(2+) ion, and a module made of a flexible domain, the Trigger Loop. Uniquely, the … TL module can be substituted by alternative modules, thus changing the catalytic properties of the active center.

Authors: , Mohammad Roghanian,

Date Published: 10th Jul 2012

Publication Type: Not specified

From steady-state to synchronized yeast glycolytic oscillations I: model construction

Abstract (Expand)

An existing detailed kinetic model for the steady-state behavior of yeast glycolysis was tested for its ability to simulate dynamic behavior. Using a small subset of experimental data, the original … model was adapted by adjusting its parameter values in three optimization steps. Only small adaptations to the original model were required for realistic simulation of experimental data for limit-cycle oscillations. The greatest changes were required for parameter values for the phosphofructokinase reaction. The importance of ATP for the oscillatory mechanism and NAD(H) for inter-and intra-cellular communications and synchronization was evident in the optimization steps and simulation experiments. In an accompanying paper [du Preez F et al. (2012) FEBS J doi:10.1111/j.1742-4658.2012.08658.x], we validate the model for a wide variety of experiments on oscillatory yeast cells. The results are important for re-use of detailed kinetic models in modular modeling approaches and for approaches such as that used in the Silicon Cell initiative. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/dupreez/index.html.

Authors: , David D van Niekerk, Bob Kooi, Johann M Rohwer,

Date Published: 21st Jun 2012

Publication Type: Not specified

From steady-state to synchronized yeast glycolytic oscillations II: model validation

Abstract (Expand)

In an accompanying paper [du Preez et al., (2012) FEBS J doi: 10.1111/j.1742-4658.2012.08665.x], we adapt an existing kinetic model for steady-state yeast glycolysis to simulate limit-cycle oscillations. … Here we validate the model by testing its capacity to simulate a wide range of experiments on dynamics of yeast glycolysis. In addition to its description of the oscillations of glycolytic intermediates in intact cells and the rapid synchronization observed when mixing out-of-phase oscillatory cell populations (see accompanying paper), the model was able to predict the Hopf bifurcation diagram with glucose as the bifurcation parameter (and one of the bifurcation points with cyanide as the bifurcation parameter), the glucose- and acetaldehyde-driven forced oscillations, glucose and acetaldehyde quenching, and cell-free extract oscillations (including complex oscillations and mixed-mode oscillations). Thus, the model was compliant, at least qualitatively, with the majority of available experimental data for glycolytic oscillations in yeast. To our knowledge, this is the first time that a model for yeast glycolysis has been tested against such a wide variety of independent data sets. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/dupreez/index.html.

Authors: , David D van Niekerk,

Date Published: 13th Jun 2012

Publication Type: Not specified

Optimized submerged batch fermentation strategy for systems scale studies of metabolic switching in Streptomyces coelicolor A3(2)

Abstract (Expand)

BACKGROUND: Systems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth … and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2). RESULTS: By a step-wise approach, cultivation conditions and two fully defined cultivation media were developed and evaluated using strain M145 of S. coelicolor A3(2), providing a high degree of cultivation reproducibility and enabling reliable studies of the effect of phosphate depletion and L-glutamate depletion on the metabolic transition to antibiotic production phase. Interestingly, both of the two carbon sources provided, D-glucose and L-glutamate, were found to be necessary in order to maintain high growth rates and prevent secondary metabolite production before nutrient depletion. Comparative analysis of batch cultivations with (i) both L-glutamate and D-glucose in excess, (ii) L-glutamate depletion and D-glucose in excess, (iii) L-glutamate as the sole source of carbon and (iv) D-glucose as the sole source of carbon, reveal a complex interplay of the two carbon sources in the bacterium's central carbon metabolism. CONCLUSIONS: The present study presents for the first time a dedicated cultivation strategy fulfilling the requirements for systems biology studies of metabolic switching in S. coelicolor A3(2). Key results from labelling and cultivation experiments on either or both of the two carbon sources provided indicate that in the presence of D-glucose, L-glutamate was the preferred carbon source, while D-glucose alone appeared incapable of maintaining culture growth, likely due to a metabolic bottleneck at the oxidation of pyruvate to acetyl-CoA.

Authors: A. Wentzel, P. Bruheim, A. Overby, O. M. Jakobsen, H. Sletta, W. A. Omara, D. A. Hodgson, T. E. Ellingsen

Date Published: 9th Jun 2012

Publication Type: Not specified

Synthesis, release, and recapture of compatible solute proline by osmotically stressed Bacillus subtilis cells

Abstract (Expand)

Bacillus subtilis synthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm … and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, an opuE mutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of the opuE mutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of the opuE mutant was considerably lower than that of its opuE(+) parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels of B. subtilis participated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesized de novo and subsequently released by salt-stressed B. subtilis cells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered.

Authors: , Carsten von Blohn, Agnieszka Stanek, Susanne Moses, Helena Barzantny,

Date Published: 8th Jun 2012

Publication Type: Not specified

My data are your data

Abstract (Expand)

Encouraging more broad and inclusive data sharing in today's world will involve concerted community efforts to overcome technical barriers and human foibles. Vivien Marx investigates. (includes …s comments from Carole Goble, and mentions SysMO, SEEK and RightField).

Author: Vivien Marx

Date Published: 7th Jun 2012

Publication Type: Not specified

Sustained glycolytic oscillations in individual isolated yeast cells

Abstract (Expand)

Yeast glycolytic oscillations have been studied since the 1950s in cell-free extracts and intact cells. For intact cells, sustained oscillations have so far only been observed at the population level, … i.e. for synchronized cultures at high biomass concentrations. Using optical tweezers to position yeast cells in a microfluidic chamber, we were able to observe sustained oscillations in individual isolated cells. Using a detailed kinetic model for the cellular reactions, we simulated the heterogeneity in the response of the individual cells, assuming small differences in a single internal parameter. This is the first time that sustained limit-cycle oscillations have been demonstrated in isolated yeast cells. Database The mathematical model described here has been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/gustavsson/index.html free of charge.

Authors: Anna-Karin Gustavsson, David D van Niekerk, Caroline B Adiels, , Mattias Goksör,

Date Published: 23rd May 2012

Publication Type: Not specified

RNA degradation in Bacillus subtilis: an interplay of essential endo- and exoribonucleases

Abstract (Expand)

RNA processing and degradation are key processes in the control of transcript accumulation and thus in the control of gene expression. In Escherichia coli, the underlying mechanisms and components of … RNA decay are well characterized. By contrast, Gram-positive bacteria do not possess several important players of E. coli RNA degradation, most notably the essential enzyme RNase E. Recent research on the model Gram-positive organism, Bacillus subtilis, has identified the essential RNases J1 and Y as crucial enzymes in RNA degradation. While RNase J1 is the first bacterial exoribonuclease with 5'-to-3' processivity, RNase Y is the founding member of a novel class of endoribonucleases. Both RNase J1 and RNase Y have a broad impact on the stability of B. subtilis mRNAs; a depletion of either enzyme affects more than 25% of all mRNAs. RNases J1 and Y as well as RNase J2, the polynucleotide phosphorylase PNPase, the RNA helicase CshA and the glycolytic enzymes enolase and phosphofructokinase have been proposed to form a complex, the RNA degradosome of B. subtilis. This review presents a model, based on recent published data, of RNA degradation in B. subtilis. Degradation is initiated by RNase Y-dependent endonucleolytic cleavage, followed by processive exoribonucleolysis of the generated fragments both in 3'-to-5' and in 5'-to-3' directions. The implications of these findings for pathogenic Gram-positive bacteria are also discussed.

Authors: Martin Lehnik-Habrink, , ,

Date Published: 8th May 2012

Publication Type: Not specified

Proliferating bloodstream-form Trypanosoma brucei use a negligible part of consumed glucose for anabolic processes

Abstract (Expand)

Our quantitative knowledge of carbon fluxes in the long slender bloodstream form (BSF) Trypanosoma brucei is mainly based on non-proliferating parasites, isolated from laboratory animals and kept in … buffers. In this paper we present a carbon balance for exponentially growing bloodstream form trypanosomes. The cells grew with a doubling time of 5.3h, contained 46 mu mol of carbon (10(8) cells)(-1) and had a glucose consumption flux of 160 nmol min(-1) (10(8) cells)(-1). The molar ratio of pyruvate excreted versus glucose consumed was 2.1. Furthermore, analysis of the (13)C label distribution in pyruvate in (13)C-glucose incubations of exponentially growing trypanosomes showed that glucose was the sole substrate for pyruvate production. We conclude that the glucose metabolised in glycolysis was hardly, if at all, used for biosynthetic processes. Carbon flux through glycolysis in exponentially growing trypanosomes was 10 times higher than the incorporation of carbon into biomass. This biosynthetic carbon is derived from other precursors present in the nutrient rich growth medium. Furthermore, we found that the glycolytic flux was unaltered when the culture went into stationary phase, suggesting that most of the ATP produced in glycolysis is used for processes other than growth.

Authors: , A. van Tuijl, J. van Dam, W. van Winden, A. G. Tielens, J. J. van Hellemond,

Date Published: 8th May 2012

Publication Type: Not specified

Hypothesis: emergence of translation as a result of RNA helicase evolution

Abstract (Expand)

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had … already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.

Editor:

Date Published: 28th Apr 2012

Publication Type: Not specified

CcpA forms complexes with CodY and RpoA in Bacillus subtilis

Abstract (Expand)

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator which is controlled by interactions with the phosphoproteins HPrSer46P and CrhP and with low molecular … weight effectors depending on the availability of preferred carbon sources like glucose. Distinct point mutations in CcpA abolish regulation of some but not all target genes suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and mass spectrometry were applied to identify CcpA complexes active in repression and activation. To compensate for the excess of promoters only repressed by CcpA, this experiment was accomplished with cells with multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase (RNAP) subunits and the global regulator CodY were observed. Bacterial two-hybrid assays combining each RNAP subunit with CcpA localized CcpA binding at the α-subunit (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance (SPR) analyses confirmed binding of CcpA to the N- (αNTD) and C-terminal domains (αCTD) of RpoA as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by SPR. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are between 5 and 8μM. CcpA and CodY form a loose complex with a K(D) of 60μM. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Authors: Andrea Wünsche, Elke Hammer, , , Andreas Burkovski, ,

Date Published: 20th Apr 2012

Publication Type: Not specified

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

Abstract (Expand)

How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type … Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.

Authors: Katrin Beilharz, Linda Nováková, Daniela Fadda, Pavel Branny, Orietta Massidda,

Date Published: 21st Mar 2012

Publication Type: Not specified

The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach

Abstract (Expand)

BACKGROUND: Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made … biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures. RESULTS: We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g.(g.h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system. CONCLUSION: This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.

Authors: , I. F. Escapa, C. Jager, J. Puchalka, , , ,

Date Published: 20th Mar 2012

Publication Type: Not specified

Extracting regulator activity profiles by integration of de novo motifs and expression data: characterizing key regulators of nutrient depletion responses in Streptomyces coelicolor

Abstract (Expand)

Determining transcriptional regulator activities is a major focus of systems biology, providing key insight into regulatory mechanisms and co-regulators. For organisms such as Escherichia coli, … transcriptional regulator binding site data can be integrated with expression data to infer transcriptional regulator activities. However, for most organisms there is only sparse data on their transcriptional regulators, while their associated binding motifs are largely unknown. Here, we address the challenge of inferring activities of unknown regulators by generating de novo (binding) motifs and integrating with expression data. We identify a number of key regulators active in the metabolic switch, including PhoP with its associated directed repeat PHO box, candidate motifs for two SARPs, a CRP family regulator, an iron response regulator and that for LexA. Experimental validation for some of our predictions was obtained using gel-shift assays. Our analysis is applicable to any organism for which there is a reasonable amount of complementary expression data and for which motifs (either over represented or evolutionary conserved) can be identified in the genome.

Authors: M. Iqbal, Y. Mast, R. Amin, D. A. Hodgson, W. Wohlleben, N. J. Burroughs

Date Published: 13th Mar 2012

Publication Type: Not specified

Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis

Abstract (Expand)

Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report … the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Authors: Pierre Nicolas, , Etienne Dervyn, Tatiana Rochat, Aurélie Leduc, Nathalie Pigeonneau, Elena Bidnenko, Elodie Marchadier, Mark Hoebeke, Stéphane Aymerich, Dörte Becher, Paola Bisicchia, Eric Botella, Olivier Delumeau, Geoff Doherty, Emma L Denham, Mark J Fogg, Vincent Fromion, Anne Goelzer, Annette Hansen, Elisabeth Härtig, , Georg Homuth, Hanne Jarmer, Matthieu Jules, Edda Klipp, Ludovic Le Chat, François Lecointe, , Wolfram Liebermeister, Anika March, , , David Noone, Susanne Pohl, Bernd Rinn, Frank Rügheimer, , Franck Samson, Marc Schaffer, Benno Schwikowski, , , Thomas Wiegert, Kevin M Devine, Anthony J Wilkinson, , , , Philippe Bessières, Philippe Noirot

Date Published: 3rd Mar 2012

Publication Type: Not specified

A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum–cellular behavior in adaptation to n-butanol

Abstract (Expand)

To gain more insight into the butanol stress response of Clostridium acetobutylicum the transcriptional response of a steady state acidogenic culture to different levels of n-butanol (0.25-1%) was … investigated. No effect was observed on the fermentation pattern and expression of typical solvent genes (aad, ctfA/B, adc, bdhA/B, ptb, buk). Elevated levels of butanol mainly affected class I heat-shock genes (hrcA, grpE, dnaK, dnaJ, groES, groEL, hsp90), which were upregulated in a dose- and time-dependent manner, and genes encoding proteins involved in the membrane composition (fab and fad or glycerophospholipid related genes) and various ABC-transporters of unknown specificity. Interestingly, fab and fad genes were embedded in a large, entirely repressed cluster (CAC1988-CAC2019), which inter alia encoded an iron-specific ABC-transporter and molybdenum-cofactor synthesis proteins. Of the glycerophospholipid metabolism, the glycerol-3-phosphate dehydrogenase (glpA) gene was highly upregulated, whereas a glycerophosphodiester ABC-transporter (ugpAEBC) and a phosphodiesterase (ugpC) were repressed. On the megaplasmid, only a few genes showed differential expression, e.g. a rare lipoprotein (CAP0058, repressed) and a membrane protein (CAP0102, upregulated) gene. Observed transcriptional responses suggest that C. acetobutylicum reacts to butanol stress by induction of the general stress response and changing its cell envelope and transporter composition, but leaving the central catabolism unaffected. --------------------------------------------------------------------------------

Authors: , , Christina Grimmler, ,

Date Published: 1st Mar 2012

Publication Type: Not specified

Role of phosphate in the central metabolism of two lactic acid bacteria - a comparative systems biology approach

Abstract (Expand)

Lactic acid-producing bacteria survive in distinct environments, but show common metabolic characteristics. Here we studied the dynamic interactions of the central metabolism in Lactococcus lactis, … extensively used as starter in dairy industry, and Streptococcus pyogenes, a human pathogen. Glucose-pulse experiments and enzymatic measurements were performed to parameterize kinetic models of glycolysis. Significant improvements were made to existing kinetic models for L. lactis, which subsequently accelerated the development of the first kinetic model of S. pyogenes glycolysis. The models revealed an important role for extracellular phosphate in regulation of central metabolism and the efficient use of glucose. Thus, phosphate which is rarely taken into account as an independent species in models of central metabolism has to be considered more thoroughly in the analysis of metabolic systems in the future. Insufficient phosphate supply can lead to a strong inhibition of glycolysis at high glucose concentration in both species, but more severely in S. pyogenes. S. pyogenes is more efficient in converting glucose to ATP, showing a higher tendency towards heterofermentative energy metabolism than L. lactis. Our comparative systems biology approach revealed that the glycolysis of L. lactis and S. pyogenes have similar characteristics, but are adapted to their individual natural habitats with respect to phosphate regulation. The mathematical models described here have been submitted to the Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/levering/index.html free of charge.

Editor:

Date Published: 14th Feb 2012

Publication Type: Not specified

Toward interoperable bioscience data

Abstract (Expand)

To make full use of research data, the bioscience community needs to adopt technologies and reward mechanisms that support interoperability and promote the growth of an open 'data commoning' … culture. Here we describe the prerequisites for data commoning and present an established and growing ecosystem of solutions using the shared 'Investigation-Study-Assay' framework to support that vision.

Authors: Susanna-Assunta Sansone, Philippe Rocca-Serra, Dawn Field, Eamonn Maguire, Chris Taylor, Oliver Hofmann, Hong Fang, Steffen Neumann, Weida Tong, Linda Amaral-Zettler, Kimberly Begley, Tim Booth, Lydie Bougueleret, Gully Burns, Brad Chapman, Tim Clark, Lee-Ann Coleman, Jay Copeland, Sudeshna Das, Antoine de Daruvar, Paula de Matos, Ian Dix, Scott Edmunds, Chris T Evelo, Mark J Forster, Pascale Gaudet, Jack Gilbert, , Julian L Griffin, Daniel Jacob, Jos Kleinjans, Lee Harland, Kenneth Haug, Henning Hermjakob, Shannan J Ho Sui, Alain Laederach, Shaoguang Liang, Stephen Marshall, Annette McGrath, Emily Merrill, Dorothy Reilly, Magali Roux, Caroline E Shamu, Catherine A Shang, Christoph Steinbeck, Anne Trefethen, Bryn Williams-Jones, , Ioannis Xenarios, Winston Hide

Date Published: 28th Jan 2012

Publication Type: Not specified

Populous: a tool for building OWL ontologies from templates

Abstract (Expand)

BACKGROUND: Ontologies are being developed for the life sciences to standardise the way we describe and interpret the wealth of data currently being generated. As more ontology based applications begin … to emerge, tools are required that enable domain experts to contribute their knowledge to the growing pool of ontologies. There are many barriers that prevent domain experts engaging in the ontology development process and novel tools are needed to break down these barriers to engage a wider community of scientists. RESULTS: We present Populous, a tool for gathering content with which to construct an ontology. Domain experts need to add content, that is often repetitive in its form, but without having to tackle the underlying ontological representation. Populous presents users with a table based form in which columns are constrained to take values from particular ontologies. Populated tables are mapped to patterns that can then be used to automatically generate the ontology's content. These forms can be exported as spreadsheets, providing an interface that is much more familiar to many biologists. CONCLUSIONS: Populous's contribution is in the knowledge gathering stage of ontology development; it separates knowledge gathering from the conceptualisation and axiomatisation, as well as separating the user from the standard ontology authoring environments. Populous is by no means a replacement for standard ontology editing tools, but instead provides a useful platform for engaging a wider community of scientists in the mass production of ontology content.

Authors: Simon Jupp, Matthew Horridge, Luigi Iannone, Julie Klein, , Joost Schanstra, , Robert Stevens

Date Published: 25th Jan 2012

Publication Type: Not specified

Dynamic Modelling under Uncertainty: The Case of Trypanosoma brucei Energy Metabolism

Abstract (Expand)

Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the … parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki (http://silicotryp.ibls.gla.ac.uk/wiki/Gl​ycolysis). Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies.

Editor:

Date Published: 19th Jan 2012

Publication Type: Not specified

In vitro experimental system for analysis of transcription-translation coupling

Abstract (Expand)

Transcription and translation are coupled in bacteria, meaning that translation takes place co-transcriptionally. During transcription-translation, both machineries mutually affect each others' … functions, which is important for regulation of gene expression. Analysis of interactions between RNA polymerase (RNAP) and the ribosome, however, are limited due to the lack of an in vitro experimental system. Here, we report the development of an in vitro transcription coupled to translation system assembled from purified components. The system allows controlled stepwise transcription and simultaneous stepwise translation of the nascent RNA, and permits investigation of the interactions of RNAP with the ribosome, as well as the effects of translation on transcription and transcription on translation. As an example of usage of this experimental system, we uncover complex effects of transcription-translation coupling on pausing of transcription.

Authors: Daniel Castro-Roa,

Date Published: 3rd Jan 2012

Publication Type: Not specified

Proteolysis of beta-galactosidase following SigmaB activation in Bacillus subtilis

Abstract (Expand)

In Bacillus subtilis the σB mediated general stress response provides protection against various environmental and energy related stress conditions. To better understand the general stress response, … we need to explore the mechanism by which the components interact. Here, we performed experiments in B. subtilis wild type and mutant strains to test and validate a mathematical model of the dynamics of σB activity. In the mutant strain BSA115, σB transcription is inducible by the addition of IPTG and negative control of σB activity by the anti-sigma factor RsbW is absent. In contrast to our expectations of a continuous β-galactosidase activity from a ctc::lacZ fusion, we observed a transient activity in the mutant. To explain this experimental finding, we constructed mathematical models reflecting different hypotheses regarding the regulation of σB and β-galactosidase dynamics. Only the model assuming instability of either ctc::lacZ mRNA or β-galactosidase protein is able to reproduce the experiments in silico. Subsequent Northern blot experiments revealed stable high-level ctc::lacZ mRNA concentrations after the induction of the σB response. Therefore, we conclude that protein instability following σB activation is the most likely explanation for the experimental observations. Our results thus support the idea that B. subtilis increases the cytoplasmic proteolytic degradation to adapt the proteome in face of environmental challenges following activation of the general stress response. The findings also have practical implications for the analysis of stress response dynamics using lacZ reporter gene fusions, a frequently used strategy for the σB response.

Authors: , , , , Georg Homuth, ,

Date Published: 2012

Publication Type: Not specified

Modelling complex biological systems using an agent-based approach

Abstract (Expand)

Many of the complex systems found in biology are comprised of numerous components, where interactions between individual agents result in the emergence of structures and function, typically in a highly … dynamic manner. Often these entities have limited lifetimes but their interactions both with each other and their environment can have profound biological consequences. We will demonstrate how modelling these entities, and their interactions, can lead to a new approach to experimental biology bringing new insights and a deeper understanding of biological systems.

Authors: , Salem Adra, Mesude Bicak, Shawn Chin, Simon Coakley, , , Chris Greenough, Duncan Jackson, Mariam Kiran, Sheila MacNeil, , Phil McMinn, Mark Pogson, , Eva Qwarnstrom, Francis Ratnieks, , Rod Smallwood, Tao Sun, David Worth

Date Published: 2012

Publication Type: Not specified

Intracellular Metabolite Pool Changes in Response to Nutrient Depletion Induced Metabolic Switching in Streptomyces coelicolor

Abstract (Expand)

A metabolite profiling study of the antibiotic producing bacterium Streptomyces coelicolor A3(2) has been performed. The aim of this study was to monitor intracellular metabolite pool changes occurring … as strains of S. coelicolor react to nutrient depletion with metabolic re-modeling, so-called metabolic switching, and transition from growth to secondary metabolite production phase. Two different culture media were applied, providing depletion of the key nutrients phosphate and L-glutamate, respectively, as the triggers for metabolic switching. Targeted GC-MS and LC-MS methods were employed to quantify important primary metabolite groups like amino acids, organic acids, sugar phosphates and other phosphorylated metabolites, and nucleotides in time-course samples withdrawn from fully-controlled batch fermentations. A general decline, starting already in the early growth phase, was observed for nucleotide pools and phosphorylated metabolite pools for both the phosphate and glutamate limited cultures. The change in amino acid and organic acid pools were more scattered, especially in the phosphate limited situation while a general decrease in amino acid and non-amino organic acid pools was observed in the L-glutamate limited situation. A phoP deletion mutant showed basically the same metabolite pool changes as the wild-type strain M145 when cultivated on phosphate limited medium. This implies that the inactivation of the phoP gene has only little effect on the detected metabolite levels in the cell. The energy charge was found to be relatively constant during growth, transition and secondary metabolite production phase. The results of this study and the employed targeted metabolite profiling methodology are directly relevant for the evaluation of precursor metabolite and energy supply for both natural and heterologous production of secondary metabolites in S. coelicolor.

Authors: A. Wentzel, H. Sletta, T. E. Ellingsen, P. Bruheim

Date Published: 2012

Publication Type: Not specified

Dissection of the network of interactions that links RNA processing with glycolysis in the Bacillus subtilis degradosome

Abstract (Expand)

The RNA degradosome is a multiprotein macromolecular complex that is involved in the degradation of messenger RNA in bacteria. The composition of this complex has been found to display a high degree … of evolutionary divergence, which may reflect the adaptation of species to different environments. Recently, a degradosome-like complex identified in Bacillus subtilis was found to be distinct from those found in proteobacteria, the degradosomes of which are assembled around the unstructured C-terminus of ribonuclease E, a protein not present in B. subtilis. In this report, we have investigated in vitro the binary interactions between degradosome components and have characterized interactions between glycolytic enzymes, RNA-degrading enzymes, and those that appear to link these two cellular processes. The crystal structures of the glycolytic enzymes phosphofructokinase and enolase are presented and discussed in relation to their roles in the mediation of complex protein assemblies. Taken together, these data provide valuable insights into the structure and dynamics of the RNA degradosome, a fascinating and complex macromolecular assembly that links RNA degradation with central carbon metabolism.

Authors: , Lorraine Hewitt, Cecilia Rodrigues, Alexandra S Solovyova, ,

Date Published: 16th Dec 2011

Publication Type: Not specified

A high-frequency mutation in Bacillus subtilis: requirements for the decryptification of the gudB glutamate dehydrogenase gene

Abstract (Expand)

Common laboratory strains of Bacillus subtilis encode two glutamate dehydrogenases: the enzymatically active protein RocG and the cryptic enzyme GudB that is inactive due to a duplication of three amino … acids in its active center. The inactivation of the rocG gene results in poor growth of the bacteria on complex media due to the accumulation of toxic intermediates. Therefore, rocG mutants readily acquire suppressor mutations that decryptify the gudB gene. This decryptification occurs by a precise deletion of one part of the 9-bp direct repeat that causes the amino acid duplication. This mutation occurs at the extremely high frequency of 10(-4). Mutations affecting the integrity of the direct repeat result in a strong reduction of the mutation frequency; however, the actual sequence of the repeat is not essential. The mutation frequency of gudB was not affected by the position of the gene on the chromosome. When the direct repeat was placed in the completely different context of an artificial promoter, the precise deletion of one part of the repeat was also observed, but the mutation frequency was reduced by 3 orders of magnitude. Thus, transcription of the gudB gene seems to be essential for the high frequency of the appearance of the gudB1 mutation. This idea is supported by the finding that the transcription-repair coupling factor Mfd is required for the decryptification of gudB. The Mfd-mediated coupling of transcription to mutagenesis might be a built-in precaution that facilitates the accumulation of mutations preferentially in transcribed genes.

Authors: Katrin Gunka, Stefan Tholen, Jan Gerwig, Christina Herzberg, , Fabian M Commichau

Date Published: 16th Dec 2011

Publication Type: Not specified

Reproducible computational biology experiments with SED-ML--the Simulation Experiment Description Markup Language

Abstract (Expand)

The increasing use of computational simulation experiments to inform modern biological research creates new challenges to annotate, archive, share and reproduce such experiments. The recently published … Minimum Information About a Simulation Experiment (MIASE) proposes a minimal set of information that should be provided to allow the reproduction of simulation experiments among users and software tools.

Authors: Dagmar Waltemath, Richard Adams, Frank T Bergmann, Michael Hucka, Fedor Kolpakov, Andrew K Miller, Ion I Moraru, David Nickerson, Sven Sahle, , Nicolas Le Novère

Date Published: 15th Dec 2011

Publication Type: Not specified

Metabolic switches and adaptations deduced from the proteomes of Streptomyces coelicolor wild type and phoP mutant grown in batch culture

Abstract (Expand)

Bacteria in the genus Streptomyces are soil-dwelling oligotrophs and important producers of secondary metabolites. Previously, we showed that global messenger RNA expression was subject to a series of … metabolic and regulatory switches during the lifetime of a fermentor batch culture of Streptomyces coelicolor M145. Here we analyze the proteome from eight time points from the same fermentor culture and, because phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (DeltaphoP), defective in the control of phosphate utilization. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index, which also revealed the importance of stress pathway proteins in both cultures. As expected, the DeltaphoP mutant was deficient in expression of PhoP-dependent genes, and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that coordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures.

Authors: L. Thomas, D. A. Hodgson, A. Wentzel, K. Nieselt, T. E. Ellingsen, J. Moore, E. R. Morrissey, R. Legaie, W. Wohlleben, A. Rodriguez-Garcia, J. F. Martin, N. J. Burroughs, E. M. Wellington, M. C. Smith

Date Published: 8th Dec 2011

Publication Type: Not specified

Simplified absolute metabolite quantification by gas chromatography–isotope dilution mass spectrometry on the basis of commercially available source material

Abstract (Expand)

In the field of metabolomics, GC–MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or … extracellular metabolites is nowadays mostly accomplished by application of diverse LC–MS techniques. Only few groups have so far adopted GC–MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography–isotope dilution mass spectrometry (GC–IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available 13C-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed.

Authors: Oliver Vielhauer, , Thomas Horn, Ralf Takors,

Date Published: 1st Dec 2011

Publication Type: Not specified

Factor-independent transcription pausing caused by recognition of the RNA-DNA hybrid sequence

Abstract (Expand)

Pausing of transcription is an important step of regulation of gene expression in bacteria and eukaryotes. Here we uncover a factor-independent mechanism of transcription pausing, which is determined … by the ability of the elongating RNA polymerase to recognize the sequence of the RNA-DNA hybrid. We show that, independently of thermodynamic stability of the elongation complex, RNA polymerase directly 'senses' the shape and/or identity of base pairs of the RNA-DNA hybrid. Recognition of the RNA-DNA hybrid sequence delays translocation by RNA polymerase, and thus slows down the nucleotide addition cycle through 'in pathway' mechanism. We show that this phenomenon is conserved among bacterial and eukaryotic RNA polymerases, and is involved in regulatory pauses, such as a pause regulating the production of virulence factors in some bacteria and a pause regulating transcription/replication of HIV-1. The results indicate that recognition of RNA-DNA hybrid sequence by multi-subunit RNA polymerases is involved in transcription regulation and may determine the overall rate of transcription elongation.

Authors: Aleksandra Bochkareva, , Vasisht R Tadigotla,

Date Published: 29th Nov 2011

Publication Type: Not specified

Carbon source control of the phosphorylation state of the Bacillus subtilis carbon-flux regulator Crh in vivo

Abstract (Expand)

Bacillus subtilis possesses carbon-flux regulating histidine protein (Crh), a paralog of the histidine protein (HPr) of the phosphotransferase system (PTS). Like HPr, Crh becomes (de)phosphorylated in … vitro at residue Ser46 by the metabolite-controlled HPr kinase/phosphorylase HPrK/P. Depending on its phosphorylation state, Crh exerts regulatory functions in connection with carbohydrate metabolism. So far, knowledge on phosphorylation of Crh in vivo has been limited and derived from indirect evidence. Here, we studied the dynamics of Crh phosphorylation directly by non-denaturing gel electrophoresis followed by Western analysis. The results confirm that HPrK/P is the single kinase catalyzing phosphorylation of Crh in vivo. Accordingly, phosphorylation of Crh is triggered by the carbon source as observed previously for HPr, but with some differences. Phosphorylation of both proteins occurred during exponential growth and disappeared upon exhaustion of the carbon source. During exponential growth, ~80% of the Crh molecules were phosphorylated when cells utilized a preferred carbon source. The reverse distribution, i.e. around 20% of Crh molecules phosphorylated, was obtained upon utilization of less favorable substrates. This clear-cut classification of the substrates into two groups has not previously been observed for HPr(Ser)~P formation. The likely reason for this difference is the additional PTS-dependent phosphorylation of HPr at His15, which limits accumulation of HPr(Ser)~P.

Authors: Jens J Landmann, Susanne Werner, , , Boris Görke

Date Published: 28th Nov 2011

Publication Type: Not specified

SABIO-RK--database for biochemical reaction kinetics.

Abstract (Expand)

SABIO-RK (http://sabio.h-its.org/) is a web-accessible database storing comprehensive information about biochemical reactions and their kinetic properties. SABIO-RK offers standardized data manually … extracted from the literature and data directly submitted from lab experiments. The database content includes kinetic parameters in relation to biochemical reactions and their biological sources with no restriction on any particular set of organisms. Additionally, kinetic rate laws and corresponding equations as well as experimental conditions are represented. All the data are manually curated and annotated by biological experts, supported by automated consistency checks. SABIO-RK can be accessed via web-based user interfaces or automatically via web services that allow direct data access by other tools. Both interfaces support the export of the data together with its annotations in SBML (Systems Biology Markup Language), e.g. for import in modelling tools.

Authors: Ulrike Wittig, , Martin Golebiewski, , Lei Shi, Lenneke Jong, Enkhjargal Algaa, Andreas Weidemann, Heidrun Sauer-Danzwith, Saqib Mir, , Meik Bittkowski, Elina Wetsch, ,

Date Published: 22nd Nov 2011

Publication Type: Journal

SubtiWiki--a comprehensive community resource for the model organism Bacillus subtilis

Abstract (Expand)

In the post-genomic era, most components of a cell are known and they can be quantified by large-scale functional genomics approaches. However, genome annotation is the bottleneck that hampers our … understanding of living cells and organisms. Up-to-date functional annotation is of special importance for model organisms that provide a frame of reference for studies with other relevant organisms. We have generated a Wiki-type database for the Gram-positive model bacterium Bacillus subtilis, SubtiWiki (http://subtiwiki.uni-goettingen.de/). This Wiki is centered around the individual genes and gene products of B. subtilis and provides information on each aspect of gene function and expression as well as protein activity and its control. SubtiWiki is accompanied by two companion databases SubtiPathways and SubtInteract that provide graphical representations of B. subtilis metabolism and its regulation and of protein-protein interactions, respectively. The diagrams of both databases are easily navigatable using the popular Google maps API, and they are extensively linked with the SubtiWiki gene pages. Moreover, each gene/gene product was assigned to one or more functional categories and transcription factor regulons. Pages for the specific categories and regulons provide a rapid overview of functionally related genes/proteins. Today, SubtiWiki can be regarded as one of the most complete inventories of knowledge on a living organism in one single resource.

Authors: , Arne G Schmeisky, ,

Date Published: 16th Nov 2011

Publication Type: Not specified

Growth rate dependent control in Enterococcus faecalis: effects on the transcriptome, proteome and strong regulation of lactate dehydrogenase

Abstract (Expand)

Enterococcus faecalis V583 was grown in a glucose-limited chemostat at three different (0.05 h(-1), 0.15 h(-1) and 0.4 h(-1)) growth rates. The fermentation pattern changed with growth rate, from a … mostly homolactic profile at high growth rate to a fermentation dominated by formate, acetate and ethanol production at low growth rate. A number of amino acids were consumed at the lower growth rates but not by fast growing cells. The change in metabolic profile was mainly caused by decreased flux through lactate dehydrogenase. Transcription of ldh-1, encoding the principal lactate dehydrogenase, showed very strong growth rate dependence and differed by three orders of magnitude between the highest and the lowest growth rates. Despite the increase in ldh-1 transcript, the content of the Ldh-1 protein was the same under all conditions. Using microarrays and qPCR the levels of 227 gene transcript were found to be affected by the growth rate, and 56 differentially expressed proteins were found by proteomic analyses. Few genes or proteins showed a growth rate-dependent increase or decrease in expression over the whole range of conditions, and many showed at maximum or minimum at the middle growth rate (D=0.15h(-1)). For many gene products a discrepancy between transcriptomic and proteomic data were seen, indicating post-transcriptional regulation of expression.

Authors: , Ellen M Faergestad, , Lars Snipen, ,

Date Published: 1st Nov 2011

Publication Type: Not specified

The PII protein GlnK is a pleiotropic regulator for morphological differentiation and secondary metabolism in Streptomyces coelicolor

Abstract (Expand)

GlnK is an important nitrogen sensor protein in Streptomyces coelicolor. Deletion of glnK results in a medium-dependent failure of aerial mycelium and spore formation and loss of antibiotic production. … Thus, GlnK is not only a regulator of nitrogen metabolism but also of morphological differentiation and secondary metabolite production. Through a comparative transcriptomic approach between the S. coelicolor wild-type and a S. coelicolor glnK mutant strain, 142 genes were identified that are differentially regulated in both strains. Among these are genes of the ram and rag operon, which are involved in S. coelicolor morphogenesis, as well as genes involved in gas vesicle biosynthesis and ectoine biosynthesis. Surprisingly, no relevant nitrogen genes were found to be differentially regulated, revealing that GlnK is not an important nitrogen sensor under the tested conditions.

Authors: E. Waldvogel, A. Herbig, F. Battke, R. Amin, M. Nentwich, K. Nieselt, T. E. Ellingsen, A. Wentzel, D. A. Hodgson, W. Wohlleben, Y. Mast

Date Published: 29th Oct 2011

Publication Type: Not specified

Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway

Abstract (Expand)

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as … the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate.

Authors: Frederik M Meyer, Matthieu Jules, Felix M P Mehne, Dominique Le Coq, Jens J Landmann, Boris Görke, Stéphane Aymerich,

Date Published: 14th Oct 2011

Publication Type: Not specified

The SEEK: a platform for sharing data and models in systems biology.

Abstract (Expand)

Systems biology research is typically performed by multidisciplinary groups of scientists, often in large consortia and in distributed locations. The data generated in these projects tend to be … heterogeneous and often involves high-throughput "omics" analyses. Models are developed iteratively from data generated in the projects and from the literature. Consequently, there is a growing requirement for exchanging experimental data, mathematical models, and scientific protocols between consortium members and a necessity to record and share the outcomes of experiments and the links between data and models. The overall output of a research consortium is also a valuable commodity in its own right. The research and associated data and models should eventually be available to the whole community for reuse and future analysis. The SEEK is an open-source, Web-based platform designed for the management and exchange of systems biology data and models. The SEEK was originally developed for the SysMO (systems biology of microorganisms) consortia, but the principles and objectives are applicable to any systems biology project. The SEEK provides an index of consortium resources and acts as gateway to other tools and services commonly used in the community. For example, the model simulation tool, JWS Online, has been integrated into the SEEK, and a plug-in to PubMed allows publications to be linked to supporting data and author profiles in the SEEK. The SEEK is a pragmatic solution to data management which encourages, but does not force, researchers to share and disseminate their data to community standard formats. It provides tools to assist with management and annotation as well as incentives and added value for following these recommendations. Data exchange and reuse rely on sufficient annotation, consistent metadata descriptions, and the use of standard exchange formats for models, data, and the experiments they are derived from. In this chapter, we present the SEEK platform, its functionalities, and the methods employed for lowering the barriers to adoption of standard formats. As the production of biological data continues to grow, in systems biology and in the life sciences in general, the need to record, manage, and exploit this wealth of information in the future is increasing. We promote the SEEK as a data and model management tool that can be adapted to the specific needs of a particular systems biology project.

Editor:

Date Published: 28th Sep 2011

Publication Type: Journal

Reprogramming of Escherichia coli K-12 metabolism during the initial phase of transition from an anaerobic to a micro-aerobic environment

Abstract (Expand)

Many bacteria undergo transitions between environments with differing O₂ availabilities as part of their natural lifestyles and during biotechnological processes. However, the dynamics of adaptation … when bacteria experience changes in O₂ availability are understudied. The model bacterium and facultative anaerobe Escherichia coli K-12 provides an ideal system for exploring this process.

Authors: Eleanor W Trotter, , Andrea M Hounslow, C Jeremy Craven, Michael P Williamson, , ,

Date Published: 27th Sep 2011

Publication Type: Not specified

The role of the 5'-3' exoribonuclease XRNA in transcriptome-wide mRNA degradation

Abstract (Expand)

The steady-state level of each mRNA in a cell is a balance between synthesis and degradation. Here, we use high-throughput RNA sequencing (RNASeq) to determine the relationship between mRNA degradation … and mRNA abundance on a transcriptome-wide scale. The model organism used was the bloodstream form of Trypanosoma brucei, a protist that lacks regulation of RNA polymerase II initiation. The mRNA half-lives varied over two orders of magnitude, with a median half-life of 13 min for total (rRNA-depleted) mRNA. Data for poly(A)+ RNA yielded shorter half-lives than for total RNA, indicating that removal of the poly(A) tail was usually the first step in degradation. Depletion of the major 5'-3' exoribonuclease, XRNA, resulted in the stabilization of most mRNAs with half-lives under 30 min. Thus, on a transcriptome-wide scale, degradation of most mRNAs is initiated by deadenylation. Trypanosome mRNA levels are strongly influenced by gene copy number and mRNA half-life: Very abundant mRNAs that are required throughout the life-cycle may be encoded by multicopy genes and have intermediate-to-long half-lives; those encoding ribosomal proteins, with one to two gene copies, are exceptionally stable. Developmentally regulated transcripts with a lower abundance in the bloodstream forms than the procyclic forms had half-lives around the median, whereas those with a higher abundance in the bloodstream forms than the procyclic forms, such as those encoding glycolytic enzymes, had longer half-lives.

Authors: Theresa Manful, ,

Date Published: 26th Sep 2011

Publication Type: Not specified

Functional analysis of the sortase YhcS in Bacillus subtilis

Abstract (Expand)

Sortases of Gram-positive bacteria catalyze the covalent C-terminal anchoring of proteins to the cell wall. Bacillus subtilis, a well-known host organism for protein production, contains two putative … sortases named YhcS and YwpE. The present studies were aimed at investigating the possible sortase function of these proteins in B. subtilis. Proteomics analyses revealed that sortase-mutant cells released elevated levels of the putative sortase substrate YfkN into the culture medium upon phosphate starvation. The results indicate that YfkN required sortase activity of YhcS for retention in the cell wall. To analyze sortase function in more detail, we focused attention on the potential sortase substrate YhcR, which is co-expressed with the sortase YhcS. Our results showed that the sortase recognition and cell-wall-anchoring motif of YhcR is functional when fused to the Bacillus pumilus chitinase ChiS, a readily detectable reporter protein that is normally secreted. The ChiS fusion protein is displayed at the cell wall surface when YhcS is co-expressed. In the absence of YhcS, or when no cell-wall-anchoring motif is fused to ChiS, the ChiS accumulates predominately in the culture medium. Taken together, these novel findings show that B. subtilis has a functional sortase for anchoring proteins to the cell wall.

Authors: Hamidreza Fasehee, Helga Westers, Albert Bolhuis, Haike Antelmann, , Wim J Quax, Agha F Mirlohi, , Gholamreza Ahmadian

Date Published: 31st Aug 2011

Publication Type: Not specified

A mathematical modelling approach to assessing the reliability of biomarkers of glutathione metabolism

Abstract (Expand)

One of the main pathways for the detoxification of reactive metabolites in the liver involves glutathione conjugation. Metabolic profiling studies have shown paradoxical responses in glutathione-related … biochemical pathways. One of these is the increase in 5-oxoproline and ophthalmic acid concentrations with increased dosage of paracetamol. Experimental studies have thus far failed to resolve these paradoxes and the robustness of how these proposed biomarkers correlate with liver glutathione levels has been questioned. To better understand how these biomarkers behave in the glutathione system a kinetic model of this pathway was made. By using metabolic control analysis and by simulating biomarker levels under a variety of conditions, we found that 5-oxoproline and ophthalmic acid concentrations may not only depend on the glutathione but also on the methionine status of the cell. We show that neither of the two potential biomarkers are reliable on their own since they need additional information about the methionine status of the system to relate them uniquely to intracellular glutathione concentration. However, when both biomarkers are measured simultaneously a direct inference of the glutathione concentration can be made, irrespective of the methionine concentration in the system.

Authors: Suzanne Geenen, , Michael Reed, H Frederik Nijhout, J Gerry Kenna, Ian D Wilson, ,

Date Published: 24th Aug 2011

Publication Type: Not specified

Live Cell Imaging of <em>Bacillus subtilis</em> and <em>Streptococcus pneumoniae</em> using Automated Time-lapse Microscopy

Abstract (Expand)

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of … single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see (1,2,3)). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy). Here, we provide a detailed description of a microscopy method used in several recent studies (4, 5, 6, 7), which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Authors: , Katrin Beilharz, ,

Date Published: 16th Aug 2011

Publication Type: Not specified

RNA processing in Bacillus subtilis: identification of targets of the essential RNase Y

Abstract (Expand)

RNA processing and degradation is initiated by endonucleolytic cleavage of the target RNAs. In many bacteria, this activity is performed by RNase E which is not present in Bacillus subtilis and other … Gram-positive bacteria. Recently, the essential endoribonuclease RNase Y has been discovered in B. subtilis. This RNase is involved in the degradation of bulk mRNA suggesting a major role in RNA metabolism. However, only a few targets of RNase Y have been identified so far. In order to assess the global impact of RNase Y, we compared the transcriptomes in response to the expression level of RNase Y. Our results demonstrate that processing by RNase Y results in accumulation of about 550 mRNAs. Some of these targets were substantially stabilized by RNase Y depletion, resulting in half-lives in the range of an hour. Moreover, about 350 mRNAs were less abundant when RNase Y was depleted among them the mRNAs of the operons required for biofilm formation. Interestingly, overexpression of RNase Y was sufficient to induce biofilm formation. The results presented in this work emphasize the importance of RNase Y as the global acting endoribonuclease for B. subtilis.

Authors: Martin Lehnik-Habrink, Marc Schaffer, , Christine Diethmaier, Christina Herzberg,

Date Published: 4th Aug 2011

Publication Type: Not specified

RNase Y in Bacillus subtilis: a Natively disordered protein that is the functional equivalent of RNase E from Escherichia coli

Abstract (Expand)

The control of mRNA stability is an important component of regulation in bacteria. Processing and degradation of mRNAs are initiated by an endonucleolytic attack, and the cleavage products are processively … degraded by exoribonucleases. In many bacteria, these RNases, as well as RNA helicases and other proteins, are organized in a protein complex called the RNA degradosome. In Escherichia coli, the RNA degradosome is assembled around the essential endoribonuclease E. In Bacillus subtilis, the recently discovered essential endoribonuclease RNase Y is involved in the initiation of RNA degradation. Moreover, RNase Y interacts with other RNases, the RNA helicase CshA, and the glycolytic enzymes enolase and phosphofructokinase in a degradosome-like complex. In this work, we have studied the domain organization of RNase Y and the contribution of the domains to protein-protein interactions. We provide evidence for the physical interaction between RNase Y and the degradosome partners in vivo. We present experimental and bioinformatic data which indicate that the RNase Y contains significant regions of intrinsic disorder and discuss the possible functional implications of this finding. The localization of RNase Y in the membrane is essential both for the viability of B. subtilis and for all interactions that involve RNase Y. The results presented in this study provide novel evidence for the idea that RNase Y is the functional equivalent of RNase E, even though the two enzymes do not share any sequence similarity.

Authors: Martin Lehnik-Habrink, , Fabian M Rothe, Alexandra S Solovyova, Cecilia Rodrigues, Christina Herzberg, Fabian M Commichau, ,

Date Published: 29th Jul 2011

Publication Type: Not specified

A new basal promoter element recognized by RNA polymerase core enzyme

Abstract (Expand)

Bacterial promoters are recognized by RNA polymerase (RNAP) σ subunit, which specifically interacts with the -10 and -35 promoter elements. Here, we provide evidence that the β' zipper, an … evolutionarily conserved loop of the largest subunit of RNAP core, interacts with promoter spacer, a DNA segment that separates the -10 and -35 promoter elements, and facilitates the formation of stable closed promoter complex. Depending on the spacer sequence, the proposed interaction of the β' zipper with the spacer can also facilitate open promoter complex formation and even substitute for interactions of the σ subunit with the -35 element. These results suggest that there exists a novel class of promoters that rely on interaction of the β' zipper with promoter spacer, along with or instead of interactions of σ subunit with the -35 element, for their activity. Finally, our data suggest that sequence-dependent interactions of the β' zipper with DNA can contribute to promoter-proximal σ-dependent RNAP pausing, a recently recognized important step of transcription control.

Authors: , Vasisht R Tadigotla, Konstantin Severinov,

Date Published: 26th Jul 2011

Publication Type: Not specified

Osmotically controlled synthesis of the compatible solute proline is critical for cellular defense of Bacillus subtilis against high osmolarity

Abstract (Expand)

Bacillus subtilis is known to accumulate large amounts of the compatible solute proline via de novo synthesis as a stress protectant when it faces high-salinity environments. We elucidated the genetic … determinants required for the osmoadaptive proline production from the precursor glutamate. This proline biosynthesis route relies on the proJ-encoded γ-glutamyl kinase, the proA-encoded γ-glutamyl phosphate reductase, and the proH-encoded Δ1-pyrroline-5-caboxylate reductase. Disruption of the proHJ operon abolished osmoadaptive proline production and strongly impaired the ability of B. subtilis to cope with high-osmolarity growth conditions. Disruption of the proA gene also abolished osmoadaptive proline biosynthesis but caused, in contrast to the disruption of proHJ, proline auxotrophy. Northern blot analysis demonstrated that the transcription of the proHJ operon is osmotically inducible, whereas that of the proBA operon is not. Reporter gene fusion studies showed that proHJ expression is rapidly induced upon an osmotic upshift. Increased expression is maintained as long as the osmotic stimulus persists and is sensitively linked to the prevalent osmolarity of the growth medium. Primer extension analysis revealed the osmotically controlled proHJ promoter, a promoter that resembles typical SigA-type promoters of B. subtilis. Deletion analysis of the proHJ promoter region identified a 126-bp DNA segment carrying all sequences required in cis for osmoregulated transcription. Our data disclose the presence of ProA-interlinked anabolic and osmoadaptive proline biosynthetic routes in B. subtilis and demonstrate that the synthesis of the compatible solute proline is a central facet of the cellular defense to high-osmolarity surroundings for this soil bacterium.

Authors: Jeanette Brill, , Monika Bleisteiner,

Date Published: 22nd Jul 2011

Publication Type: Not specified

Nonlinear fitness landscape of a molecular pathway

Abstract (Expand)

Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost factor, but the enzymatic activity … of the proteins produced can also reduce fitness, depending on the internal state and the environment of the cell. Here, we map the fitness costs of a key metabolic network, the lactose utilization pathway in Escherichia coli. We measure the growth of several regulatory lac operon mutants in different environments inducing expression of the lac genes. We find a strikingly nonlinear fitness landscape, which depends on the production rate and on the activity rate of the lac proteins. A simple fitness model of the lac pathway, based on elementary biophysical processes, predicts the growth rate of all observed strains. The nonlinearity of fitness is explained by a feedback loop: production and activity of the lac proteins reduce growth, but growth also affects the density of these molecules. This nonlinearity has important consequences for molecular function and evolution. It generates a cliff in the fitness landscape, beyond which populations cannot maintain growth. In viable populations, there is an expression barrier of the lac genes, which cannot be exceeded in any stationary growth process. Furthermore, the nonlinearity determines how the fitness of operon mutants depends on the inducer environment. We argue that fitness nonlinearities, expression barriers, and gene-environment interactions are generic features of fitness landscapes for metabolic pathways, and we discuss their implications for the evolution of regulation.

Authors: Lilia Perfeito, Stéphane Ghozzi, , Karin Schnetz, Michael Lässig

Date Published: 21st Jul 2011

Publication Type: Not specified

RightField: embedding ontology annotation in spreadsheets.

Abstract (Expand)

MOTIVATION: In the Life Sciences, guidelines, checklists and ontologies describing what metadata is required for the interpretation and reuse of experimental data are emerging. Data producers, however, … may have little experience in the use of such standards and require tools to support this form of data annotation. RESULTS: RightField is an open source application that provides a mechanism for embedding ontology annotation support for Life Science data in Excel spreadsheets. Individual cells, columns or rows can be restricted to particular ranges of allowed classes or instances from chosen ontologies. The RightField-enabled spreadsheet presents selected ontology terms to the users as a simple drop-down list, enabling scientists to consistently annotate their data. The result is 'semantic annotation by stealth', with an annotation process that is less error-prone, more efficient, and more consistent with community standards. AVAILABILITY AND IMPLEMENTATION: RightField is open source under a BSD license and freely available from http://www.rightfield.org.uk

Authors: , , Matthew Horridge, , , , ,

Date Published: 15th Jul 2011

Publication Type: Journal

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Abstract (Expand)

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes … is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.

Authors: Anita Minnen, Laetitia Attaiech, Maria Thon, Stephan Gruber,

Date Published: 22nd Jun 2011

Publication Type: Not specified

Emergence of the silicon human and network targeting drugs

Abstract (Expand)

The development of disease may be characterized as a pathological shift of homeostasis; the main goal of contemporary drug treatment is, therefore, to return the pathological homeostasis back to the … normal physiological range. From the view point of systems biology, homeostasis emerges from the interactions within the network of biomolecules (e.g. DNA, mRNA, proteins), and, hence, understanding how drugs impact upon the entire network should improve their efficacy at returning the network (body) to physiological homeostasis. Large, mechanism-based computer models, such as the anticipated human whole body models (silicon or virtual human), may help in the development of such network-targeting drugs. Using the philosophical concept of weak and strong emergence, we shall here take a more general look at the paradigm of network-targeting drugs, and propose our approaches to scale the strength of strong emergence. We apply these approaches to several biological examples and demonstrate their utility to reveal principles of bio-modeling. We discuss this in the perspective of building the silicon human.

Authors: Alexey Kolodkin, Fred C Boogerd, Nick Plant, Frank J Bruggeman, Valeri Goncharuk, Jeantine Lunshof, Rafael Moreno-Sanchez, Nilgun Yilmaz, Barbara M Bakker, , Rudi Balling,

Date Published: 16th Jun 2011

Publication Type: Not specified

Characterization of E. coli MG1655 and frdA and sdhC mutants at various aerobiosis levels

Abstract

Not specified

Authors: , S. Frixel, ,

Date Published: 1st Jun 2011

Publication Type: Not specified

Structural basis for the enhanced activity of cyclic antimicrobial peptides: The case of BPC194

Abstract (Expand)

We report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements … to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a beta-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides.

Authors: , Gemma Moiset, Anna D Cirac, Lidia Feliu, Eduard Bardají, Marta Planas, Durba Sengupta, Siewert J Marrink,

Date Published: 19th May 2011

Publication Type: Not specified

The molecular basis for antimicrobial activity of pore-forming cyclic peptides

Abstract (Expand)

The mechanism of action of antimicrobial peptides is, to our knowledge, still poorly understood. To probe the biophysical characteristics that confer activity, we present here a molecular-dynamics and … biophysical study of a cyclic antimicrobial peptide and its inactive linear analog. In the simulations, the cyclic peptide caused large perturbations in the bilayer and cooperatively opened a disordered toroidal pore, 1-2 nm in diameter. Electrophysiology measurements confirm discrete poration events of comparable size. We also show that lysine residues aligning parallel to each other in the cyclic but not linear peptide are crucial for function. By employing dual-color fluorescence burst analysis, we show that both peptides are able to fuse/aggregate liposomes but only the cyclic peptide is able to porate them. The results provide detailed insight on the molecular basis of activity of cyclic antimicrobial peptides.

Authors: Anna D Cirac, Gemma Moiset, , Armagan Koçer, Pedro Salvador, , Siewert J Marrink, Durba Sengupta

Date Published: 18th May 2011

Publication Type: Not specified

A metabolomics and proteomics study of the adaptation of Staphylococcus aureus to glucose starvation

Abstract (Expand)

As a versatile pathogen Staphylococcus aureus can cause various disease patterns, which are influenced by strain specific virulence factor repertoires but also by S. aureus physiological adaptation … capacity. Here, we present metabolomic descriptions of S. aureus central metabolic pathways and demonstrate the potential for combined metabolomics- and proteomics-based approaches for the basic research of this important pathogen. This study provides a time-resolved picture of more than 500 proteins and 94 metabolites during the transition from exponential growth to glucose starvation. Under glucose excess, cells exhibited higher levels of proteins involved in glycolysis and protein-synthesis, whereas entry into the stationary phase triggered an increase of enzymes of TCC and gluconeogenesis. These alterations in levels of metabolic enzymes were paralleled by more pronounced changes in the concentrations of associated metabolites, in particular, intermediates of the glycolysis and several amino acids.

Authors: Manuel Liebeke, Kirsten Dörries, Daniela Zühlke, Jörg Bernhardt, Stephan Fuchs, Jan Pané-Farré, Susanne Engelmann, , Rüdiger Bode, Thomas Dandekar, Ulrike Lindequist, ,

Date Published: 1st Apr 2011

Publication Type: Not specified

Environmental salinity determines the specificity and need for Tat-dependent secretion of the YwbN protein in Bacillus subtilis

Abstract (Expand)

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into … a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described "minimal Tat translocase" consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate.

Authors: René van der Ploeg, , Georg Homuth, Marc Schaffer, Emma L Denham, Carmine G Monteferrante, Marcus Miethke, Mohamed A Marahiel, , Theresa Winter, , Haike Antelmann,

Date Published: 30th Mar 2011

Publication Type: Not specified

A technical platform for generating reproducible expression data from Streptomyces coelicolor batch cultivations

Abstract (Expand)

Streptomyces coelicolor, the model species of the genus Streptomyces, presents a complex life cycle of successive morphological and biochemical changes involving the formation of substrate and aerial … mycelium, sporulation and the production of antibiotics. The switch from primary to secondary metabolism can be triggered by nutrient starvation and is of particular interest as some of the secondary metabolites produced by related Streptomycetes are commercially relevant. To understand these events on a molecular basis, a reliable technical platform encompassing reproducible fermentation as well as generation of coherent transcriptomic data is required. Here, we investigate the technical basis of a previous study as reported by Nieselt et al. (BMC Genomics 11:10, 2010) in more detail, based on the same samples and focusing on the validation of the custom-designed microarray as well as on the reproducibility of the data generated from biological replicates. We show that the protocols developed result in highly coherent transcriptomic measurements. Furthermore, we use the data to predict chromosomal gene clusters, extending previously known clusters as well as predicting interesting new clusters with consistent functional annotations.

Authors: F. Battke, A. Herbig, A. Wentzel, O. M. Jakobsen, M. Bonin, D. A. Hodgson, W. Wohlleben, T. E. Ellingsen, K. Nieselt

Date Published: 25th Mar 2011

Publication Type: Not specified

Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics

Abstract (Expand)

Knowledge on absolute protein concentrations is mandatory for the simulation of biological processes in the context of systems biology. A novel approach for the absolute quantification of proteins at … a global scale has been developed and its applicability demonstrated using glucose starvation of the Gram-positive model bacterium Bacillus subtilis and the pathogen Staphylococcus aureus as proof-of-principle examples. Absolute intracellular protein concentrations were initially determined for a preselected set of anchor proteins by employing a targeted mass spectrometric method and isotopically labeled internal standard peptides. Known concentrations of these anchor proteins were then used to calibrate two-dimensional (2-D) gels allowing the calculation of absolute abundance of all detectable proteins on the 2-D gels. Using this approach, concentrations of the majority of metabolic enzymes were determined, and thus a quantification of the players of metabolism was achieved. This new strategy is fast, cost-effective, applicable to any cell type, and thus of value for a broad community of laboratories with experience in 2-D gel-based proteomics and interest in quantitative approaches. Particularly, this approach could also be utilized to quantify existing data sets with the aid of a few standard anchor proteins.

Authors: , Susanne Sievers, Daniela Zühlke, Judith Kuzinski, , Jan Muntel, Bernd Hessling, Jörg Bernhardt, Rabea Sietmann, , , Dörte Becher

Date Published: 11th Mar 2011

Publication Type: Not specified

Functional genomics of the initial phase of cold adaptation of Pseudomonas putida KT2440

Abstract (Expand)

The cold stress response of Pseudomonas putida KT2440 was investigated by genomewide deep cDNA sequencing and gel-free MS-based protein profiling. Transcriptome and proteome profiles were assessed at … 30 degrees C and 2 h after a downshift from 30 to 10 degrees C. Pseudomonas putida adapted to lower ambient temperature by the activation of ribosome-associated functional modules that facilitate translational efficiency. The outer membrane profile was reorganized, anabolic pathways and core as well as energy metabolism were repressed and the alginate regulon and sugar catabolism were activated. At the investigated early time point of cold adaptation, the transcriptome was reprogrammed in almost all functional categories, but the protein profile had still not adapted to the change of living conditions in the cold.

Authors: , F. Schmidt, , C. F. Davenport, M. Gesell Salazar, U. Volker,

Date Published: 1st Mar 2011

Publication Type: Not specified

Pseudomonas putida KT2440 genome update by cDNA sequencing and microarray transcriptomics

Abstract (Expand)

Pseudomonas putida KT2440 is a completely sequenced biosafety strain that has retained its capability to survive and function in the environment. The global mRNA expression profiles of the KT2440 strain … grown at 10 degrees C and 30 degrees C were determined by deep cDNA sequencing to refine the genome annotation. Transcriptome sequencing identified 36 yet unknown small non-coding RNAs, 143 novel ORFs in 106 intergenic regions, 42 unclassified genes and eight highly expressed leaderless mRNA transcripts. The genome coordinates of eight genes and the organization of 57 operons were corrected. No overrepresented sequence motifs were detected in the 5'-untranslated regions. The 50 most highly expressed genes made up 60% of the total mRNA pool. Comparison of cDNA sequencing, Affymetrix and Progenika microarray data from the same mRNA preparation revealed a higher sensitivity and specificity of cDNA sequencing, a relatively poor correlation between the normalized cDNA reads and microarray signal intensities, and a systematic signal-dependent bias of microarrays in the detection of differentially regulated genes. The study demonstrates the power of next-generation cDNA sequencing for the quantitation of mRNA transcripts and the refinement of bacterial genome annotation.

Authors: , , P. Hagendorf, R. Geffers, U. Schock, T. Pohl, C. F. Davenport,

Date Published: 28th Feb 2011

Publication Type: Not specified

Transcriptome, proteome and metabolite analysis of a lactate dehydrogenase negative mutant of Enterococcus faecalis V583

Abstract (Expand)

A constructed lactate dehydrogenase-negative mutant of Enterococcus faecalis V583 grows at the same rate as the wild type, but ferments glucose to ethanol, formate, and acetoin. Microrray analysis … showed that LDH deficiency had profound transcriptional effects, 43 genes in the mutant were found to be upregulated and 45 to be downregulated. Most of the upregulated genes encode enzymes of energy metabolism or transport. By 2D gel analysis 45 differentially expressed proteins were identified. A comparison of transcriptomic and proteomic data suggests that for several proteins the level of expression is regulated beyond the level of transcription. Pyruvate catabolic genes, including the truncated ldh, showed highly increased transcription in the mutant. These genes, along with a number of other differentially expressed genes, are preceded by sequences with homology to binding sites for the global redox-sensing repressor, Rex, of Staphylococcus aureus. The data indicate that the genes are transcriptionally regulated by the NADH/NAD ratio and that this ratio plays an important role in the regulatory network controlling energy metabolism in E. faecalis.

Authors: , , Ellen M Fergestad, Geir Mathiesen, ,

Date Published: 8th Feb 2011

Publication Type: Not specified

Alkali-metal-cation influx and efflux systems in nonconventional yeast species

Abstract (Expand)

To maintain optimal intracellular concentrations of alkali-metal-cations, yeast cells use a series of influx and efflux systems. Nonconventional yeast species have at least three different types of … efficient transporters that ensure potassium uptake and accumulation in cells. Most of them have Trk uniporters and Hak K(+) -H(+) symporters and a few yeast species also have the rare K(+) (Na(+) )-uptake ATPase Acu. To eliminate surplus potassium or toxic sodium cations, various yeast species use highly conserved Nha Na(+) (K(+) )/H(+) antiporters and Na(+) (K(+) )-efflux Ena ATPases. The potassium-specific yeast Tok1 channel is also highly conserved among various yeast species and its activity is important for the regulation of plasma membrane potential.

Editor:

Date Published: 1st Feb 2011

Publication Type: Not specified

Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre

Abstract (Expand)

The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. … Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes.

Authors: Mohammad Roghanian, ,

Date Published: 27th Jan 2011

Publication Type: Not specified

Transcript profiling and inference of Escherichia coli K-12 ArcA activity across the range of physiologically relevant oxygen concentrations

Abstract (Expand)

Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Whilst much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic … conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here for the first time the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundances of cytochrome bd and bo and outer membrane protein W. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself, or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFinfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis, but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. Measurement of the amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.

Authors: , , , Eleanor W Trotter, H M Shahzad Asif, Guido Sanguinetti, , ,

Date Published: 22nd Jan 2011

Publication Type: Not specified

Bet hedging or not? A guide to proper classification of microbial survival strategies

Abstract (Expand)

Bacteria have developed an impressive ability to survive and propagate in highly diverse and changing environments by evolving phenotypic heterogeneity. Phenotypic heterogeneity ensures that a subpopulation … is well prepared for environmental changes. The expression bet hedging is commonly (but often incorrectly) used by molecular biologists to describe any observed phenotypic heterogeneity. In evolutionary biology, however, bet hedging denotes a risk-spreading strategy displayed by isogenic populations that evolved in unpredictably changing environments. Opposed to other survival strategies, bet hedging evolves because the selection environment changes and favours different phenotypes at different times. Consequently, in bet hedging populations all phenotypes perform differently well at any time, depending on the selection pressures present. Moreover, bet hedging is the only strategy in which temporal variance of offspring numbers per individual is minimized. Our paper aims to provide a guide for the correct use of the term bet hedging in molecular biology.

Authors: , Patsy Haccou,

Date Published: 21st Jan 2011

Publication Type: Not specified

T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

Abstract (Expand)

Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ-ProA-ProH route is responsible for the production of proline as an osmoprotectant, and the ProB-ProA-ProI route … provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB-treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine.

Authors: Jeanette Brill, , Harald Putzer,

Date Published: 13th Jan 2011

Publication Type: Not specified

SPABBATS: A pathway-discovery method based on Boolean satisfiability that facilitates the characterization of suppressor mutants

Abstract (Expand)

Several computational methods exist to suggest rational genetic interventions that improve the productivity of industrial strains. Nonetheless, these methods are less effective to predict possible … genetic responses of the strain after the intervention. This problem requires a better understanding of potential alternative metabolic and regulatory pathways able to counteract the targeted intervention.

Authors: , Katrin Gunka, Rafael Polanía, Stefan Tholen,

Date Published: 11th Jan 2011

Publication Type: Not specified

Genome-wide gene expression analysis of the switch between acidogenesis and solventogenesis in continuous cultures of Clostridium acetobutylicum

Abstract (Expand)

Clostridium acetobutylicum is able to switch from acidogenic growth to solventogenic growth. We used phosphate-limited continuous cultures that established acidogenic growth at pH 5.8 and solventogenic … growth at pH 4.5. These cultures allowed a detailed transcriptomic study of the switch from acidogenesis to solventogenesis that is not superimposed by sporulation and other growth phase-dependent parameters. These experiments led to new insights into the physiological role of several genes involved in solvent formation. The adc gene for acetone decarboxylase is upregulated well before the rest of the sol locus during the switch, and pyruvate decarboxylase is induced exclusively for the period of this switch. The aldehyde-alcohol dehydrogenase gene adhE1 located in the sol operon is regulated antagonistically to the paralog adhE2 that is expressed during acidogenic conditions. A similar antagonistic pattern can be seen with the two paralogs of thiolase genes, thlA and thlB. Interestingly, the genes coding for the putative cellulosome in C. acetobutylicum are exclusively transcribed throughout solventogenic growth. The genes for stress response are only induced during the shift but not in the course of solventogenesis when butanol is present in the culture. Finally, the data clearly indicate that solventogenesis is independent from sporulation.

Authors: Christina Grimmler, , , , , , Wolfgang Liebl,

Date Published: 6th Jan 2011

Publication Type: Not specified

A mathematical analysis of nuclear intensity dynamics for Mig1-GFP under consideration of bleaching effects and background noise in Saccharomyces cerevisiae

Abstract (Expand)

Fluorescence microscopy is an imaging technique that provides insights into signal transduction pathways through the generation of quantitative data, such as the spatiotemporal distribution of GFP-tagged … proteins in signaling pathways. The data acquired are, however, usually a composition of both the GFP-tagged proteins of interest and of an autofluorescent background, which both undergo photobleaching during imaging. We here present a mathematical model based on ordinary differential equations that successfully describes the shuttling of intracellular Mig1-GFP under changing environmental conditions regarding glucose concentration. Our analysis separates the different bleaching rates of Mig1-GFP and background, and the background-to-Mig1-GFP ratio. By applying our model to experimental data, we can thus extract the Mig1-GFP signal from the overall acquired signal and investigate the influence of kinase and phosphatase on Mig1. We found a stronger regulation of Mig1 through its kinase than through its phosphatase when controlled by the glucose concentration, with a constant (de)phosphorylation rate independent of the glucose concentration. By replacing the term for decreasing excited Mig1-GFP concentration with a constant, we were able to reconstruct the dynamics of Mig1-GFP, as it would occur without bleaching and background noise. Our model effectively demonstrates how data, acquired with an optical microscope, can be processed and used for a systems biology analysis of signal transduction pathways.

Authors: Simone Frey, Kristin Sott, Maria Smedh, , Peter Dahl, , Mattias Goksör

Date Published: 2011

Publication Type: Not specified

A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture

Abstract (Expand)

Background: Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly … efficient biofuel. For butanol production by C. acetobutylicum to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by C. acetobutylicum in continuous culture must be understood as fully as possible. Results: We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7) acids are the dominant product while at low pH (pH 4.5) this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of C. acetobutylicum could be exploited to increase butanol yield in a continuous culture fermentation. Conclusions: Incorporating gene regulation into the mathematical model of solvent production by C. acetobutylicum enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required.

Editor:

Date Published: 2011

Publication Type: Not specified

Cellular Adjustments of Bacillus subtilis and Other Bacilli to Fluctuating Salinities

Abstract

Not specified

Authors: Marco Pittelkow,

Date Published: 2011

Publication Type: Not specified

Requirement of the agr locus for colony spreading of Staphylococcus aureus

Abstract (Expand)

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be … related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.

Authors: Eleni Tsompanidou, Mark J J B Sibbald, Monika A Chlebowicz, Annette Dreisbach, Jaap Willem Back, , Girbe Buist, Emma L Denham

Date Published: 17th Dec 2010

Publication Type: Not specified

Insights into Streptococcus pyogenes pathogenesis from transcriptome studies

Abstract (Expand)

Streptococcus pyogenes (group A Streptococcus [GAS]) is a major human pathogen, causing diseases ranging from mild superficial infections of the skin and pharyngeal mucosal membrane, up to severe … systemic and invasive diseases and autoimmune sequelae. The capability of GAS to cause this wide variety of infections is due to the expression of a large set of virulence factors, their concerted transcriptional regulation, and bacterial adaptation mechanisms to various host niches, which we are now beginning to understand on a molecular level. The addition of -omics technologies for GAS pathogenesis investigation, on top of traditional molecular methods, led to fast progress in understanding GAS pathogenesis mechanisms. This article focuses on differential transcriptional analysis performed on the bacterial side as well as on the host cell side. The microarray studies discussed provide new insight into the following five topics: gene-expression patterns under infection-relevant conditions, gene-expression patterns in mutant strains compared with wild-type strains, emergence of exceptionally fit GAS clones, gene-expression patterns of eukaryotic target and immune cells in response to GAS infection, and mechanisms underlying shifts from a pharyngeal to invasive GAS lifestyle.

Authors: , Venelina Sugareva, Nadja Patenge,

Date Published: 8th Dec 2010

Publication Type: Not specified

Structures of carbon catabolite protein A-(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators

Abstract (Expand)

In Gram-positive bacteria, carbon catabolite protein A (CcpA) is the master regulator of carbon catabolite control, which ensures optimal energy usage under diverse conditions. Unlike other LacI-GalR … proteins, CcpA is activated for DNA binding by first forming a complex with the phosphoprotein HPr-Ser46-P. Bacillus subtilis CcpA functions as both a transcription repressor and activator and binds to more than 50 operators called catabolite response elements (cres). These sites are highly degenerate with the consensus, WTGNNARCGNWWWCAW. How CcpA-(HPr-Ser46-P) binds such diverse sequences is unclear. To gain insight into this question, we solved the structures of the CcpA-(HPr-Ser46-P) complex bound to three different operators, the synthetic (syn) cre, ackA2 cre and gntR-down cre. Strikingly, the structures show that the CcpA-bound operators display different bend angles, ranging from 31° to 56°. These differences are accommodated by a flexible linkage between the CcpA helix-turn-helix-loop-helix motif and hinge helices, which allows independent docking of these DNA-binding modules. This flexibility coupled with an abundance of non-polar residues capable of non-specific nucleobase interactions permits CcpA-(HPr-Ser46-P) to bind diverse operators. Indeed, biochemical data show that CcpA-(HPr-Ser46-P) binds the three cre sites with similar affinities. Thus, the data reveal properties that license this protein to function as a global transcription regulator.

Authors: Maria A Schumacher, Mareen Sprehe, , , Richard G Brennan

Date Published: 26th Nov 2010

Publication Type: Not specified

Physiological characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows that these organisms are optimized for Yatp and not ATP formation rates at their physiological pH

Abstract (Expand)

Several lactic acid bacteria use homolactic fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid … bacteria Lactococcus lactis, Enterococcus faecalis and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium in microaerophilic conditions. However, growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed in glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and dependent on the pH for E. faecalis. In S. pyogenes and L. lactis a change in pH resulted in a clear change in Yatp. The pH that showed the highest Yatp corresponded to the pH of the natural habitat of the organisms.

Authors: , , , , Anja Pritzschke, Nikolai Siemens, , ,

Date Published: 25th Nov 2010

Publication Type: Not specified

Modeling of cellular processes: methods, data, and requirements

Abstract (Expand)

Systems biology is a comprehensive quantitative analysis how the components of a biological system interact over time which requires an interdisciplinary team of investigators. System-theoretic methods … are applied to investigate the system's behavior. Using known information about the considered system, a conceptual model is defined. It is transferred in a mathematical model that can be simulated (analytically or numerically) and analyzed using system-theoretic tools. Finally, simulation results are compared with experimental data. However, assumptions, approximations, and requirements to available experimental data are crucial ingredients of this systems biology workflow. Consequently, the modeling of cellular processes creates special demands on the design of experiments: the quality, the amount, and the completeness of data. The relation between models and data is discussed in this chapter. Thereby, we focus on the requirements on experimental data from the perspective of systems biology projects.

Editor:

Date Published: 11th Nov 2010

Publication Type: Not specified

Comprehensive identification and quantification of microbial transcriptomes by genome-wide unbiased methods

Abstract (Expand)

Genomic tiling array transcriptomics and RNA-seq are two powerful and rapidly developing approaches for unbiased transcriptome analysis. Providing comprehensive identification and quantification of … transcripts with an unprecedented resolution, they are leading to major breakthroughs in systems biology. Here we review each step of the analysis from library preparation to the interpretation of the data, with particular attention paid to the possible sources of artifacts. Methodological requirements and statistical frameworks are often similar in both the approaches despite differences in the nature of the data. Tiling array analysis does not require rRNA depletion and benefits from a more mature computational workflow, whereas RNA-Seq has a clear lead in terms of background noise and dynamic range with a considerable potential for evolution with the improvements of sequencing technologies. Being independent of prior sequence knowledge, RNA-seq will boost metatranscriptomics and evolutionary transcriptomics applications.

Authors: , Pierre Nicolas, Hugues Richard, Philippe Bessières, Stéphane Aymerich

Date Published: 10th Nov 2010

Publication Type: Not specified

Macromolecule diffusion and confinement in prokaryotic cells

Abstract (Expand)

We review recent observations on the mobility of macromolecules and their spatial organization in live bacterial cells. We outline the major fluorescence microscopy-based methods to determine the … mobility and thus the diffusion coefficients (D) of molecules, which is not trivial in small cells. The extremely high macromolecule crowding of prokaryotes is used to rationalize the reported lower diffusion coefficients as compared to eukaryotes, and we speculate on the nature of the barriers for diffusion observed for proteins (and mRNAs) in vivo. Building on in vitro experiments and modeling studies, we evaluate the size dependence of diffusion coefficients for macromolecules in vivo, in case of both water-soluble and integral membrane proteins. We comment on the possibilities of anomalous diffusion and provide examples where the macromolecule mobility may be limiting biological processes.

Editor:

Date Published: 16th Oct 2010

Publication Type: Not specified

CellPublisher: a web platform for the intuitive visualization and sharing of metabolic, signalling and regulatory pathways

Abstract (Expand)

Systems biology relies increasingly on collaborations between several groups with different expertise. Therefore, the systems biology community is adopting standards that allow effective communication … of concepts, as well as transmission and processing of pathway information. The Systems Biology Graphical Notation (SBGN) is a graphical language for biological pathways that has both a biological as well as a computational meaning. The program CellDesigner allows the codification of biological phenomena in an SBGN compliant form. CellPublisher is a web server that allows the conversion of CellDesigner files to web-based navigatable diagrams based on the user interface of Google maps. Thus, CellPublisher complements CellDesigner by facilitating the understanding of complex diagrams and by providing the possibility to share any CellDesigner diagram online with collaborators and get their feedback. Due to the intuitive interface of the online diagrams, CellPublisher serves as a basis for discovery of novel properties of the modelled networks.

Authors: , Christoph R Lammers, Raphael Michna,

Date Published: 14th Oct 2010

Publication Type: Not specified

Metabolic fluxes and beyond-systems biology understanding and engineering of microbial metabolism

Abstract (Expand)

The recent years have seen tremendous progress towards the understanding of microbial metabolism on a higher level of the entire functional system. Hereby, huge achievements including the sequencing … of complete genomes and efficient post-genomic approaches provide the basis for a new, fascinating era of research-analysis of metabolic and regulatory properties on a global scale. Metabolic flux (fluxome) analysis displays the first systems oriented approach to unravel the physiology of microorganisms since it combines experimental data with metabolic network models and allows determining absolute fluxes through larger networks of central carbon metabolism. Hereby, fluxes are of central importance for systems level understanding because they fundamentally represent the cellular phenotype as integrated output of the cellular components, i.e. genes, transcripts, proteins, and metabolites. A currently emerging and promising area of research in systems biology and systems metabolic engineering is therefore the integration of fluxome data in multi-omics studies to unravel the multiple layers of control that superimpose the flux network and enable its optimal operation under different environmental conditions.

Authors: , Judith Becker,

Date Published: 7th Sep 2010

Publication Type: Not specified

DnaA and ORC: more than DNA replication initiators

Abstract (Expand)

Mutations in DNA replication initiator genes in both prokaryotes and eukaryotes lead to a pleiotropic array of phenotypes, including defects in chromosome segregation, cytokinesis, cell cycle regulation … and gene expression. For years, it was not clear whether these diverse effects were indirect consequences of perturbed DNA replication, or whether they indicated that DNA replication initiator proteins had roles beyond their activity in initiating DNA synthesis. Recent work from a range of organisms has demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities. The aim of this review is to highlight these new findings, focusing on the pathways and mechanisms utilized by DNA replication initiator proteins to carry out a diverse array of cellular functions.

Authors: Graham Scholefield, , Heath Murray

Date Published: 27th Aug 2010

Publication Type: Not specified

TFInfer: a tool for probabilistic inference of transcription factor activities

Abstract (Expand)

SUMMARY: TFInfer is a novel open access, standalone tool for genome-wide inference of transcription factor activities from gene expression data. Based on an earlier MATLAB version, the software has now … been extended in a number of ways. It has been significantly optimised in terms of performance, and it was given novel functionality, by allowing the user to model both time series and data from multiple independent conditions. With a full documentation and intuitive graphical user interface, together with an in-built data base of yeast and Escherichia coli transcription factors, the software does not require any mathematical or computational expertise to be used effectively. AVAILABILITY: http://homepages.inf.ed.ac.uk/gsanguin/TFInfer.html CONTACT: gsanguin@staffmail.ed.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Authors: H M Shahzad Asif, , , Neil D Lawrence, Magnus Rattray,

Date Published: 24th Aug 2010

Publication Type: Not specified

Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: Evidence for a metabolon

Abstract (Expand)

The majority of all proteins of a living cell is active in complexes rather than in an isolated way. These protein-protein interactions are of high relevance for many biological functions. In addition … to many well established protein complexes an increasing number of protein-protein interactions, which form rather transient complexes has recently been discovered. The formation of such complexes seems to be a common feature especially for metabolic pathways. In the Gram-positive model organism Bacillus subtilis, we identified a protein complex of three citric acid cycle enzymes. This complex consists of the citrate synthase, the isocitrate dehydrogenase, and the malate dehydrogenase. Moreover, fumarase and aconitase interact with malate dehydrogenase and with each other. These five enzymes catalyze sequential reaction of the TCA cycle. Thus, this interaction might be important for a direct transfer of intermediates of the TCA cycle and thus for elevated metabolic fluxes via substrate channeling. In addition, we discovered a link between the TCA cycle and gluconeogenesis through a flexible interaction of two proteins: the association between the malate dehydrogenase and phosphoenolpyruvate carboxykinase is directly controlled by the metabolic flux. The phosphoenolpyruvate carboxykinase links the TCA cycle with gluconeogenesis and is essential for B. subtilis growing on gluconeogenic carbon sources. Only under gluconeogenic growth conditions an interaction of these two proteins is detectable and disappears under glycolytic growth conditions.

Authors: Frederik M Meyer, Jan Gerwig, Elke Hammer, Christina Herzberg, Fabian M Commichau, ,

Date Published: 20th Aug 2010

Publication Type: Not specified

Integrative responses to high pH stress in S. cerevisiae

Abstract (Expand)

The budding yeast Saccharomyces cerevisiae grows far better at acidic than at neutral or alkaline pH. Consequently, even a modest alkalinization of the medium represents a stressful situation for this … yeast. In the past few years, data generated by a combination of genome-wide techniques has demonstrated that adaptive responses of S. cerevisiae to high pH stress involves extensive gene remodeling as a result of the fast activation of a number of stress-related signaling pathways, such as the Rim101, the Wsc1-Pkc1-Slt2 MAP kinase, and the calcium-activated calcineurin pathways. Alkalinization of the environment also disturbs nutrient homeostasis, as deduced from its impact on iron/copper, phosphate, and glucose uptake/utilization pathways. In this review we will examine these responses, their possible interactions, and the role that they play in tolerance to high pH stress.

Editor:

Date Published: 20th Aug 2010

Publication Type: Not specified

A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture

Abstract (Expand)

The complex changes in the life cycle of Clostridium acetobutylicum, a promising biofuel producer, are not well understood. During exponential growth, sugars are fermented to acetate and butyrate, and … in the transition phase, the metabolism switches to the production of the solvents acetone and butanol accompanied by the initiation of endospore formation. Using phosphate-limited chemostat cultures at pH 5.7, C. acetobutylicum was kept at a steady state of acidogenic metabolism, whereas at pH 4.5, the cells showed stable solvent production without sporulation. Novel proteome reference maps of cytosolic proteins from both acidogenesis and solventogenesis with a high degree of reproducibility were generated. Yielding a 21% coverage, 15 protein spots were specifically assigned to the acidogenic phase, and 29 protein spots exhibited a significantly higher abundance in the solventogenic phase. Besides well-known metabolic proteins, unexpected proteins were also identified. Among these, the two proteins CAP0036 and CAP0037 of unknown function were found as major striking indicator proteins in acidogenic cells. Proteome data were confirmed by genome-wide DNA microarray analyses of the identical cultures. Thus, a first systematic study of acidogenic and solventogenic chemostat cultures is presented, and similarities as well as differences to previous studies of batch cultures are discussed.

Authors: , , , Birgit Voigt, Michael Hecker, ,

Date Published: 1st Aug 2010

Publication Type: Not specified

How mathematical modelling elucidates signalling in B. subtilis

Abstract (Expand)

Appropriate stimulus perception, signal processing and transduction ensure optimal adaptation of bacteria to environmental challenges. In the Gram-positive model bacterium Bacillus subtilis signalling …g networks and molecular interactions therein are well-studied, making this species a suitable candidate for the application of mathematical modelling. Here, we review systems biology approaches, focusing on chemotaxis, sporulation, σB-dependent general stress response and competence. Processes like chemotaxis and Z-ring assembly depend critically on the subcellular localization of proteins. Environmental response strategies, including sporulation and competence, are characterized by phenotypic heterogeneity in isogenic cultures. The examples of mathematical modelling also include investigations that have demonstrated how operon structure and signalling dynamics are intricately interwoven to establish optimal responses. Our review illustrates that these interdisciplinary approaches offer new insights into the response of B. subtilis to environmental challenges. These case studies reveal modelling as a tool to increase the understanding of complex systems, to help formulating hypotheses and to guide the design of more directed experiments that test predictions.

Editor:

Date Published: 1st Jul 2010

Publication Type: Not specified

Molecular sieving properties of the cytoplasm of Escherichia coli and consequences of osmotic stress

Abstract (Expand)

We determined the diffusion coefficients (D) of (macro)molecules of different sizes (from ∼0.5 to 600 kDa) in the cytoplasm of live Escherichia coli cells under normal osmotic conditions and osmotic … upshift. D values decreased with increasing molecular weight of the molecules. Upon osmotic upshift, the decrease in D of NBD-glucose was much smaller than that of macromolecules. Barriers for diffusion were found in osmotically challenged cells only for GFP and larger proteins. These barriers are likely formed by the nucleoid and crowding of the cytoplasm. The cytoplasm of E. coli appears as a meshwork allowing the free passage of small molecules while restricting the diffusion of bigger ones.

Authors: , Geert Van Den Bogaart, Liesbeth Veenhoff, Victor Krasnikov,

Date Published: 1st Jul 2010

Publication Type: Not specified

Central role of the RNA polymerase trigger loop in intrinsic RNA hydrolysis

Abstract (Expand)

The active center of RNA polymerase can hydrolyze phosphodiester bonds in nascent RNA, a reaction thought to be important for proofreading of transcription. The reaction proceeds via a general two … Mg(2+) mechanism and is assisted by the 3' end nucleotide of the transcript. Here, by using Thermus aquaticus RNA polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (TL). We show that the invariant histidine (beta' His1242) of the TL is essential for hydrolysis/proofreading and participates in the reaction in two distinct ways: by positioning the 3' end nucleotide of the transcript that assists catalysis and/or by directly participating in the reaction as a general base. We also show that participation of the beta' His1242 of the TL in phosphodiester bond hydrolysis does not depend on the extent of elongation complex backtracking. We obtained similar results with Escherichia coli RNA polymerase, indicating that the function of the TL in phosphodiester bond hydrolysis is conserved among bacteria.

Authors: Yulia Yuzenkova,

Date Published: 1st Jun 2010

Publication Type: Not specified

Lack of main K(+) uptake systems in Saccharomyces cerevisiae cells affects yeast performance in both potassium-sufficient and potassium-limiting conditions

Abstract (Expand)

Abstract A new YNB medium containing very low concentrations of alkali metal cations has been developed to carry out experiments to study potassium homoeostasis. Physiological characterization of … Saccharomyces cerevisiae BY4741 strain and the corresponding mutant lacking the main potassium uptake systems (trk1 trk2) under potassium nonlimiting and limiting concentrations was performed, and novel important differences between both strains were found. At nonlimiting concentrations of KCl, the two strains had a comparable cell size and potassium content. Nevertheless, mutants were hyperpolarized, had lower pH and extruded fewer protons compared with the BY4741 strain. Upon transfer to K(+)-limiting conditions, cells of both strains became hyperpolarized and their cell volume and K(+) content diminished; however, the decrease was more relevant in BY4741. In low potassium, trk1 trk2 cells were not able to accomplish the cell cycle to the same extent as in BY4741. Moreover, K(+) limitation triggered a high-affinity K(+)/Rb(+) uptake process only in BY4741, with the highest affinity being reached as soon as 30 min after transfer to potassium-limiting conditions. By establishing basic cellular parameters under standard growth conditions, this work aims to establish a basis for the investigation of potassium homoeostasis at the system level.

Authors: , , , José L Martínez, , , ,

Date Published: 25th May 2010

Publication Type: Not specified

SensSB: A software toolbox for the development and sensitivity analysis of systems biology models

Abstract (Expand)

SUMMARY: SensSB (Sensitivity Analysis for Systems Biology) is an easy to use, MATLAB-based software toolbox, which integrates several local and global sensitivity methods that can be applied to a wide … variety of biological models. In addition to addressing the sensitivity analysis problem, SensSB aims to cover all the steps involved during the modeling process. The main features of SensSB are: (i) derivative and variance based global sensitivity analysis, (ii) pseudo-global identifiability analysis, (iii) optimal experimental design based on global sensitivities, (iv) robust parameter estimation, (v) local sensitivity and identifiability analysis, (vi) confidence intervals of the estimated parameters, and (vii) optimal experimental design based on the Fisher Information Matrix (FIM). SensSB is also able to import models in the Systems Biology Mark-up Language (SBML) format. Several examples from simple analytical functions to more complex biological pathways have been implemented and can be downloaded together with the toolbox. The importance of using sensitivity analysis techniques for identifying unessential parameters and designing new experiments is quantified by increased identifiability metrics of the models and decreased confidence intervals of the estimated parameters. AVAILABILITY: SensSB is a software toolbox freely downloadable from http://www.iim.csic.es/~gingproc/SensSB.html. The web site also contains several examples and an extensive documentation. CONTACT: mrodriguez@iim.csic.es.

Editor:

Date Published: 7th May 2010

Publication Type: Not specified

The silicon trypanosome

Abstract (Expand)

African trypanosomes have emerged as promising unicellular model organisms for the next generation of systems biology. They offer unique advantages, due to their relative simplicity, the availability … of all standard genomics techniques and a long history of quantitative research. Reproducible cultivation methods exist for morphologically and physiologically distinct life-cycle stages. The genome has been sequenced, and microarrays, RNA-interference and high-accuracy metabolomics are available. Furthermore, the availability of extensive kinetic data on all glycolytic enzymes has led to the early development of a complete, experiment-based dynamic model of an important biochemical pathway. Here we describe the achievements of trypanosome systems biology so far and outline the necessary steps towards the ambitious aim of creating a 'Silicon Trypanosome', a comprehensive, experiment-based, multi-scale mathematical model of trypanosome physiology. We expect that, in the long run, the quantitative modelling enabled by the Silicon Trypanosome will play a key role in selecting the most suitable targets for developing new anti-parasite drugs.

Authors: , , , , , , Paul A M Michels, ,

Date Published: 6th May 2010

Publication Type: Not specified

Penicillin-binding protein folding is dependent on the PrsA peptidyl-prolyl cis-trans isomerase in Bacillus subtilis

Abstract (Expand)

Summary The PrsA protein is a membrane-anchored peptidyl-prolyl cis-trans isomerase in Bacillus subtilis and most other Gram-positive bacteria. It catalyses the post-translocational folding of exported … proteins and is essential for normal growth of B. subtilis. We studied the mechanism behind this indispensability. We could construct a viable prsA null mutant in the presence of a high concentration of magnesium. Various changes in cell morphology in the absence of PrsA suggested that PrsA is involved in the biosynthesis of the cylindrical lateral wall. Consistently, four penicillin-binding proteins (PBP2a, PBP2b, PBP3 and PBP4) were unstable in the absence of PrsA, while muropeptide analysis revealed a 2% decrease in the peptidoglycan cross-linkage index. Misfolded PBP2a was detected in PrsA-depleted cells, indicating that PrsA is required for the folding of this PBP either directly or indirectly. Furthermore, strongly increased uniform staining of cell wall with a fluorescent vancomycin was observed in the absence of PrsA. We also demonstrated that PrsA is a dimeric or oligomeric protein which is localized at distinct spots organized in a helical pattern along the cell membrane. These results suggest that PrsA is essential for normal growth most probably as PBP folding is dependent on this PPIase.

Authors: Hanne-Leena Hyyryläinen, , Kathleen Dahncke, Milla Pietiäinen, Pascal Courtin, Marika Vitikainen, Raili Seppala, Andreas Otto, Dörte Becher, Marie-Pierre Chapot-Chartier, , Vesa P Kontinen

Date Published: 4th May 2010

Publication Type: Not specified

In vitro phosphorylation of key metabolic enzymes from Bacillus subtilis: PrkC phosphorylates enzymes from different branches of basic metabolism

Abstract (Expand)

Phosphorylation is an important mechanism of protein modification. In the Gram-positive soil bacterium Bacillus subtilis, about 5% of all proteins are subject to phosphorylation, and a significant … portion of these proteins is phosphorylated on serine or threonine residues. We were interested in the regulation of the basic metabolism in B. subtilis. Many enzymes of the central metabolic pathways are phosphorylated in this organism. In an attempt to identify the responsible protein kinase(s), we identified four candidate kinases, among them the previously studied kinase PrkC. We observed that PrkC is indeed able to phosphorylate several metabolic enzymes in vitro. Determination of the phosphorylation sites revealed a remarkable preference of PrkC for threonine residues. Moreover, PrkC often used several phosphorylation sites in one protein. This feature of PrkC-dependent protein phosphorylation resembles the multiple phosphorylations often observed in eukaryotic proteins. The HPr protein of the phosphotransferase system is one of the proteins phosphorylated by PrkC, and PrkC phosphorylates a site (Ser-12) that has recently been found to be phosphorylated in vivo. The agreement between in vivo and in vitro phosphorylation of HPr on Ser-12 suggests that our in vitro observations reflect the events that take place in the cell.

Authors: Nico Pietack, Dörte Becher, Sebastian R Schmidl, Milton H Saier, , Fabian M Commichau,

Date Published: 13th Apr 2010

Publication Type: Not specified

Stepwise mechanism for transcription fidelity

Abstract (Expand)

Transcription is the first step of gene expression and is characterized by a high fidelity of RNA synthesis. During transcription, the RNA polymerase active centre discriminates against not just … non-complementary ribo NTP substrates but also against complementary 2'- and 3'-deoxy NTPs. A flexible domain of the RNA polymerase active centre, the Trigger Loop, was shown to play an important role in this process, but the mechanisms of this participation remained elusive.

Authors: , Aleksandra Bochkareva, Vasisht R Tadigotla, Mohammad Roghanian, Savva Zorov, Konstantin Severinov,

Date Published: 1st Apr 2010

Publication Type: Not specified

Graphical analysis and experimental evaluation of Saccharomyces cerevisiae PTRK1|2 and PBMH1|2 promoter region

Abstract (Expand)

We designed a simple graphical presentation for the results of a transcription factor (TF) pattern matching analysis. The TF analysis algorithm utilized known sequence signature motifs from several … databases. The graphical presentation enabled a quick overview of potential TF binding sites, their frequency and spacing on both DNA strands and thus straight forward identification of promising candidates for further experimental investigations. The developed tool was applied on in total four Saccharomyces cerevisiae gene promoter regions. The selected differentially expressed genes belong to functionally different families and encode duplicate functions, TRK1 and TRK2 as ion transporters and BMH1 and BMH2 as multiple regulators. Output evaluation revealed a number of TFs with promising differences in the promoter regions of each gene pair. Experimental investigations were performed by using corresponding TF yeast mutants for either phenotypic analysis of ion transport mediated growth or expression analysis of BMH1,2 genes. Upon phenotypic testing one TF mutant exhibited severely impaired growth under non-permissive conditions. This TF, Mot3p was identified as of most abundant potential binding sites and distinctive patterns among the TRK promoter regions.

Editor:

Date Published: 19th Mar 2010

Publication Type: Not specified

Gene position within a long transcript as a determinant for stochastic switching in bacteria

Abstract (Expand)

How cultures of genetically identical cells bifurcate into distinct phenotypic subpopulations under uniform growth conditions is an important question in developmental biology of relevance even to … relatively simple developmental systems, such as spore formation in bacteria. A growing Bacillus subtilis culture consists of either cells that are motile and can swim or cells that are non-motile and are chained together. In this issue of Molecular Microbiology, Cozy and Kearns show that the probability of a cell to become motile depends on the position of the sigD gene within the long (27 kb) motility operon. sigD encodes the alternative sigma factor sigma(D) that, together with RNA polymerase, drives expression of genes required for cell separation and the assembly of flagella. sigD is the penultimate gene of the B. subtilis motility operon and, in the control strain approximately, 70% of the cells are motile. When sigD was moved upstream within the operon, a larger fraction of cells became motile (up to 100%). This study highlights that the position of a gene within an operon can have a large impact on the control of gene expression. Furthermore, it suggests that RNA polymerase processivity or mRNA turnover can play important roles as sources of noise in bacterial development, and that gene position might be an unrecognized and possibly widespread mechanism to regulate phenotypic variation.

Editor:

Date Published: 10th Mar 2010

Publication Type: Not specified

Alkali metal cation transport and homeostasis in yeasts

Abstract (Expand)

The maintenance of appropriate intracellular concentrations of alkali metal cations, principally K(+) and Na(+), is of utmost importance for living cells, since they determine cell volume, intracellular … pH, and potential across the plasma membrane, among other important cellular parameters. Yeasts have developed a number of strategies to adapt to large variations in the concentrations of these cations in the environment, basically by controlling transport processes. Plasma membrane high-affinity K(+) transporters allow intracellular accumulation of this cation even when it is scarce in the environment. Exposure to high concentrations of Na(+) can be tolerated due to the existence of an Na(+), K(+)-ATPase and an Na(+), K(+)/H(+)-antiporter, which contribute to the potassium balance as well. Cations can also be sequestered through various antiporters into intracellular organelles, such as the vacuole. Although some uncertainties still persist, the nature of the major structural components responsible for alkali metal cation fluxes across yeast membranes has been defined within the last 20 years. In contrast, the regulatory components and their interactions are, in many cases, still unclear. Conserved signaling pathways (e.g., calcineurin and HOG) are known to participate in the regulation of influx and efflux processes at the plasma membrane level, even though the molecular details are obscure. Similarly, very little is known about the regulation of organellar transport and homeostasis of alkali metal cations. The aim of this review is to provide a comprehensive and up-to-date vision of the mechanisms responsible for alkali metal cation transport and their regulation in the model yeast Saccharomyces cerevisiae and to establish, when possible, comparisons with other yeasts and higher plants.

Editor:

Date Published: 4th Mar 2010

Publication Type: Not specified

Novel components of an active mitochondrial K(+)/H(+) exchange

Abstract (Expand)

Defects of the mitochondrial K(+)/H(+) exchanger (KHE) result in increased matrix K(+) content, swelling, and autophagic decay of the organelle. We have previously identified the yeast Mdm38 and its … human homologue LETM1, the candidate gene for seizures in Wolf-Hirschhorn syndrome, as essential components of the KHE. In a genome-wide screen for multicopy suppressors of the pet(-) (reduced growth on nonfermentable substrate) phenotype of mdm38Delta mutants, we now characterized the mitochondrial carriers PIC2 and MRS3 as moderate suppressors and MRS7 and YDL183c as strong suppressors. Like Mdm38p, Mrs7p and Ydl183cp are mitochondrial inner membrane proteins and constituents of approximately 500-kDa protein complexes. Triple mutant strains (mdm38Delta mrs7Delta ydl183cDelta) exhibit a remarkably stronger pet(-) phenotype than mdm38Delta and a general growth reduction. They totally lack KHE activity, show a dramatic drop of mitochondrial membrane potential, and heavy fragmentation of mitochondria and vacuoles. Nigericin, an ionophore with KHE activity, fully restores growth of the triple mutant, indicating that loss of KHE activity is the underlying cause of its phenotype. Mdm38p or overexpression of Mrs7p, Ydl183cp, or LETM1 in the triple mutant rescues growth and KHE activity. A LETM1 human homologue, HCCR-1/LETMD1, described as an oncogene, partially suppresses the yeast triple mutant phenotype. Based on these results, we propose that Ydl183p and the Mdm38p homologues Mrs7p, LETM1, and HCCR-1 are involved in the formation of an active KHE system.

Authors: Ludmila Zotova, Markus Aleschko, Gerhard Sponder, Roland Baumgartner, Siegfried Reipert, Monika Prinz, ,

Date Published: 2nd Mar 2010

Publication Type: Not specified

Functional dissection of a trigger enzyme: mutations of the bacillus subtilis glutamate dehydrogenase RocG that affect differentially its catalytic activity and regulatory properties

Abstract (Expand)

Any signal transduction requires communication between a sensory component and an effector. Some enzymes engage in signal perception and transduction, as well as in catalysis, and these proteins are … known as "trigger" enzymes. In this report, we detail the trigger properties of RocG, the glutamate dehydrogenase of Bacillus subtilis. RocG not only deaminates the key metabolite glutamate to form alpha-ketoglutarate but also interacts directly with GltC, a LysR-type transcription factor that regulates glutamate biosynthesis from alpha-ketoglutarate, thus linking the two metabolic pathways. We have isolated mutants of RocG that separate the two functions. Several mutations resulted in permanent inactivation of GltC as long as a source of glutamate was present. These RocG proteins have lost their ability to catabolize glutamate due to a strongly reduced affinity for glutamate. The second class of mutants is exemplified by the replacement of aspartate residue 122 by asparagine. This mutant protein has retained enzymatic activity but has lost the ability to control the activity of GltC. Crystal structures of glutamate dehydrogenases that permit a molecular explanation of the properties of the various mutants are presented. Specifically, we may propose that D122N replacement affects the surface of RocG. Our data provide evidence for a correlation between the enzymatic activity of RocG and its ability to inactivate GltC, and thus give insights into the mechanism that couples the enzymatic activity of a trigger enzyme to its regulatory function.

Authors: Katrin Gunka, , Fabian M Commichau, Christina Herzberg, Cecilia Rodrigues, Lorraine Hewitt, , Jörg Stülke

Date Published: 22nd Feb 2010

Publication Type: Not specified

Metabolomic systems biology of trypanosomes

Abstract (Expand)

Metabolomics analysis, which aims at the systematic identification and quantification of all metabolites in biological systems, is emerging as a powerful new tool to identify biomarkers of disease, … report on cellular responses to environmental perturbation, and to identify the targets of drugs. Here we discuss recent developments in metabolomic analysis, from the perspective of trypanosome research, highlighting remaining challenges and the most promising areas for future research.

Editor:

Date Published: 17th Feb 2010

Publication Type: Not specified

Heterochronic phosphorelay gene expression as a source of heterogeneity in Bacillus subtilis spore formation

Abstract (Expand)

In response to limiting nutrient sources and cell density signals, Bacillus subtilis can differentiate and form highly resistant endospores. Initiation of spore development is governed by the master … regulator Spo0A, which is activated by phosphorylation via a multicomponent phosphorelay. Interestingly, only part of a clonal population will enter this developmental pathway, a phenomenon known as sporulation bistability or sporulation heterogeneity. How sporulation heterogeneity is established is largely unknown. To investigate the origins of sporulation heterogeneity, we constructed promoter-green fluorescent protein (GFP) fusions to the main phosphorelay genes and perturbed their expression levels. Using time-lapse fluorescence microscopy and flow cytometry, we showed that expression of the phosphorelay genes is distributed in a unimodal manner. However, single-cell trajectories revealed that phosphorelay gene expression is highly dynamic or "heterochronic" between individual cells and that stochasticity of phosphorelay gene transcription might be an important regulatory mechanism for sporulation heterogeneity. Furthermore, we showed that artificial induction or depletion of the phosphorelay phosphate flow results in loss of sporulation heterogeneity. Our data suggest that sporulation heterogeneity originates from highly dynamic and variable gene activity of the phosphorelay components, resulting in large cell-to-cell variability with regard to phosphate input into the system. These transcriptional and posttranslational differences in phosphorelay activity appear to be sufficient to generate a heterogeneous sporulation signal without the need of the positive-feedback loop established by the sigma factor SigH.

Editor:

Date Published: 12th Feb 2010

Publication Type: Not specified

Saccharomyces cerevisiae BY4741 and W303-1A laboratory strains differ in salt tolerance

Abstract (Expand)

Saccharomyces cerevisiae yeast cells serve as a model to elucidate the bases of salt tolerance and potassium homeostasis regulation in eukaryotic cells. In this study, we show that two widely used … laboratory strains, BY4741 and W303-1A, differ not only in cell size and volume but also in their relative plasma-membrane potential (estimated with a potentiometric fluorescent dye diS-C3(3) and as Hygromycin B sensitivity) and tolerance to alkali-metal cations. W303-1A cells and their mutant derivatives lacking either uptake (trk1 trk2) or efflux (nha1) systems for alkali-metal cations are more tolerant to toxic sodium and lithium cations but also more sensitive to higher external concentrations of potassium than BY4741 cells and their mutants. Moreover, our results suggest that though the two strains do not differ in the total potassium content, the regulation of intracellular potassium homeostasis is probably not the same in BY4741 and W303-1A cells.

Editor:

Date Published: 1st Feb 2010

Publication Type: Not specified

Miniaturized device for agitating a high-density yeast suspension that is suitable for in vivo nuclear magnetic resonance applications

Abstract (Expand)

In vivo nuclear magnetic resonance (NMR) monitoring requires a high-density cell suspension, where cell precipitation should be avoided. We have designed a miniaturized cell agitator that fits entirely … into an 8-mm NMR probe but that, being mounted into the instrument, is situated outside of the sensitive area. The device consists of two glass tubes connected in a way that, when gas flow is blown through them, creates influx of cell suspension into the device that returns through apertures. This flow creates continuous circular vortex of the cell suspension in the whole sample volume, whereas there are no moving mechanical parts or gas bubbles crossing the instrument’s sensitive area. The gas flow controls conditions of the cell suspension and removes volatile waste metabolites.

Authors: , Christian Bock

Date Published: 1st Feb 2010

Publication Type: Not specified

Transformation of environmental Bacillus subtilis isolates by transiently inducing genetic competence

Abstract (Expand)

Domesticated laboratory strains of Bacillus subtilis readily take up and integrate exogenous DNA. In contrast, "wild" ancestors or Bacillus strains recently isolated from the environment can … only be genetically modified by phage transduction, electroporation or protoplast transformation. Such methods are laborious, have a variable yield or cannot efficiently be used to alter chromosomal DNA. A major disadvantage of using laboratory strains is that they have often lost, or do not display ecologically relevant physiologies such as the ability to form biofilms. Here we present a method that allows genetic transformation by natural competence in several environmental isolates of B. subtilis. Competence in these strains was established by expressing the B. subtilis competence transcription factor ComK from an IPTG-inducible promoter construct present on an unstable plasmid. This transiently activates expression of the genes required for DNA uptake and recombination in the host strain. After transformation, the comK encoding plasmid is lost easily because of its intrinsic instability and the transformed strain returns to its wild state. Using this method, we have successfully generated mutants and introduced foreign DNA into a number of environmental isolates and also B. subtilis strain NCIB3610, which is widely used to study biofilm formation. Application of the same method to strains of B. licheniformis was unsuccessful. The efficient and rapid approach described here may facilitate genetic studies in a wider array of environmental B. subtilis strains.

Authors: Reindert Nijland, J Grant Burgess, Jeff Errington,

Date Published: 11th Jan 2010

Publication Type: Not specified

Identification and characterization of the PhhR regulon inPseudomonas putida

Abstract (Expand)

Pseudomonas putida is a soil microorganism that utilizes aromatic amino acids present in root exudates as a nitrogen source. We have previously shown that the PhhR transcriptional regulator induces … phhAB genes encoding a phenylalanine hydroxylase. In this study we show, using microarray assays and promoter fusions, that PhhR is a global regulator responsible for the activation of genes essential for phenylalanine degradation, phenylalanine homeostasis and other genes of unknown function. Recently, it has been shown that phenylalanine catabolism occurs through more than one pathway. One of these possible pathways involves the metabolism of phenylalanine via tyrosine, p-hydroxyphenylpyruvate, and homogentisate. We identified two genes within this pathway that encode an acyl-CoA transferase involved in the metabolism of acetoacetate. All genes in this pathway were induced in response to phenylalanine in a PhhR-proficient background. The second potential degradative pathway involves the degradation of phenylalanine to produce phenylpyruvate, which seems to be degraded via phenylacetyl-CoA. A number of mutants in the paa genes encoding phenylacetyl-CoA degradation enzymes fail to grow on phenylpyruvate or phenylacetate, further supporting the existence of this second pathway. We found that the PhhR regulon also includes genes involved in the biosynthesis of aromatic amino acids that are repressed in the presence of phenylalanine, suggesting the possibility of feedback at the transcriptional level. In addition, we found that PhhR modulates the level of expression of the broad-substrate-specificity MexEF/OprN efflux pump. Expression from this pump is under the control of mexT gene product because phenylalanine-dependent transcription from the mexE promoter does not occur in a mexT mutant background. These results place PhhR as an important regulator in the control of bacterial responses to aromatic amino acids.

Authors: M. Carmen Herrera, , José J. Rodríguez-Herva, Ana M. Fernández-Escamilla,

Date Published: 2010

Publication Type: Not specified

Metabolic modeling and analysis of the metabolic switch in Streptomyces coelicolor

Abstract (Expand)

Background The transition from exponential to stationary phase in Streptomyces coelicolor is accompanied by a major metabolic switch and results in a strong activation of secondary metabolism. Here we … have explored the underlying reorganization of the metabolome by combining computational predictions based on constraint-based modeling and detailed transcriptomics time course observations. Results We reconstructed the stoichiometric matrix of S. coelicolor, including the major antibiotic biosynthesis pathways, and performed flux balance analysis to predict flux changes that occur when the cell switches from biomass to antibiotic production. We defined the model input based on observed fermenter culture data and used a dynamically varying objective function to represent the metabolic switch. The predicted fluxes of many genes show highly significant correlation to the time series of the corresponding gene expression data. Individual mispredictions identify novel links between antibiotic production and primary metabolism. Conclusion Our results show the usefulness of constraint-based modeling for providing a detailed interpretation of time course gene expression data. Other Sections▼

Authors: , , The STREAM Consortium (stream), , , ,

Date Published: 2010

Publication Type: Not specified

Integration of sensitivity and bifurcation analysis to detect critical processes in a model combining signalling and cell population dynamics

Abstract (Expand)

In this article we present and test a strategy to integrate, in a sequential manner, sensitivity analysis, bifurcation analysis and predictive simulations. Our strategy uses some of these methods in a … coordinated way such that information, generated in one step, feeds into the definition of further analyses and helps refining the structure of the mathematical model. The aim of the method is to help in the designing of more informative predictive simulations, which focus on critical model parameters and the biological effects of their modulation. We tested our methodology with a multilevel model, accounting for the effect of erythropoietin (Epo)-mediated JAK2-STAT5 signalling in erythropoiesis. Our analysis revealed that time-delays associated with the proliferation-differentiation process are critical to induce pathological sustained oscillations, whereas the modulation of time-delays related to intracellular signalling and hypoxia-controlled physiological dynamics is not enough to induce self-oscillations in the system. Furthermore, our results suggest that the system is able to compensate (through the physiological-level feedback loop on hypoxia) the partial impairment of intracellular signalling processes (downregulation or overexpression of Epo receptor complex and STAT5), but cannot control impairment in some critical physiological-level processes, which provoke the emergence of pathological oscillations.

Authors: S. Nikolov, X. Lai, , , J. Vera

Date Published: 2010

Publication Type: Not specified

Ref2, a regulatory subunit of the yeast protein phosphatase 1, is a novel component of cation homoeostasis

Abstract (Expand)

Maintenance of cation homoeostasis is a key process for any living organism. Specific mutations in Glc7, the essential catalytic subunit of yeast protein phosphatase 1, result in salt and alkaline pH … sensitivity, suggesting a role for this protein in cation homoeostasis. We screened a collection of Glc7 regulatory subunit mutants for altered tolerance to diverse cations (sodium, lithium and calcium) and alkaline pH. Among 18 candidates, only deletion of REF2 (RNA end formation 2) yielded increased sensitivity to these conditions, as well as to diverse organic toxic cations. The Ref2F374A mutation, which renders it unable to bind Glc7, did not rescue the salt-related phenotypes of the ref2 strain, suggesting that Ref2 function in cation homoeostasis is mediated by Glc7. The ref2 deletion mutant displays a marked decrease in lithium efflux, which can be explained by the inability of these cells to fully induce the Na+-ATPase ENA1 gene. The effect of lack of Ref2 is additive to that of blockage of the calcineurin pathway and might disrupt multiple mechanisms controlling ENA1 expression. ref2 cells display a striking defect in vacuolar morphogenesis, which probably accounts for the increased calcium levels observed under standard growth conditions and the strong calcium sensitivity of this mutant. Remarkably, the evidence collected indicates that the role of Ref2 in cation homoeostasis may be unrelated to its previously identified function in the formation of mRNA via the APT (for associated with Pta1) complex.

Authors: Jofre Ferrer-Dalmau, Asier González, Maria Platara, , José L Martínez, , , ,

Date Published: 24th Dec 2009

Publication Type: Not specified

Computer controlled automated assay for comprehensive studies of enzyme kinetic parameters

Abstract (Expand)

Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information … on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework ("robot lab assistant") that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK). A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation-sites at the enzyme or substrates critical for enzymatic activity. The developed framework is a powerful tool to investigate enzyme reaction specifics and explore biological system behaviour in a wide range of experimental conditions.

Authors: Felix Bonowski, , , Jinda Holzwarth, Igor Kitanovic, Van Ngoc Bui, Elke Lederer,

Date Published: 23rd Dec 2009

Publication Type: Not specified

Signal Transduction by Trigger Enzymes: Bifunctional Enzymes and Transporters Controlling Gene Expression

Abstract

Not specified

Authors: Fabian M. Commichau, Jrg Stlke

Date Published: 16th Dec 2009

Publication Type: Not specified

Recombination between ccrC genes in a type V (5C2&5) staphylococcal cassette chromosome mec (SCCmec) of Staphylococcus aureus ST398 leads to conversion from methicillin resistance to methicillin susceptibility in vivo

Abstract (Expand)

Various types of the staphylococcal cassette chromosome mec (SCCmec) are known to confer methicillin resistance on the human pathogen Staphylococcus aureus. Such cassettes are not always stably maintained. … The present studies were aimed at identifying the mechanism underlying the in vivo conversion of methicillin-resistant S. aureus (MRSA) to methicillin-susceptible S. aureus (MSSA) derivatives as encountered in two patients suffering from pneumonia and an umbilicus infection, respectively. All MRSA and MSSA isolates identified belong to multilocus sequence type (MLST) 398, have spa type t034, and are Panton-Valentine leukocidin positive. Sequencing of 27,616 nucleotides from the chromosomal SCCmec insertion site in orfX to the hsdR gene for a restriction enzyme revealed a type V (5C2&5) SCCmec. Sequence comparisons show that parts of the cassette are highly similar to sequences within SCCmec elements from coagulase-negative staphylococci, indicating a possible common origin. The cassette investigated contains ccrC-carrying units on either side of its class C2b mec gene complex. In vivo loss of the mec gene complex was caused by recombination between the recombinase genes ccrC1 allele 8 and ccrC1 allele 10. In vitro, the SCCmec was very stable, and low-frequency MRSA-to-MSSA conversion was only observed when MRSA isolates were cultivated at 41 degrees C for prolonged periods of time. In this case also, loss of the mec complex was due to ccrC gene recombination. Interestingly, the MRSA and MSSA isolates studied displayed no detectable differences in competitive growth and virulence, suggesting that the presence of the intact type V (5C2&5) SCCmec has no negative bearing on staphylococcal fitness under the conditions used.

Authors: Monika A Chlebowicz, Kristelle Nganou, Svitlana Kozytska, Jan P Arends, Susanne Engelmann, Hajo Grundmann, Knut Ohlsen, , Girbe Buist

Date Published: 7th Dec 2009

Publication Type: Not specified

Quantitative proteomic analysis of Sulfolobus solfataricus membrane proteins

Abstract (Expand)

A quantitative proteomic analysis of the membrane of the archaeon Sulfolobus solfataricus P2 using iTRAQ was successfully demonstrated in this technical note. The estimated number of membrane proteins … of this organism is 883 (predicted based on Gravy score), corresponding to 30% of the total number of proteins. Using a modified iTRAQ protocol for membrane protein analysis, of the 284 proteins detected, 246 proteins were identified as membrane proteins, while using an original iTRAQ protocol, 147 proteins were detected with only 133 proteins being identified as membrane proteins. Furthermore, 97.2% of proteins identified in the modified protocol contained more than 2 distinct peptides compared to the original workflow. The successful application of this modified protocol offers a potential technique for quantitatively analyzing membrane-associated proteomes of organisms in the archaeal kingdom. The combination of 3 different iTRAQ experiments resulted in the detection of 395 proteins (>or=2 distinct peptides) of which 373 had predicted membrane properties. Approximately 20% of the quantified proteins were observed to exhibit >or=1.5-fold differential expression at temperatures well below the optimum for growth.

Editor:

Date Published: 4th Dec 2009

Publication Type: Not specified

Connecting parts with processes: SubtiWiki and SubtiPathways integrate gene and pathway annotation for Bacillus subtilis

Abstract (Expand)

Bacillus subtilis is the model organism for a large group of Gram-positive bacteria, the Firmicutes. Several online databases have been established over time to manage its genetic and metabolic … information, but they differ greatly in their rate of update and their focus on B. subtilis. Therefore, a European systems biology consortium called for an integrated solution that empowers its users to enrich online content. To meet this goal we created SubtiWiki and SubtiPathways, two complementary online tools for gene and pathway information on B. subtilis 168. SubtiWiki (http://subtiwiki.uni-goettingen.de/ ) is a scientific wiki for all genes of B. subtilis and their protein or RNA products. Each gene page contains a summary of the most important information; sections on the gene, its product and expression; sections concerning biological materials and laboratories; and a list of references. SubtiWiki has been seeded with key content and can be extended by any researcher after a simple registration, thus keeping it always up to date. As a complement, SubtiPathways (http://subtipathways.uni-goettingen.de/) is an online tool for navigation of the metabolism of B. subtilis and its regulation. Each SubtiPathways diagram presents a metabolic pathway with its participating enzymes, together with the regulatory mechanisms that act on their expression and activity, in an intuitive interface that is based on Google Maps. Together, SubtiWiki and SubtiPathways provide an integrated view of the processes that make up B. subtilis and its components, making it the most comprehensive web resource for B. subtilis researchers.

Authors: Christoph R Lammers, , Arne G Schmeisky, Sebastian F Roppel, Ulrike Mäder, ,

Date Published: 3rd Dec 2009

Publication Type: Not specified

A mathematical investigation of the effects of inhibitor therapy on three putative phosphorylation cascades governing the two-component system of the agr operon

Abstract (Expand)

Two-component systems (TCSs) are widely employed by bacteria to sense specific external signals and conduct an appropriate response via a phosphorylation cascade within the cell. The TCS of the agr … operon in the bacterium Staphylococcus aureus forms part of a regulatory process termed quorum sensing, a cell-to-cell communication mechanism used to assess population density. Since S. aureus manipulates this "knowledge" in order to co-ordinate production of its armoury of exotoxin virulence factors required to promote infection, it is important to understand fully how this process works. We present three models of the agr operon, each incorporating a different phosphorylation cascade for the TCS since the precise nature of the cascade is not fully understood. Using numerical and asymptotic techniques we examine the effects of inhibitor therapy, a novel approach to controlling bacterial infection through the attenuation of virulence, on each of these three cascades. We present results which, if evaluated against appropriate experimental data, provide insights into the potential effectiveness of such therapy. Moreover, the TCS models presented here are of broad relevance given that TCSs are widely conserved throughout the bacterial kingdom.

Authors: , , Paul Williams

Date Published: 30th Nov 2009

Publication Type: Not specified

Contributions of the pre- and pro-regions of a Staphylococcus hyicus lipase to secretion of a heterologous protein by Bacillus subtilis

Abstract (Expand)

Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. … subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre(lip)) and pro (pro(lip)) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the pro(lip)-peptide, in combination with the lipase signal peptide (pre(lip)), contributes significantly to the efficient secretion of PhoA by B. subtilis and that pre(lip) directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.

Authors: Thijs R H M Kouwen, Allan K Nielsen, Emma L Denham, Jean-Yves F Dubois, Ronald Dorenbos, Michael D Rasmussen, Wim J Quax, Roland Freudl,

Date Published: 30th Nov 2009

Publication Type: Not specified

Significance analysis and statistical mechanics: an application to clustering

Abstract (Expand)

This Letter addresses the statistical significance of structures in random data: given a set of vectors and a measure of mutual similarity, how likely is it that a subset of these vectors forms a cluster … with enhanced similarity among its elements? The computation of this cluster p value for randomly distributed vectors is mapped onto a well-defined problem of statistical mechanics. We solve this problem analytically, establishing a connection between the physics of quenched disorder and multiple-testing statistics in clustering and related problems. In an application to gene expression data, we find a remarkable link between the statistical significance of a cluster and the functional relationships between its genes.

Authors: Marta Łuksza, Michael Lässig,

Date Published: 27th Nov 2009

Publication Type: Not specified

Annotation and merging of SBML models with semanticSBML

Abstract (Expand)

SUMMARY: Systems Biology Markup Language (SBML) is the leading exchange format for mathematical models in Systems Biology. Semantic annotations link model elements with external knowledge via unique … database identifiers and ontology terms, enabling software to check and process models by their biochemical meaning. Such information is essential for model merging, one of the key steps towards the construction of large kinetic models. SemanticSBML is a tool that helps users to check and edit MIRIAM annotations and SBO terms in SBML models. Using a large collection of biochemical names and database identifiers, it supports modellers in finding the right annotations and in merging existing models. Initially, an element matching is derived from the MIRIAM annotations and conflicting element attributes are categorized and highlighted. Conflicts can then be resolved automatically or manually, allowing the user to control the merging process in detail. AVAILABILITY: SemanticSBML comes as a free software written in Python and released under the GPL 3. A Debian package, a source package for other Linux distributions, a Windows installer and an online version of semanticSBML with limited functionality are available at http://www.semanticsbml.org. A preinstalled version can be found on the Linux live DVD SB.OS, available at http://www.sbos.eu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Authors: , Jannis Uhlendorf, Timo Lubitz, Marvin Schulz, , Wolfram Liebermeister

Date Published: 17th Nov 2009

Publication Type: Not specified

Staphylococcal PknB as the first prokaryotic representative of the proline-directed kinases

Abstract (Expand)

In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a … eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of eukaryotic serine/threonine kinases exist in bacteria. Moreover, various pathogenic species, such as Yersinia and Mycobacterium, require serine/threonine kinases for successful invasion of human host cells. The substrates targeted by bacterial serine/threonine kinases have remained largely unknown. Here we report that the serine/threonine kinase PknB from the important pathogen Staphylococcus aureus is released into the external milieu, which opens up the possibility that PknB does not only phosphorylate bacterial proteins but also proteins of the human host. To identify possible human targets of purified PknB, we studied in vitro phosphorylation of peptide microarrays and detected 68 possible human targets for phosphorylation. These results show that PknB is a proline-directed kinase with MAP kinase-like enzymatic activity. As the potential cellular targets for PknB are involved in apoptosis, immune responses, transport, and metabolism, PknB secretion may help the bacterium to evade intracellular killing and facilitate its growth. In apparent agreement with this notion, phosphorylation of the host-cell response coordinating transcription factor ATF-2 by PknB was confirmed by mass spectrometry. Taken together, our results identify PknB as the first prokaryotic representative of the proline-directed kinase/MAP kinase family of enzymes.

Authors: Malgorzata Miller, Stefanie Donat, Sonja Rakette, Thilo Stehle, Thijs R H M Kouwen, Sander H Diks, Annette Dreisbach, Ewoud Reilman, Katrin Gronau, Dörte Becher, Maikel P Peppelenbosch, , Knut Ohlsen

Date Published: 12th Nov 2009

Publication Type: Not specified

Systems biology: the elements and principles of life

Abstract (Expand)

Systems Biology has a mission that puts it at odds with traditional paradigms of physics and molecular biology, such as the simplicity requested by Occam's razor and minimum energy/maximal efficiency. … By referring to biochemical experiments on control and regulation, and on flux balancing in yeast, we show that these paradigms are inapt. Systems Biology does not quite converge with biology either: Although it certainly requires accurate 'stamp collecting', it discovers quantitative laws. Systems Biology is a science of its own, discovering own fundamental principles, some of which we identify here.

Authors: , Catherine Winder, , Evangelos Simeonidis, Malgorzata Adamczyk, , Frank J Bruggeman, Warwick Dunn

Date Published: 6th Nov 2009

Publication Type: Not specified

Functional analysis of new transporters involved in stress tolerance inPseudomonas putidaDOT-T1E

Abstract (Expand)

Pseudomonas putida DOT-T1E is a highly solvent-tolerant strain. Although the main mechanism that confers solvent tolerance to the strain is the TtgGHI efflux pump, a number of other proteins are also … involved in the response to toluene. Previous proteomic and transcriptomic analysis carried out in our lab with P. putida DOT-T1E, and the solvent-sensitive strain, P. putida KT2440, revealed several transporters that were induced in the presence of toluene. We prepared five mutants of the corresponding genes in P. putida DOT-T1E and analysed their phenotypes with respect to solvent tolerance, stress endurance and growth with different carbon, nitrogen and sulfur sources. The data clearly demonstrated that two transporters (Ttg2ABC and TtgK) are involved in multidrug resistance and toluene tolerance, whereas another (homologous to PP0219 of P. putida KT2440) is a sulfate/sulfite transporter. No clear function could be assigned to the other two transporters. Of the transporters shown to be involved in toluene tolerance, one (ttg2ABC) belongs to the ATP-Binding Cassette (ABC) family, and is involved in multidrug resistance in P. putida DOT-T1E, while the other belongs to the Major Facilitator Superfamily and exhibits homology to a putative transporter of the Bcr/CflA family that has not previously been reported to be involved in toluene tolerance.

Authors: Vanina García, Patricia Godoy, Craig Daniels, Ana Hurtado, , Ana Segura

Date Published: 1st Nov 2009

Publication Type: Not specified

Diamide triggers mainly S Thiolations in the cytoplasmic proteomes of Bacillus subtilis and Staphylococcus aureus

Abstract (Expand)

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. … Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.

Authors: Dierk-Christoph Pöther, Manuel Liebeke, Falko Hochgräfe, Haike Antelmann, Dörte Becher, , Ulrike Lindequist, Ilya Borovok, Gerald Cohen, Yair Aharonowitz,

Date Published: 16th Oct 2009

Publication Type: Not specified

Stress-responsive systems set specific limits to the overproduction of membrane proteins in Bacillus subtilis

Abstract (Expand)

Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins … for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the sigma(W) regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production.

Authors: Jessica C Zweers, Thomas Wiegert,

Date Published: 9th Oct 2009

Publication Type: Not specified

The large mechanosensitive channel MscL determines bacterial susceptibility to the bacteriocin sublancin 168

Abstract (Expand)

Bacillus subtilis strain 168 produces the extremely stable and broad-spectrum lantibiotic sublancin 168. Known sublancin 168-susceptible organisms include important pathogens, such as Staphylococcus … aureus. Nevertheless, since its discovery, the mode of action of sublancin 168 has remained elusive. The present studies were, therefore, aimed at the identification of cellular determinants for bacterial susceptibility toward sublancin 168. Growth inhibition and competition assays on plates and in liquid cultures revealed that sublancin 168-mediated growth inhibition of susceptible B. subtilis and S. aureus cells is affected by the NaCl concentration in the growth medium. Added NaCl did not influence the production, activity, or stability of sublancin 168 but, instead, lowered the susceptibility of sensitive cells toward this lantibiotic. Importantly, the susceptibility of B. subtilis and S. aureus cells toward sublancin 168 was shown to depend on the presence of the large mechanosensitive channel of conductance MscL. In contrast, MscL was not involved in susceptibility toward the bacteriocin nisin or Pep5. Taken together, our unprecedented results demonstrate that MscL is a critical and specific determinant in bacterial sublancin 168 susceptibility that may serve either as a direct target for this lantibiotic or as a gate of entry to the cytoplasm.

Authors: Thijs R H M Kouwen, Erik N Trip, Emma L Denham, Mark J J B Sibbald, Jean-Yves F Dubois,

Date Published: 8th Sep 2009

Publication Type: Not specified

Cellular localization of choline-utilization proteins in Streptococcus pneumoniae using novel fluorescent reporter systems

Abstract (Expand)

The molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to … investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp-fusion gene expression and for protein depletion experiments in S. pneumoniae. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.

Authors: Alice Eberhardt, Ling J Wu, Jeff Errington, Waldemar Vollmer,

Date Published: 8th Sep 2009

Publication Type: Not specified

Mechanisms and evolution of control logic in prokaryotic transcriptional regulation

Abstract (Expand)

A major part of organismal complexity and versatility of prokaryotes resides in their ability to fine-tune gene expression to adequately respond to internal and external stimuli. Evolution has been … very innovative in creating intricate mechanisms by which different regulatory signals operate and interact at promoters to drive gene expression. The regulation of target gene expression by transcription factors (TFs) is governed by control logic brought about by the interaction of regulators with TF binding sites (TFBSs) in cis-regulatory regions. A factor that in large part determines the strength of the response of a target to a given TF is motif stringency, the extent to which the TFBS fits the optimal TFBS sequence for a given TF. Advances in high-throughput technologies and computational genomics allow reconstruction of transcriptional regulatory networks in silico. To optimize the prediction of transcriptional regulatory networks, i.e., to separate direct regulation from indirect regulation, a thorough understanding of the control logic underlying the regulation of gene expression is required. This review summarizes the state of the art of the elements that determine the functionality of TFBSs by focusing on the molecular biological mechanisms and evolutionary origins of cis-regulatory regions.

Authors: Sacha A F T van Hijum, Marnix H Medema,

Date Published: 2nd Sep 2009

Publication Type: Not specified

"Hot standards" for the thermoacidophilic archaeon Sulfolobus solfataricus

Abstract (Expand)

Within the archaea, the thermoacidophilic crenarchaeote Sulfolobus solfataricus has become an important model organism for physiology and biochemistry, comparative and functional genomics, as well as, … more recently also for systems biology approaches. Within the Sulfolobus Systems Biology ("SulfoSYS")-project the effect of changing growth temperatures on a metabolic network is investigated at the systems level by integrating genomic, transcriptomic, proteomic, metabolomic and enzymatic information for production of a silicon cell-model. The network under investigation is the central carbohydrate metabolism. The generation of high-quality quantitative data, which is critical for the investigation of biological systems and the successful integration of the different datasets, derived for example from high-throughput approaches (e.g., transcriptome or proteome analyses), requires the application and compliance of uniform standard protocols, e.g., for growth and handling of the organism as well as the "-omics" approaches. Here, we report on the establishment and implementation of standard operating procedures for the different wet-lab and in silico techniques that are applied within the SulfoSYS-project and that we believe can be useful for future projects on Sulfolobus or (hyper)thermophiles in general. Beside established techniques, it includes new methodologies like strain surveillance, the improved identification of membrane proteins and the application of crenarchaeal metabolomics.

Authors: , Dominik Esser, , , , , , Julia Reimann, , , Daniela Teichmann, Marleen van Wolferen, , , , , , , , , ,

Date Published: 31st Aug 2009

Publication Type: Not specified

A mechanism for cell cycle regulation of sporulation initiation in Bacillus subtilis

Abstract (Expand)

Coordination of DNA replication with cellular development is a crucial problem in most living organisms. Bacillus subtilis cells switch from vegetative growth to sporulation when starved. Sporulation … normally occurs in cells that have stopped replicating DNA and have two completed chromosomes: one destined for the prespore and the other for the mother cell. It has long been recognized that there is a sensitive period in the cell cycle during which the initiation of spore development can be triggered, presumably to allow for the generation of exactly two complete chromosomes. However, the mechanism responsible for this has remained unclear. Here we show that the sda gene, previously identified as a checkpoint factor preventing sporulation in response to DNA damage, exerts cell cycle control over the initiation of sporulation. Expression of sda occurs in a pulsatile manner, with a burst of expression each cell cycle at the onset of DNA replication. Up-regulation of the intrinsically unstable Sda protein, which is dependent on the active form of the DNA replication initiator protein, DnaA, transiently inhibits the initiation of sporulation. This regulation avoids the generation of spore formers with replicating chromosomes, which would result in diploid or polyploid spores that we show have reduced viability.

Authors: , Heath Murray, Jeff Errington

Date Published: 18th Aug 2009

Publication Type: Not specified

Mathematical Modelling of the Sporulation-Initiation Network in Bacillus Subtilis Revealing the Dual Role of the Putative Quorum-Sensing Signal Molecule PhrA

Abstract (Expand)

Bacillus subtilis cells may opt to forgo normal cell division and instead form spores if subjected to certain environmental stimuli, for example nutrient deficiency or extreme temperature. The resulting … spores are extremely resilient and can survive for extensive periods of time, importantly under particularly harsh conditions such as those mentioned above. The sporulation process is highly time and energy consuming and essentially irreversible. The bacteria must therefore ensure that this route is only undertaken under appropriate circumstances. The gene regulation network governing sporulation initiation accordingly incorporates a variety of signals and is of significant complexity. We present a model of this network that includes four of these signals: nutrient levels, DNA damage, the products of the competence genes, and cell population size. Our results can be summarised as follows: (i) the model displays the correct phenotypic behaviour in response to these signals; (ii) a basal level of sda expression may prevent sporulation in the presence of nutrients; (iii) sporulation is more likely to occur in a large population of cells than in a small one; (iv) finally, and of most interest, PhrA can act simultaneously as a quorum-sensing signal and as a timing mechanism, delaying sporulation when the cell has damaged DNA, possibly thereby allowing the cell time to repair its DNA before forming a spore.

Editor:

Date Published: 21st Jul 2009

Publication Type: Not specified

Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase

Abstract (Expand)

The phosphatase calcineurin and the kinases Hal4/Hal5 regulate high-affinity potassium uptake in Saccharomyces cerevisiae through the Trk1 transporter. We demonstrate that calcineurin is necessary for … high-affinity potassium uptake even in the absence of Na(+) stress. HAL5 expression is induced in response to stress in a calcineurin-dependent manner through a newly identified functional CDRE (nt -195/-189). Lack of calcineurin decreases Hal5 protein levels, although with little effect on Trk1 amounts. However, the growth defect of cnb1 cells at K(+)-limiting conditions can be rescued in part by overexpression of HAL5, and this mutation further aggravates the potassium requirements of a hal4 strain. This suggests that the control exerted by calcineurin on Hal5 expression may be biologically relevant for Trk1 regulation.

Authors: Carlos Casado, Lynne Yenush, Carmen Melero, María del Carmen Ruiz, Raquel Serrano, Jorge Pérez-Valle, ,

Date Published: 3rd Jul 2009

Publication Type: Not specified

Protein transport across and into cell membranes in bacteria and archaea

Abstract (Expand)

In the three domains of life, the Sec, YidC/Oxa1, and Tat translocases play important roles in protein translocation across membranes and membrane protein insertion. While extensive studies have been … performed on the endoplasmic reticular and Escherichia coli systems, far fewer studies have been done on archaea, other Gram-negative bacteria, and Gram-positive bacteria. Interestingly, work carried out to date has shown that there are differences in the protein transport systems in terms of the number of translocase components and, in some cases, the translocation mechanisms and energy sources that drive translocation. In this review, we will describe the different systems employed to translocate and insert proteins across or into the cytoplasmic membrane of archaea and bacteria.

Authors: Jijun Yuan, Jessica C Zweers, , Ross E Dalbey

Date Published: 16th Jun 2009

Publication Type: Not specified

Applications of thiol-disulfide oxidoreductases for optimized in vivo production of functionally active proteins in Bacillus

Abstract (Expand)

Bacillus subtilis is a well-established cellular factory for proteins and fine chemicals. In particular, the direct secretion of proteinaceous products into the growth medium greatly facilitates their … downstream processing, which is an important advantage of B. subtilis over other biotechnological production hosts, such as Escherichia coli. The application spectrum of B. subtilis is, however, often confined to proteins from Bacillus or closely related species. One of the major reasons for this (current) limitation is the inefficient formation of disulfide bonds, which are found in many, especially eukaryotic, proteins. Future exploitation of B. subtilis to fulfill the ever-growing demand for pharmaceutical and other high-value proteins will therefore depend on overcoming this particular hurdle. Recently, promising advances in this area have been achieved, which focus attention on the need to modulate the cellular levels and activity of thiol-disulfide oxidoreductases (TDORs). These TDORs are enzymes that control the cleavage or formation of disulfide bonds. This review will discuss readily applicable approaches for TDOR modulation and aims to provide leads for further improvement of the Bacillus cell factory for production of disulfide bond-containing proteins.

Authors: Thijs R H M Kouwen,

Date Published: 11th Jun 2009

Publication Type: Not specified

The role of biomacromolecular crowding, ionic strength, and physicochemical gradients in the complexities of life's emergence

Abstract (Expand)

We have developed a general scenario of prebiotic physicochemical evolution during the Earth's Hadean eon and reviewed the relevant literature. We suggest that prebiotic chemical evolution started … in microspaces with membranous walls, where external temperature and osmotic gradients were coupled to free-energy gradients of potential chemical reactions. The key feature of this scenario is the onset of an emergent evolutionary transition within the microspaces that is described by the model of complex vectorial chemistry. This transition occurs at average macromolecular crowding of 20 to 30% of the cell volume, when the ranges of action of stabilizing colloidal forces (screened electrostatic forces, hydration, and excluded volume forces) become commensurate. Under these conditions, the macromolecules divide the interior of microspaces into dynamically crowded macromolecular regions and topologically complementary electrolyte pools. Small ions and ionic metabolites are transported vectorially between the electrolyte pools and through the (semiconducting) electrolyte pathways of the crowded macromolecular regions from their high electrochemical potential (where they are biochemically produced) to their lower electrochemical potential (where they are consumed). We suggest a sequence of tentative transitions between major evolutionary periods during the Hadean eon as follows: (i) the early water world, (ii) the appearance of land masses, (iii) the pre-RNA world, (iv) the onset of complex vectorial chemistry, and (v) the RNA world and evolution toward Darwinian thresholds. We stress the importance of high ionic strength of the Hadean ocean (short Debye's lengths) and screened electrostatic interactions that enabled the onset of the vectorial structure of the cytoplasm and the possibility of life's emergence.

Authors: Jan Spitzer,

Date Published: 3rd Jun 2009

Publication Type: Not specified

14-3-3 Proteins: insights from genome-wide studies in yeast

Abstract (Expand)

14-3-3 proteins form a family of highly conserved, acidic, dimeric proteins. These proteins have been identified in all eukaryotic species investigated, often in multiple isoforms, up to 13 in the plant … Arabidopsis thaliana. Hundreds of proteins, from diverse eukaryotic organisms, implicated in numerous cellular processes, have been identified as binding partners of 14-3-3 proteins. Therefore, the major activity of 14-3-3 proteins seems to be its ability to bind other intracellular proteins. Binding to 14-3-3 proteins may result in a conformational change of the protein required for its full activity or for inhibition of its activity, in interaction between two binding partners or in a different subcellular localization. Most of these interactions take place after phosphorylation of the binding partners. These observations suggest a major role of 14-3-3 proteins in regulatory networks. Here, the information on 14-3-3 proteins gathered from several genome- and proteome-wide studies in the yeast Saccharomyces cerevisiae is reviewed. In particular, the protein kinases responsible for the phosphorylation of 14-3-3 binding partners, phosphorylation of 14-3-3 proteins themselves, the transcriptional regulation of the 14-3-3 genes, and the role of 14-3-3 proteins in transcription are addressed. These large scale studies may help understand the function of 14-3-3 proteins at a cellular level rather than at the level of a single process.

Editor:

Date Published: 29th May 2009

Publication Type: Not specified

The dynamic architecture of the metabolic switch in Streptomyces coelicolor

Abstract (Expand)

BACKGROUND: During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene … expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples. RESULTS: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis. CONCLUSIONS: Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.

Authors: , Florian Battke, Alexander Herbig, , , , , , , , , Edward R Morrissey, Miguel A Juarez-Hermosillo, , Merle Nentwich, , Mudassar Iqbal, , , , , , , , Michael Bonin, , , , , , , , , ,

Date Published: 28th May 2009

Publication Type: Not specified

A community-curated consensual annotation that is continuously updated: the Bacillus subtilis centred wiki SubtiWiki

Abstract (Expand)

Bacillus subtilis is the model organism for Gram-positive bacteria, with a large amount of publications on all aspects of its biology. To facilitate genome annotation and the collection of comprehensive … information on B. subtilis, we created SubtiWiki as a community-oriented annotation tool for information retrieval and continuous maintenance. The wiki is focused on the needs and requirements of scientists doing experimental work. This has implications for the design of the interface and for the layout of the individual pages. The pages can be accessed primarily by the gene designations. All pages have a similar flexible structure and provide links to related gene pages in SubtiWiki or to information in the World Wide Web. Each page gives comprehensive information on the gene, the encoded protein or RNA as well as information related to the current investigation of the gene/protein. The wiki has been seeded with information from key publications and from the most relevant general and B. subtilis-specific databases. We think that SubtiWiki might serve as an example for other scientific wikis that are devoted to the genes and proteins of one organism.Database URL: The wiki can be accessed at http://subtiwiki.uni-goettingen.de/

Authors: , Sebastian F Roppel, Arne G Schmeisky, Christoph R Lammers,

Date Published: 26th May 2009

Publication Type: Not specified

Construction and characterization of three lactate dehydrogenase-negative Enterococcus faecalis V583 mutants

Abstract (Expand)

The roles of the two ldh genes of Enterococcus faecalis were studied using knockout mutants. Deletion of ldh-1 causes a metabolic shift from homolactic fermentation to ethanol, formate, and acetoin … production, with a high level of formate production even under aerobic conditions. Ldh-2 plays only a minor role in lactate production.

Authors: , Zhian Saleihan, ,

Date Published: 22nd May 2009

Publication Type: Not specified

Chapter 6. Regulation of antibiotic production by bacterial hormones

Abstract (Expand)

Antibiotic production is regulated by numerous signals, including the so-called bacterial hormones found in antibiotic producing organisms such as Streptomyces. These signals, the gamma-butyrolactones, … are produced in very small quantities, which has hindered their structural elucidation and made it difficult to assess whether they are being produced. In this chapter, we describe a rapid small-scale extraction method from either solid or liquid cultures in scales of one plate or 50 ml of medium. Also described is a bioassay to detect the gamma-butyrolactones by determining either the production of pigmented antibiotic of Streptomyces coelicolor or kanamycin resistant growth on addition of the gamma-butyrolactones. We also describe some insights into the identification of the gamma-butyrolactone receptor and its targets and also the gel retardation conditions with three differently labeled probes.

Authors: Nai-Hua Hsiao, Marco Gottelt,

Date Published: 21st Apr 2009

Publication Type: Not specified

SemanticSBML: a tool for annotating, checking, and merging of biochemical models in SBML format

Abstract (Expand)

Semantic annotations in SBML (systems biology markup language) enable computer programs to check and process biochemical models based on their biochemical meaning. Annotations are an important prerequisite … for model merging, which would be a major step towards the construction of large-scale cell models. The software tool semanticSBML allows users to check and edit MIRIAM annotations and SBO terms, the most common forms of annotation in SBML models. It uses a large collection of biochemical names and database identifiers to support modellers in finding the right annotations. Annotated SBML models can also be built from lists of chemical reactions. In model merging, semanticSBML suggests a preliminary merged model based on MIRIAM annotations in the original models. This model provides a starting point for manually aligning the elements of all input models. To resolve conflicting element properties, conflicts are highlighted and categorised. The user can navigate through the models, change the matching of model elements, check the conflicts between them and decide how they should be resolved. Alternatively, the software can resolve all conflicts automatically, selecting each time the attribute value from the input model with highest priority. URL: http://www.semanticsbml.org/

Authors: Wolfram Liebermeister, , Jannis Uhlendorf, Timo Lubitz,

Date Published: 20th Apr 2009

Publication Type: Not specified

The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis

Abstract (Expand)

Mycobacterium tuberculosis can utilize various nutrients including nitrate as a source of nitrogen. Assimilation of nitrate requires the reduction of nitrate via nitrite to ammonium, which is then … incorporated into metabolic pathways. This study was undertaken to define the molecular mechanism of nitrate assimilation in M. tuberculosis. Homologues to a narGHJI-encoded nitrate reductase and a nirBD-encoded nitrite reductase have been found on the chromosome of M. tuberculosis. Previous studies have implied a role for NarGHJI in nitrate respiration rather than nitrate assimilation. Here, we show that a narG mutant of M. tuberculosis failed to grow on nitrate. A nirB mutant of M. tuberculosis failed to grow on both nitrate and nitrite. Mutant strains of Mycobacterium smegmatis mc(2)155 that are unable to grow on nitrate were isolated. The mutants were rescued by screening a cosmid library from M. tuberculosis, and a gene with homology to the response regulator gene glnR of Streptomyces coelicolor was identified. A DeltaglnR mutant of M. tuberculosis was generated, which also failed to grow on nitrate, but regained its ability to utilize nitrate when nirBD was expressed from a plasmid, suggesting a role of GlnR in regulating nirBD expression. A specific binding site for GlnR within the nirB promoter was identified and confirmed by electrophoretic mobility shift assay using purified recombinant GlnR. Semiquantitative reverse transcription PCR, as well as microarray analysis, demonstrated upregulation of nirBD expression in response to GlnR under nitrogen-limiting conditions. In summary, we conclude that NarGHJI and NirBD of M. tuberculosis mediate the assimilatory reduction of nitrate and nitrite, respectively, and that GlnR acts as a transcriptional activator of nirBD.

Authors: Sven Malm, Yvonne Tiffert, Julia Micklinghoff, Sonja Schultze, Insa Joost, Isabel Weber, Sarah Horst, Birgit Ackermann, , , Stefan Ehlers, Robert Geffers, , Franz-Christoph Bange

Date Published: 1st Apr 2009

Publication Type: Not specified

Phosphate control over nitrogen metabolism in Streptomyces coelicolor: direct and indirect negative control of glnR, glnA, glnII and amtB expression by the response regulator PhoP

Abstract (Expand)

Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate … control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxes composed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB-glnK-glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. 'Information theory' quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussed.

Authors: , Alberto Sola-Landa, Kristian Apel, Fernando Santos-Beneit,

Date Published: 24th Mar 2009

Publication Type: Not specified

The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion

Abstract (Expand)

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O(2), FNR binds a … [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O(2), FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S](2+) cluster. The reaction of the [4Fe-4S](2+) cluster with O(2) has been shown to proceed via a 2-step process, an O(2)-dependent 1-electron oxidation to yield a [3Fe-4S](+) intermediate with release of 1 Fe(2+) ion, followed by spontaneous rearrangement to the [2Fe-2S](2+) form with release of 1 Fe(3+) and 2 S(2-) ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [4Fe-4S](2+) cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [4Fe-4S](2+) cluster in the presence of O(2). Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron-sulfur cluster reaction with O(2) is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of O(2) interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo O(2) concentrations.

Authors: Adrian J Jervis, Jason C Crack, Gaye White, Peter J Artymiuk, Myles R Cheesman, Andrew J Thomson, Nick E Le Brun,

Date Published: 4th Mar 2009

Publication Type: Not specified

Bacterial sensors of oxygen

Abstract (Expand)

The concentration of molecular oxygen (O(2)) began to increase in the Earth's atmosphere approximately two billion years ago. Its presence posed a threat to anaerobes but also offered opportunities … for improved energy conservation via aerobic respiration. The ability to sense environmental O(2) thus became, and remains, important for many bacteria, both for protection and switching between anaerobic and aerobic respiration. Utilizing an iron-sulfur cluster as the sensor of O(2) exploits the ability of O(2) to oxidize the iron-sulfur cluster, ultimately resulting in cluster disassembly. When utilizing heme as the sensor, the capacity of O(2) to form a reversible Fe-O(2) bond or alternatively the oxidation of the heme iron atom itself is used to detect O(2) and switch regulators between active and inactive forms.

Authors: , Jason C Crack, Andrew J Thomson, Nick E LeBrun

Date Published: 24th Feb 2009

Publication Type: Not specified

Physiological proteomics and stress/starvation responses in Bacillus subtilis and Staphylococcus aureus

Abstract (Expand)

Gel-based proteomics is a useful approach for visualizing the responses of bacteria to stress and starvation stimuli. In order to face stress/starvation, bacteria have developed very complicated gene … expression networks. A proteomic view of stress/starvation responses, however, is only a starting point which should promote follow-up studies aimed at the comprehensive description of single regulons, their signal transduction pathways on the one hand, and their adaptive functions on the other, and finally their integration into complex gene expression networks. This "road map of physiological proteomics" will be demonstrated for the general stress regulon controlled by sigma(B) in Bacillus subtilis and the oxygen starvation response with Rex as a master regulator in Staphylococcus aureus.

Authors: , Alexander Reder, Stephan Fuchs, Martin Pagels, Susanne Engelmann

Date Published: 20th Feb 2009

Publication Type: Not specified

Mathematical modelling of the agr operon in Staphylococcus aureus

Abstract (Expand)

Staphylococcus aureus is a pathogenic bacterium that utilises quorum sensing (QS), a cell-to-cell signalling mechanism, to enhance its ability to cause disease. QS allows the bacteria to monitor their … surroundings and the size of their population, and S. aureus makes use of this to regulate the production of virulence factors. Here we describe a mathematical model of this QS system and perform a detailed time-dependent asymptotic analysis in order to clarify the roles of the distinct interactions that make up the QS process, demonstrating which reactions dominate the behaviour of the system at various timepoints. We couple this analysis with numerical simulations and are thus able to gain insight into how a large population of S. aureus shifts from a relatively harmless state to a highly virulent one, focussing on the need for the three distinct phases which form the feedback loop of this particular QS system.

Authors: , , Adrian J Koerber, Paul Williams

Date Published: 19th Feb 2009

Publication Type: Not specified

Cross-talk between two global regulators in Streptomyces: PhoP and AfsR interact in the control of afsS, pstS and phoRP transcription

Abstract (Expand)

The regulatory proteins AfsR and PhoP control expression of the biosynthesis of actinorhodin and undecylprodigiosin in Streptomyces coelicolor. Electrophoretic mobility shift assays showed that PhoP(DBD) … does not bind directly to the actII-ORF4, redD and atrA promoters, but it binds to the afsS promoter, in a region overlapping with the AfsR operator. DNase I footprinting studies revealed a PhoP protected region of 26 nt (PHO box; two direct repeats of 11 nt) that overlaps with the AfsR binding sequence. Binding experiments indicated a competition between AfsR and PhoP; increasing concentrations of PhoP(DBD) resulted in the disappearance of the AfsR-DNA complex. Expression studies using the reporter luxAB gene coupled to afsS promoter showed that PhoP downregulates afsS expression probably by a competition with the AfsR activator. Interestingly, AfsR binds to other PhoP-regulated promoters including those of pstS (a component of the phosphate transport system) and phoRP (encoding the two component system itself). Analysis of the AfsR-protected sequences in each of these promoters allowed us to distinguish the AfsR binding sequence from the overlapping PHO box. The reciprocal regulation of the phoRP promoter by AfsR and of afsS by PhoP suggests a fine interplay of these regulators on the control of secondary metabolism.

Authors: Fernando Santos-Beneit, , Alberto Sola-Landa,

Date Published: 11th Feb 2009

Publication Type: Not specified

A rapid microwave-assisted derivatization of bacterial metabolome samples for gas chromatography/mass spectrometry analysis

Abstract (Expand)

Analysis of metabolome samples by gas chromatography/mass spectrometry requires a comprehensive derivatization method to afford quantitative and qualitative information of a complex biological sample. … Here we describe an extremely time-effective microwave-assisted protocol for the commonly used methoxyamine and N-methyl-N-trimethylsilylfluoracetamide silylation method of primary metabolites. Our studies show that microwave irradiation can decrease the sample preparation time from approximately 120 min to 6 min without loss of either qualitative or quantitative information for the tested synthetic metabolite mixtures and microbial-derived metabolome samples collected from Bacillus subtilis and Staphylococcus aureus. Comparisons of metabolic fingerprints and selected metabolites show no noticeable differences compared with the commonly used heating block methods.

Authors: Manuel Liebeke, Ariane Wunder,

Date Published: 4th Feb 2009

Publication Type: Not specified

Novel activities of glycolytic enzymes in Bacillus subtilis: interactions with essential proteins involved in mRNA processing

Abstract (Expand)

Glycolysis is one of the most important metabolic pathways in heterotrophic organisms. Several genes encoding glycolytic enzymes are essential in many bacteria even under conditions when neither … glycolytic nor gluconeogenic activities are required. In this study, a screening for in vivo interaction partners of glycolytic enzymes of the soil bacterium Bacillus subtilis was used to provide a rationale for essentiality of glycolytic enzymes. Glycolytic enzymes proved to be in close contact with several other proteins, among them a high proportion of essential proteins. Among these essential interaction partners, other glycolytic enzymes were most prominent. Two-hybrid studies confirmed interactions of phosphofructokinase with phosphoglyceromutase and enolase. Such a complex of glycolytic enzymes might allow direct substrate channeling of glycolytic intermediates. Moreover we found associations of glycolytic enzymes with several proteins known or suspected to be involved in RNA processing and degradation. One of these proteins, Rny (YmdA), which has so far not been functionally characterized, is required for the processing of the mRNA of the glycolytic gapA operon. Two-hybrid analyses confirmed the interactions between the glycolytic enzymes phosphofructokinase and enolase and the enzymes involved in RNA processing, RNase J1, Rny, and polynucleotide phosphorylase. Moreover RNase J1 interacts with its homologue RNase J2. We suggest that this complex of mRNA processing and glycolytic enzymes is the B. subtilis equivalent of the RNA degradosome. Our findings suggest that the functional interaction of glycolytic enzymes with essential proteins may be the reason why they are indispensable.

Authors: Fabian M Commichau, Fabian M Rothe, Christina Herzberg, Eva Wagner, Daniel Hellwig, Martin Lehnik-Habrink, Elke Hammer, ,

Date Published: 3rd Feb 2009

Publication Type: Not specified

Overflow of a hyper-produced secretory protein from the Bacillus Sec pathway into the Tat pathway for protein secretion as revealed by proteogenomics

Abstract (Expand)

Bacteria secrete numerous proteins into their environment for growth and survival under complex and ever-changing conditions. The highly different characteristics of secreted proteins pose major … challenges to the cellular protein export machinery and, accordingly, different pathways have evolved. While the main secretion (Sec) pathway transports proteins in an unfolded state, the twin-arginine translocation (Tat) pathway transports folded proteins. To date, these pathways were believed to act in strictly independent ways. Here, we have employed proteogenomics to investigate the secretion mechanism of the esterase LipA of Bacillus subtilis, using a serendipitously obtained hyper-producing strain. While LipA is secreted Sec-dependently under standard conditions, hyper-produced LipA is secreted predominantly Tat-dependently via an unprecedented overflow mechanism. Two previously identified B. subtilis Tat substrates, PhoD and YwbN, require each a distinct Tat translocase for secretion. In contrast, hyper-produced LipA is transported by both Tat translocases of B. subtilis, showing that they have distinct but overlapping specificities. The identified overflow secretion mechanism for LipA focuses interest on the possibility that secretion pathway choice can be determined by environmental and intracellular conditions. This may provide an explanation for the previous observation that many Sec-dependently transported proteins have potential twin-arginine signal peptides for export via the Tat pathway.

Authors: Thijs R H M Kouwen, René van der Ploeg, Haike Antelmann, , Georg Homuth, Ulrike Mäder,

Date Published: 31st Jan 2009

Publication Type: Not specified

MscL of Bacillus subtilis prevents selective release of cytoplasmic proteins in a hypotonic environment

Abstract (Expand)

Bacillus subtilis serves as an excellent model to study protein secretion at a proteomic scale. Most of the extracellular proteins are exported from the cytoplasm via the secretory (Sec) pathway. Despite … extensive studies, the secretion mechanisms of about 25% of the extracellular proteins are unknown. This suggests that B. subtilis makes use of alternative mechanisms to release proteins into its environment. In search for novel pathways, which contribute to biogenesis of the B. subtilis exoproteome, we investigated a possible role of the large conductance mechanosensitive channel protein MscL. We compared protein secretion by MscL deficient and proficient B. subtilis cells. MscL did not contribute to secretion under standard growth conditions. Unexpectedly, we discovered that under hypo-osmotic shock conditions specific, normally cytoplasmic proteins were released by mscL mutant cells. This protein release was selective since not all cytoplasmic proteins were equally well released. We established that this protein release by mscL mutant cells cannot be attributed to cell death or lysis. The presence of MscL, therefore, seems to prevent the specific release of cytoplasmic proteins by B. subtilis during hypo-osmotic shock. Our unprecedented findings imply that an unidentified system for selective release of cytoplasmic proteins is active in B. subtilis.

Authors: Thijs R H M Kouwen, Haike Antelmann, René van der Ploeg, Emma L Denham, ,

Date Published: 23rd Jan 2009

Publication Type: Not specified

Genome-wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol-dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR-family regulator YraB (AdhR)

Abstract (Expand)

Quinones and alpha,beta-unsaturated carbonyls are naturally occurring electrophiles that target cysteine residues via thiol-(S)-alkylation. We analysed the global expression profile of Bacillus subtilis … to the toxic carbonyls methylglyoxal (MG) and formaldehyde (FA). Both carbonyl compounds cause a stress response characteristic for thiol-reactive electrophiles as revealed by the induction of the Spx, CtsR, CymR, PerR, ArsR, CzrA, CsoR and SigmaD regulons. MG and FA triggered also a SOS response which indicates DNA damage. Protection against FA is mediated by both the hxlAB operon, encoding the ribulose monophosphate pathway for FA fixation, and a thiol-dependent formaldehyde dehydrogenase (AdhA) and DJ-1/PfpI-family cysteine proteinase (YraA). The adhA-yraA operon and the yraC gene, encoding a gamma-carboxymuconolactone decarboxylase, are positively regulated by the MerR-family regulator, YraB(AdhR). AdhR binds specifically to its target promoters which contain a 7-4-7 inverted repeat (CTTAAAG-N4-CTTTAAG) between the -35 and -10 elements. Activation of adhA-yraA transcription by AdhR requires the conserved Cys52 residue in vivo. We speculate that AdhR is redox-regulated via thiol-(S)-alkylation by aldehydes and that AdhA and YraA are specifically involved in reduction of aldehydes and degradation or repair of damaged thiol-containing proteins respectively.

Authors: Thi Thu Huyen Nguyen, Warawan Eiamphungporn, Ulrike Mäder, Manuel Liebeke, , , John D Helmann, Haike Antelmann

Date Published: 23rd Dec 2008

Publication Type: Not specified

Probabilistic assignment of formulas to mass peaks in metabolomics experiments

Abstract (Expand)

MOTIVATION: High-accuracy mass spectrometry is a popular technology for high-throughput measurements of cellular metabolites (metabolomics). One of the major challenges is the correct identification … of the observed mass peaks, including the assignment of their empirical formula, based on the measured mass. RESULTS: We propose a novel probabilistic method for the assignment of empirical formulas to mass peaks in high-throughput metabolomics mass spectrometry measurements. The method incorporates information about possible biochemical transformations between the empirical formulas to assign higher probability to formulas that could be created from other metabolites in the sample. In a series of experiments, we show that the method performs well and provides greater insight than assignments based on mass alone. In addition, we extend the model to incorporate isotope information to achieve even more reliable formula identification. AVAILABILITY: A supplementary document, Matlab code, data and further information are available from http://www.dcs.gla.ac.uk/inference/metsamp.

Authors: Simon Rogers, Richard A Scheltema, Mark Girolami,

Date Published: 18th Dec 2008

Publication Type: Not specified

Prevalence of sulfonamide resistance genes in bacterial isolates from manured agricultural soils and pig slurry in the United Kingdom

Abstract (Expand)

The prevalences of three sulfonamide resistance genes, sul1, sul2, and sul3 and sulfachloropyridazine (SCP) resistance were determined in bacteria isolated from manured agricultural clay soils and … slurry samples in the United Kingdom over a 2-year period. Slurry from tylosin-fed pigs amended with SCP and oxytetracycline was used for manuring. Isolates positive for sul genes were further screened for the presence of class 1 and 2 integrons. Phenotypic resistance to SCP was significantly higher in isolates from pig slurry and postapplication soil than in those from preapplication soil. Of 531 isolates, 23% carried sul1, 18% sul2, and 9% sul3 only. Two percent of isolates contained all three sul genes. Class 1 and class 2 integrons were identified in 5% and 11.7%, respectively, of sul-positive isolates. In previous reports, sul1 was linked to class 1 integrons, but in this study only 8% of sul1-positive isolates carried the intI1 gene. Sulfonamide-resistant pathogens, including Shigella flexneri, Aerococcus spp., and Acinetobacter baumannii, were identified in slurry-amended soil and soil leachate, suggesting a potential environmental reservoir. Sulfonamide resistance in Psychrobacter, Enterococcus, and Bacillus spp. is reported for the first time, and this study also provides the first description of the genotypes sul1, sul2, and sul3 outside the Enterobacteriaceae and in the soil environment.

Authors: K G Byrne-Bailey, , P Kay, A B A Boxall, P M Hawkey,

Date Published: 8th Dec 2008

Publication Type: Not specified

Immunity to the bacteriocin sublancin 168 Is determined by the SunI (YolF) protein of Bacillus subtilis

Abstract (Expand)

Bacillus subtilis strain 168 produces the extremely stable lantibiotic sublancin 168, which has a broad spectrum of bactericidal activity. Both sublancin 168 production and producer immunity are … determined by the SPbeta prophage. While the sunA and sunT genes for sublancin 168 production have been known for several years, the genetic basis for sublancin 168 producer immunity has remained elusive. Therefore, the present studies were aimed at identifying an SPbeta gene(s) for sublancin 168 immunity. By systematic deletion analysis, we were able to pinpoint one gene, named yolF, as the sublancin 168 producer immunity gene. Growth inhibition assays performed using plates and liquid cultures revealed that YolF is both required and sufficient for sublancin 168 immunity even when heterologously produced in the sublancin-sensitive bacterium Staphylococcus aureus. Accordingly, we propose to rename yolF to sunI (for sublancin immunity). Subcellular localization studies indicate that the SunI protein is anchored to the membrane with a single N-terminal membrane-spanning domain that has an N(out)-C(in) topology. Thus, the bulk of the protein faces the cytoplasm of B. subtilis. This topology has not yet been reported for known bacteriocin producer immunity proteins, which implies that SunI belongs to a novel class of bacteriocin antagonists.

Authors: Jean-Yves F Dubois, Thijs R H M Kouwen, Anna K C Schurich, Carlos R Reis, Hendrik T Ensing, Erik N Trip, Jessica C Zweers,

Date Published: 1st Dec 2008

Publication Type: Not specified

An approach to pathway reconstruction using whole genome metabolic models and sensitive sequence searching

Abstract (Expand)

Metabolic models have the potential to impact on genome annotation and on the interpretation of gene expression and other high throughput genome data. The genome of Streptomyces coelicolor genome has … been sequenced and some 30% of the open reading frames (ORFs) lack any functional annotation. A recently constructed metabolic network model for S. coelicolor highlights biochemical functions which should exist to make the metabolic model complete and consistent. These include 205 reactions for which no ORF is associated. Here we combine protein functional predictions for the unannotated open reading frames in the genome with \'missing but expected\' functions inferred from the metabolic model. The approach allows function predictions to be evaluated in the context of the biochemical pathway reconstruction, and feed back iteratively into the metabolic model. We describe the approach and discuss a few illustrative examples.

Authors: Mansoor Saqi, Richard J B Dobson, Preben Kraben, ,

Date Published: 13th Nov 2008

Publication Type: Not specified

Gel-based proteomics of Gram-positive bacteria: a powerful tool to address physiological questions

Abstract (Expand)

In this review, we demonstrate the power of gel-based proteomics to address physiological questions of bacteria. Although gel-based proteomics covers a subpopulation of proteins only, fundamental issues … of a bacterial cell such as almost all metabolic pathways or the main signatures of stress and starvation responses can be analyzed. The analysis of the synthesis pattern of single proteins, e.g., in response to environmental changes, requires gel-based proteomics because only this technique can compare protein synthesis and amount in the same 2-D gel. Moreover, highly sophisticated software packages facilitate the analysis of the regulation of the main metabolic enzymes or the stress/starvation responses, PTMs, protein damage/repair, and degradation and finally protein secretion mechanisms at a proteome-wide scale. The challenge of proteomics whose panorama view shows events never seen before is to select the most interesting issues for detailed follow up studies. This "road map of proteomics" from proteome data via new hypothesis and finally novel molecular mechanisms should lead to exciting information on bacterial physiology. However, many proteins escape detection by gel-based procedures, such as membrane or low abundance proteins. The smart combination of gel-free and gel-based approaches is the "state of the art" for physiological proteomics of bacteria.

Authors: , Haike Antelmann, Knut Büttner, Jörg Bernhardt

Date Published: 13th Nov 2008

Publication Type: Not specified

MINOMICS: visualizing prokaryote transcriptomics and proteomics data in a genomic context

Abstract (Expand)

We have developed MINOMICS, a tool that allows facile and in-depth visualization of prokaryotic transcriptomic and proteomic data in conjunction with genomics data. MINOMICS generates interactive linear … genome maps in which multiple experimental datasets are displayed together with operon, regulatory motif, transcriptional promoter and transcriptional terminator information. AVAILABILITY: MINOMICS is freely accessible at http://www.minomics.nl

Authors: Rutger W W Brouwer, Sacha A F T van Hijum,

Date Published: 12th Nov 2008

Publication Type: Not specified

Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis

Abstract (Expand)

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in … bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.

Authors: Thijs R H M Kouwen, Jean-Yves F Dubois, Roland Freudl, Wim J Quax,

Date Published: 24th Oct 2008

Publication Type: Not specified

Increasing the mass accuracy of high-resolution LC-MS data using background ions: a case study on the LTQ-Orbitrap

Abstract (Expand)

With the advent of a new generation of high-resolution mass spectrometers, the fields of proteomics and metabolomics have gained powerful new tools. In this paper, we demonstrate a novel computational … method that improves the mass accuracy of the LTQ-Orbitrap mass spectrometer from an initial +/- 1-2 ppm, obtained by the standard software, to an absolute median of 0.21 ppm (SD 0.21 ppm). With the increased mass accuracy it becomes much easier to match mass chromatograms in replicates and different sample types, even if compounds are detected at very low intensities. The proposed method exploits the ubiquitous presence of background ions in LC-MS profiles for accurate alignment and internal mass calibration, making it applicable for all types of MS equipment. The accuracy of this approach will facilitate many downstream systems biology applications, including mass-based molecule identification, ab initio metabolic network reconstruction, and untargeted metabolomics in general.

Authors: Richard A Scheltema, Anas Kamleh, David Wildridge, Charles Ebikeme, David G Watson, Michael P Barrett, ,

Date Published: 22nd Oct 2008

Publication Type: Not specified

Localization of general and regulatory proteolysis in Bacillus subtilis cells

Abstract (Expand)

Protein degradation mediated by ATP-dependent proteases, such as Hsp100/Clp and related AAA+ proteins, plays an important role in cellular protein homeostasis, protein quality control and the regulation … of, e.g. heat shock adaptation and other cellular differentiation processes. ClpCP with its adaptor proteins and other related proteases, such as ClpXP or ClpEP of Bacillus subtilis, are involved in general and regulatory proteolysis. To determine if proteolysis occurs at specific locations in B. subtilis cells, we analysed the subcellular distribution of the Clp system together with adaptor and general and regulatory substrate proteins, under different environmental conditions. We can demonstrate that the ATPase and the proteolytic subunit of the Clp proteases, as well as the adaptor or substrate proteins, form visible foci, representing active protease clusters localized to the polar and to the mid-cell region. These clusters could represent a compartmentalized place for protein degradation positioned at the pole close to where most of the cellular protein biosynthesis and also protein quality control are taking place, thereby spatially separating protein synthesis and degradation.

Authors: Janine Kirstein, Henrik Strahl, Noël Molière, , Kürşad Turgay

Date Published: 10th Sep 2008

Publication Type: Not specified

Carbon catabolite repression in Bacillus subtilis: quantitative analysis of repression exerted by different carbon sources

Abstract (Expand)

In many bacteria glucose is the preferred carbon source and represses the utilization of secondary substrates. In Bacillus subtilis, this carbon catabolite repression (CCR) is achieved by the global … transcription regulator CcpA, whose activity is triggered by the availability of its phosphorylated cofactors, HPr(Ser46-P) and Crh(Ser46-P). Phosphorylation of these proteins is catalyzed by the metabolite-controlled kinase HPrK/P. Recent studies have focused on glucose as a repressing substrate. Here, we show that many carbohydrates cause CCR. The substrates form a hierarchy in their ability to exert repression via the CcpA-mediated CCR pathway. Of the two cofactors, HPr is sufficient for complete CCR. In contrast, Crh cannot substitute for HPr on substrates that cause a strong repression. Determination of the phosphorylation state of HPr in vivo revealed a correlation between the strength of repression and the degree of phosphorylation of HPr at Ser46. Sugars transported by the phosphotransferase system (PTS) cause the strongest repression. However, the phosphorylation state of HPr at its His15 residue and PTS transport activity have no impact on the global CCR mechanism, which is a major difference compared to the mechanism operative in Escherichia coli. Our data suggest that the hierarchy in CCR exerted by the different substrates is exclusively determined by the activity of HPrK/P.

Authors: Kalpana D Singh, Matthias H Schmalisch, , Boris Görke

Date Published: 29th Aug 2008

Publication Type: Not specified

Phosphate-dependent regulation of the low- and high-affinity transport systems in the model actinomycete Streptomyces coelicolor

Abstract (Expand)

The transport of inorganic phosphate (P(i)) is essential for the growth of all organisms. The metabolism of soil-dwelling Streptomyces species, and their ability to produce antibiotics and other secondary … metabolites, are strongly influenced by the availability of phosphate. The transcriptional regulation of the SCO4138 and SCO1845 genes of Streptomyces coelicolor was studied. These genes encode the two putative low-affinity P(i) transporters PitH1 and PitH2, respectively. Expression of these genes and that of the high-affinity transport system pstSCAB follows a sequential pattern in response to phosphate deprivation, as shown by coupling their promoters to a luciferase reporter gene. Expression of pitH2, but not that of pap-pitH1 (a bicistronic transcript), is dependent upon the response regulator PhoP. PhoP binds to specific sequences consisting of direct repeats of 11 nt in the promoter of pitH2, but does not bind to the pap-pitH1 promoter, which lacks these direct repeats for PhoP recognition. The transcription start point of the pitH2 promoter was identified by primer extension analyses, and the structure of the regulatory sequences in the PhoP-protected DNA region was established. It consists of four central direct repeats flanked by two other less conserved repeats. A model for PhoP regulation of this promoter is proposed based on the four promoter DNA-PhoP complexes detected by electrophoretic mobility shift assays and footprinting studies.

Authors: Fernando Santos-Beneit, , Etelvina Franco-Domínguez,

Date Published: 1st Aug 2008

Publication Type: Not specified

Depletion of thiol-containing proteins in response to quinones in Bacillus subtilis

Abstract (Expand)

SUMMARY: Quinones are highly toxic naturally occurring thiol-reactive compounds. We have previously described novel pathways for quinone detoxification in the Gram-positive bacterium Bacillus subtilis. … In this study, we have investigated the extent of irreversible and reversible thiol modifications caused in vivo by electrophilic quinones. Exposure to toxic benzoquinone (BQ) concentrations leads to depletion of numerous Cys-rich cytoplasmic proteins in the proteome of B. subtilis. Mass spectrometry and immunoblot analyses demonstrated that these BQ-depleted proteins represent irreversibly damaged BQ aggregates that escape the two-dimensional gel separation. This enabled us to quantify the depletion of thiol-containing proteins which are the in vivo targets for thiol-(S)-alkylation by toxic quinone compounds. Metabolomic approaches confirmed that protein depletion is accompanied by depletion of the low-molecular-weight (LMW) thiol cysteine. Finally, no increased formation of disulphide bonds was detected in the thiol-redox proteome in response to sublethal quinone concentrations. The glyceraldehyde-3-phosphate dehydrogenase (GapA) was identified as the only new target for reversible thiol modifications after exposure to toxic quinones. Together our data show that the thiol-(S)-alkylation reaction with protein and non-protein thiols is the in vivo mechanism for thiol depletion and quinone toxicity in B. subtilis and most likely also in other bacteria.

Authors: Manuel Liebeke, Dierk-Christoph Pöther, Nguyen van Duy, Dirk Albrecht, Dörte Becher, Falko Hochgräfe, , , Haike Antelmann

Date Published: 30th Jul 2008

Publication Type: Not specified

Carbon catabolite repression in bacteria: many ways to make the most out of nutrients

Abstract (Expand)

Most bacteria can selectively use substrates from a mixture of different carbon sources. The presence of preferred carbon sources prevents the expression, and often also the activity, of catabolic … systems that enable the use of secondary substrates. This regulation, called carbon catabolite repression (CCR), can be achieved by different regulatory mechanisms, including transcription activation and repression and control of translation by an RNA-binding protein, in different bacteria. Moreover, CCR regulates the expression of virulence factors in many pathogenic bacteria. In this Review, we discuss the most recent findings on the different mechanisms that have evolved to allow bacteria to use carbon sources in a hierarchical manner.

Authors: Boris Görke,

Date Published: 17th Jul 2008

Publication Type: Not specified

The Spx paralogue MgsR (YqgZ) controls a subregulon within the general stress response of Bacillus subtilis

Abstract (Expand)

The alternative sigma factor sigma(B) of Bacillus subtilis is responsible for the induction of the large general stress regulon comprising approximately 150-200 genes. YqgZ, a member of the sigma(B) … regulon, resembles the global regulator Spx of the diamide stress regulon in B. subtilis. In this work we conducted a comprehensive transcriptome and proteome analysis of the B. subtilis wild-type 168 and its isogenic DeltasigB and DeltayqgZ mutants following exposure to 4% (v/v) ethanol stress, which led to the characterization of a 'subregulon' within the general stress response that is regulated by YqgZ. Activation and induction of sigma(B) are necessary but not sufficient for a full expression of all general stress genes. Expression of 53 genes was found to be positively regulated and the expression of 18 genes was negatively affected by YqgZ. The identification of the negatively regulated group represents a so far uncharacterized regulatory phenomenon observed in the DeltasigB mutant background that can now be attributed to the function of YqgZ. Due to the strict sigma(B)-dependent expression of YqgZ it was renamed to MgsR (modulator of the general stress response).

Authors: Alexander Reder, Dirk Höper, Christin Weinberg, Ulf Gerth, Martin Fraunholz,

Date Published: 14th Jul 2008

Publication Type: Not specified

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Abstract (Expand)

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for … use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.

Authors: Lidia Westers, Helga Westers, Geeske Zanen, Haike Antelmann, , David Noone, Kevin M Devine, , Wim J Quax

Date Published: 12th Jun 2008

Publication Type: Not specified

Interchangeable modules in bacterial thiol-disulfide exchange pathways

Abstract (Expand)

Thiol-disulfide oxidoreductases (TDORs) catalyze thiol-disulfide exchange reactions that are crucial for protein activity and stability. Specifically, they can function as thiol oxidases, disulfide … reductases or disulfide isomerases. The generally established view is that particular TDORs act unidirectionally within a fixed cascade of specific, sequentially arranged reactions. However, recent studies on both Gram-negative and Gram-positive bacteria imply that this view needs to be expanded, at least for thiol-disulfide exchanges in proteins that are exported from the cytoplasm. Here, we present our opinion that various TDORs can function as interchangeable modules in different thiol-disulfide exchange pathways. Such TDOR modules, thus, fulfil important functions in generating the diversity in activity and specificity that is needed in productive extracytoplasmic thiol-disulfide exchange.

Authors: Thijs R H M Kouwen,

Date Published: 30th May 2008

Publication Type: Not specified

Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques

Abstract (Expand)

Bacillus subtilis has been developed as a model system for physiological proteomics. However, thus far these studies have mainly been limited to cytoplasmic, extracellular, and cell-wall attached … proteins. Although being certainly important for cell physiology, the membrane protein fraction has not been studied in comparable depth due to inaccessibility by traditional 2-DE-based workflows and limitations in reliable quantification. In this study, we now compare the potential of stable isotope labeling with amino acids (SILAC) and (14)N/(15)N-labeling for the analysis of bacterial membrane fractions in physiology-driven proteomic studies. Using adaptation of B. subtilis to amino acid (lysine) and glucose starvation as proof of principle scenarios, we show that both approaches provide similarly valuable data for the quantification of bacterial membrane proteins. Even if labeling with stable amino acids allows a more straightforward analysis of data, the (14)N/(15)N-labeling has some advantages in general such as labeling of all amino acids and thereby increasing the number of peptides for quantification. Both, SILAC as well as (14)N/(15)N-labeling are compatible with 2-DE, 2-D LC-MS/MS, and GeLC-MS/MS and thus will allow comprehensive simultaneous interrogation of cytoplasmic and enriched membrane proteomes.

Authors: Annette Dreisbach, Andreas Otto, Dörte Becher, Elke Hammer, Alexander Teumer, Joost W Gouw, ,

Date Published: 21st May 2008

Publication Type: Not specified

A bistable gene switch for antibiotic biosynthesis: the butyrolactone regulon in Streptomyces coelicolor

Abstract (Expand)

Many microorganisms, including bacteria of the class Streptomycetes, produce various secondary metabolites including antibiotics to gain a competitive advantage in their natural habitat. The production … of these compounds is highly coordinated in a population to expedite accumulation to an effective concentration. Furthermore, as antibiotics are often toxic even to their producers, a coordinated production allows microbes to first arm themselves with a defense mechanism to resist their own antibiotics before production commences. One possible mechanism of coordination among individuals is through the production of signaling molecules. The gamma-butyrolactone system in Streptomyces coelicolor is a model of such a signaling system for secondary metabolite production. The accumulation of these signaling molecules triggers antibiotic production in the population. A pair of repressor-amplifier proteins encoded by scbA and scbR mediates the production and action of one particular gamma-butyrolactone, SCB1. Based on the proposed interactions of scbA and scbR, a mathematical model was constructed and used to explore the ability of this system to act as a robust genetic switch. Stability analysis shows that the butyrolactone system exhibits bistability and, in response to a threshold SCB1 concentration, can switch from an OFF state to an ON state corresponding to the activation of genes in the cryptic type I polyketide synthase gene cluster, which are responsible for production of the hypothetical polyketide. The switching time is inversely related to the inducer concentration above the threshold, such that short pulses of low inducer concentration cannot switch on the system, suggesting its possible role in noise filtering. In contrast, secondary metabolite production can be triggered rapidly in a population of cells producing the butyrolactone signal due to the presence of an amplification loop in the system. S. coelicolor was perturbed experimentally by varying concentrations of SCB1, and the model simulations match the experimental data well. Deciphering the complexity of this butyrolactone switch will provide valuable insights into how robust and efficient systems can be designed using "simple" two-protein networks.

Authors: Sarika Mehra, Salim Charaniya, , Wei-Shou Hu

Date Published: 2nd May 2008

Publication Type: Not specified

Ein Beitrag zu einer Theorie lebender Zellen (A Contribution towards a Theory of Living Cells)

Abstract (Expand)

The following article describes systems biology as a merger of systems theory with cell biology. The role of modelling in the description of living cells is discussed. As an example, an abstract … multiple-level model of a cell is developed. It is shown that a level of elementary cellular processes, realising cell functions, and a coordination-level are sufficient to create a system that is closed with respect to efficient causation. This form of self-organisation is thereby considered as basic criterion by which living systems, such as cells and organisms, are distinguished from machines and computers. Die causal closure of the cell is possible through the definition of the cell model as a cartesian closed category. It follows the conclusion that computer simulations of differential equations may be able to reproduce cellular processes but not this aspect of causal closure. The article ends with a discussion about the role of systems theory in the life sciences.

Authors: , Jan-Hendrik S. Hofmeyr

Date Published: 1st May 2008

Publication Type: Not specified

Antibiotic resistance in the environment, with particular reference to MRSA

Abstract

Not specified

Authors: , Colette O'Neill, , Peter Hawkey

Date Published: 9th Apr 2008

Publication Type: Not specified

Genome-scale reconstruction and analysis of the Pseudomonas putida KT2440 metabolic network facilitates applications in biotechnology

Abstract (Expand)

A cornerstone of biotechnology is the use of microorganisms for the efficient production of chemicals and the elimination of harmful waste. Pseudomonas putida is an archetype of such microbes due to … its metabolic versatility, stress resistance, amenability to genetic modifications, and vast potential for environmental and industrial applications. To address both the elucidation of the metabolic wiring in P. putida and its uses in biocatalysis, in particular for the production of non-growth-related biochemicals, we developed and present here a genome-scale constraint-based model of the metabolism of P. putida KT2440. Network reconstruction and flux balance analysis (FBA) enabled definition of the structure of the metabolic network, identification of knowledge gaps, and pin-pointing of essential metabolic functions, facilitating thereby the refinement of gene annotations. FBA and flux variability analysis were used to analyze the properties, potential, and limits of the model. These analyses allowed identification, under various conditions, of key features of metabolism such as growth yield, resource distribution, network robustness, and gene essentiality. The model was validated with data from continuous cell cultures, high-throughput phenotyping data, (13)C-measurement of internal flux distributions, and specifically generated knock-out mutants. Auxotrophy was correctly predicted in 75% of the cases. These systematic analyses revealed that the metabolic network structure is the main factor determining the accuracy of predictions, whereas biomass composition has negligible influence. Finally, we drew on the model to devise metabolic engineering strategies to improve production of polyhydroxyalkanoates, a class of biotechnologically useful compounds whose synthesis is not coupled to cell survival. The solidly validated model yields valuable insights into genotype-phenotype relationships and provides a sound framework to explore this versatile bacterium and to capitalize on its vast biotechnological potential.

Authors: Jacek Puchałka, Matthew A Oberhardt, Miguel Godinho, Agata Bielecka, Daniela Regenhardt, , Jason A Papin,

Date Published: 27th Mar 2008

Publication Type: Not specified

Glutamate metabolism in Bacillus subtilis: gene expression and enzyme activities evolved to avoid futile cycles and to allow rapid responses to perturbations of the system

Abstract (Expand)

Glutamate is a central metabolite in all organisms since it provides the link between carbon and nitrogen metabolism. In Bacillus subtilis, glutamate is synthesized exclusively by the glutamate synthase, … and it can be degraded by the glutamate dehydrogenase. In B. subtilis, the major glutamate dehydrogenase RocG is expressed only in the presence of arginine, and the bacteria are unable to utilize glutamate as the only carbon source. In addition to rocG, a second cryptic gene (gudB) encodes an inactive glutamate dehydrogenase. Mutations in rocG result in the rapid accumulation of gudB1 suppressor mutations that code for an active enzyme. In this work, we analyzed the physiological significance of this constellation of genes and enzymes involved in glutamate metabolism. We found that the weak expression of rocG in the absence of the inducer arginine is limiting for glutamate utilization. Moreover, we addressed the potential ability of the active glutamate dehydrogenases of B. subtilis to synthesize glutamate. Both RocG and GudB1 were unable to catalyze the anabolic reaction, most probably because of their very high K(m) values for ammonium. In contrast, the Escherichia coli glutamate dehydrogenase is able to produce glutamate even in the background of a B. subtilis cell. B. subtilis responds to any mutation that interferes with glutamate metabolism with the rapid accumulation of extragenic or intragenic suppressor mutations, bringing the glutamate supply into balance. Similarly, with the presence of a cryptic gene, the system can flexibly respond to changes in the external glutamate supply by the selection of mutations.

Authors: Fabian M Commichau, Katrin Gunka, Jens J Landmann,

Date Published: 7th Mar 2008

Publication Type: Not specified

The Streptomyces coelicolor GlnR regulon: identification of new GlnR targets and evidence for a central role of GlnR in nitrogen metabolism in actinomycetes

Abstract (Expand)

Streptomyces coelicolor GlnR is a global regulator that controls genes involved in nitrogen metabolism. By genomic screening 10 new GlnR targets were identified, including enzymes for ammonium assimilation … (glnII, gdhA), nitrite reduction (nirB), urea cleavage (ureA) and a number of biochemically uncharacterized proteins (SCO0255, SCO0888, SCO2195, SCO2400, SCO2404, SCO7155). For the GlnR regulon, a GlnR binding site which comprises the sequence gTnAc-n(6)-GaAAc-n(6)-GtnAC-n(6)-GAAAc-n(6) has been found. Reverse transcription analysis of S. coelicolor and the S. coelicolor glnR mutant revealed that GlnR activates or represses the expression of its target genes. Furthermore, glnR expression itself was shown to be nitrogen-dependent. Physiological studies of S. coelicolor and the S. coelicolor glnR mutant with ammonium and nitrate as the sole nitrogen source revealed that GlnR is not only involved in ammonium assimilation but also in ammonium supply. blast analysis demonstrated that GlnR-homologous proteins are present in different actinomycetes containing the glnA gene with the conserved GlnR binding site. By DNA binding studies, it was furthermore demonstrated that S. coelicolor GlnR is able to interact with these glnA upstream regions. We therefore suggest that GlnR-mediated regulation is not restricted to Streptomyces but constitutes a regulon conserved in many actinomycetes.

Authors: Yvonne Tiffert, Petra Supra, Reinhild Wurm, , Rolf Wagner,

Date Published: 7th Jan 2008

Publication Type: Not specified

Dynamics of receptor and protein transducer homodimerisation

Abstract (Expand)

Background Signalling pathways are complex systems in which not only simple monomeric molecules interact, but also more complex structures that include constitutive or induced protein assemblies. In … particular, the hetero-and homo-dimerisation of proteins is a commonly encountered motif in signalling pathways. Several authors have suggested in recent times that dimerisation relates to a series of physical and biological outcomes used by the cell in the regulation of signal transduction. Results In this paper we investigate the role of homodimerisation in receptor-protein transducer interactions. Towards this end, mathematical modelling is used to analyse the features of such kind of interactions and to predict the behaviour of the system under different experimental conditions. A kinetic model in which the interaction between homodimers provokes a dual mechanism of activation (single and double protein transducer activation at the same time) is proposed. In addition, we analyse under which conditions the use of a power-law representation for the system is useful. Furthermore, we investigate the dynamical consequences of this dual mechanism and compare the performance of the system in different simulated experimental conditions. Conclusion The analysis of our mathematical model suggests that in receptor-protein interacting systems with dual mechanism there may be a shift between double and single activation in a way that intense double protein transducer activation could initiate and dominate the signal in the short term (getting a fast intense signal), while single protein activation could control the system in the medium and long term (when input signal is weaker and decreases slowly). Our investigation suggests that homodimerisation and oligomerisation are mechanisms used to enhance and regulate the dynamic properties of the initial steps in signalling pathways.

Authors: Julio Vera, , Walter Kolch,

Date Published: 2008

Publication Type: Not specified

Trigger enzymes: bifunctional proteins active in metabolism and in controlling gene expression

Abstract (Expand)

All regulatory processes require components that sense the environmental or metabolic conditions of the cell, and sophisticated sensory proteins have been studied in great detail. During the last few … years, it turned out that enzymes can control gene expression in response to the availability of their substrates. Here, we review four different mechanisms by which these enzymes interfere with regulation in bacteria. First, some enzymes have acquired a DNA-binding domain and act as direct transcription repressors by binding DNA in the absence of their substrates. A second class is represented by aconitase, which can bind iron responsive elements in the absence of iron to control the expression of genes involved in iron homoeostasis. The third class of these enzymes is sugar permeases of the phosphotransferase system that control the activity of transcription regulators by phosphorylating them in the absence of the specific substrate. Finally, a fourth class of regulatory enzymes controls the activity of transcription factors by inhibitory protein-protein interactions. We suggest that the enzymes that are active in the control of gene expression should be designated as trigger enzymes. An analysis of the occurrence of trigger enzymes suggests that the duplication and subsequent functional specialization is a major pattern in their evolution.

Authors: Fabian M Commichau,

Date Published: 11th Dec 2007

Publication Type: Not specified

Towards the development of Bacillus subtilis as a cell factory for membrane proteins and protein complexes

Abstract (Expand)

ABSTRACT: BACKGROUND: The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into … the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. CONCLUSION: While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins.

Authors: Jessica C Zweers, Imrich Barák, Dörte Becher, Arnold Jm Driessen, , Vesa P Kontinen, Manfred J Saller, L'udmila Vavrová,

Date Published: 2nd Dec 2007

Publication Type: Not specified

New surveyor tools for charting microbial metabolic maps

Abstract (Expand)

The computational reconstruction and analysis of cellular models of microbial metabolism is one of the great success stories of systems biology. The extent and quality of metabolic network reconstructions … is, however, limited by the current state of biochemical knowledge. Can experimental high-throughput data be used to improve and expand network reconstructions to include unexplored areas of metabolism? Recent advances in experimental technology and analytical methods bring this aim an important step closer to realization. Data integration will play a particularly important part in exploiting the new experimental opportunities.

Authors: , Dennis Vitkup, Michael P Barrett

Date Published: 21st Nov 2007

Publication Type: Not specified

MetaNetter: inference and visualization of high-resolution metabolomic networks

Abstract (Expand)

SUMMARY: We present a Cytoscape plugin for the inference and visualization of networks from high-resolution mass spectrometry metabolomic data. The software also provides access to basic topological … analysis. This open source, multi-platform software has been successfully used to interpret metabolomic experiments and will enable others using filtered, high mass accuracy mass spectrometric data sets to build and analyse networks. AVAILABILITY: http://compbio.dcs.gla.ac.uk/fabien/abinitio/abinitio.html

Authors: Fabien Jourdan, , Michael P Barrett, David Gilbert

Date Published: 14th Nov 2007

Publication Type: Not specified

Storing and annotating of kinetic data.

Abstract (Expand)

This paper briefly describes the SABIO-RK database model for the storage of reaction kinetics information and the guidelines followed within the SABIO-RK project to annotate the kinetic data. Such … annotations support the definition of cross links to other related databases and augment the semantics of the data stored in the database.

Authors: , Martin Golebiewski, , , Saqib Mir, Andreas Weidemann, Ulrike Wittig

Date Published: 14th Sep 2007

Publication Type: Journal

How quantitative measures unravel design principles in multi-stage phosphorylation cascades

Abstract (Expand)

We investigate design principles of linear multi-stage phosphorylation cascades by using quantitative measures for signaling time, signal duration and signal amplitude. We compare alternative pathway … structures by varying the number of phosphorylations and the length of the cascade. We show that a model for a weakly activated pathway does not reflect the biological context well, unless it is restricted to certain parameter combinations. Focusing therefore on a more general model, we compare alternative structures with respect to a multivariate optimization criterion. We test the hypothesis that the structure of a linear multi-stage phosphorylation cascade is the result of an optimization process aiming for a fast response, defined by the minimum of the product of signaling time and signal duration. It is then shown that certain pathway structures minimize this criterion. Several popular models of MAPK cascades form the basis of our study. These models represent different levels of approximation, which we compare and discuss with respect to the quantitative measures.

Authors: Simone Frey, , Stefan Hohmann,

Date Published: 6th Sep 2007

Publication Type: Not specified

ClpP modulates the activity of the Bacillus subtilis stress response transcription factor, sigmaB

Abstract (Expand)

The general stress regulon of Bacillus subtilis is controlled by the activity state of sigmaB, a transcription factor that is switched on following exposure to either physical or nutritional stress. … ClpP is the proteolytic component of an ATP-dependent protease that is essential for the proper regulation of multiple adaptive responses in B. subtilis. Among the proteins whose abundance increases in ClpP- B. subtilis are several known to depend on sigmaB for their expression. In the current work we examine the relationship of ClpP to the activity of sigmaB. The data reveal that the loss of ClpP in otherwise wild-type B. subtilis results in a small increase in sigmaB activity during growth and a marked enhancement of sigmaB activity following its induction by either physical or nutritional stress. It appears to be the persistence of sigmaB's activity rather than its induction that is principally affected by the loss of ClpP. sigmaB-dependent reporter gene activity rose in parallel in ClpP+ and ClpP- B. subtilis strains but failed to display its normal transience in the ClpP- strain. The putative ClpP targets are likely to be stress generated and novel. Enhanced sigmaB activity in ClpP- B. subtilis was triggered by physical stress but not by the induced synthesis of the physical stress pathway's positive regulator (RsbT). In addition, Western blot analyses failed to detect differences in the levels of the principal known sigmaB regulators in ClpP+ and ClpP- B. subtilis strains. The data suggest a model in which ClpP facilitates the turnover of stress-generated factors, which persist in ClpP's absence to stimulate ongoing sigmaB activity.

Authors: Adam Reeves, Ulf Gerth, , W G Haldenwang

Date Published: 22nd Jun 2007

Publication Type: Not specified

Protein mobility and diffusive barriers in Escherichia coli: consequences of osmotic stress

Abstract (Expand)

The effect of osmotic stress on the intracellular diffusion of proteins in Escherichia coli was studied, using a pulsed version of fluorescence recovery after photo-bleaching, pulsed-FRAP. This method … employs sequences of laser pulses which only partly bleach the fluorophores in a cell. Because the cell size and geometry are taken into account, pulsed-FRAP enables to measure diffusion in very small cells of different shapes. We found that upon an osmotic upshock from 0.15 to 0.6 Osm, imposed by NaCl or sorbitol, the apparent intracellular diffusion (D) of mobile green fluorescent protein (GFP) decreased from 3.2 to 0.4 microm(2) s(-1), whereas the membrane permeable glycerol had no effect. Exposing E. coli cells to higher osmolalities (> 0.6 Osm) led to compartmentalization of the GFP into discrete pools, from where the GFP could not escape. Although free diffusion through the cell was hindered, the mobility of GFP in these pools was still relatively high (D approximately 0.4 microm(2) s(-1)). The presence of osmoprotectants restored the effect of osmotic stress on the protein mobility and apparent compartmentalization. Also, lowering the osmolality from 0.6 Osm back to 0.15 Osm restored the mobility of GFP. The implications of these findings in terms of heterogeneities and diffusive barriers inside the cell are discussed.

Authors: Geert van den Bogaart, Nicolaas Hermans, Victor Krasnikov,

Date Published: 28th Apr 2007

Publication Type: Not specified

The role of dynamic stimulation pattern in the analysis of bistable intracellular networks

Abstract (Expand)

Bistable systems play an important role in the functioning of living cells. Depending on the strength of the necessary positive feedback one can distinguish between (irreversible) "one-way … switch" or (reversible) "toggle-switch" type behavior. Besides the well- established steady-state properties, some important characteristics of bistable systems arise from an analysis of their dynamics. We demonstrate that a supercritical stimulus amplitude is not sufficient to move the system from the lower (off-state) to the higher branch (on-state) for either a step or a pulse input. A switching surface is identified for the system as a function of the initial condition, input pulse amplitude and duration (a supercritical signal). We introduce the concept of bounded autonomy for single level systems with a pulse input. Towards this end, we investigate and characterize the role of the duration of the stimulus. Furthermore we show, that a minimal signal power is also necessary to change the steady state of the bistable system. This limiting signal power is independent of the applied stimulus and is determined only by systems parameters. These results are relevant for the design of experiments, where it is often difficult to create a defined pattern for the stimulus. Furthermore, intracellular processes, like receptor internalization, do manipulate the level of stimulus such that level and duration of the stimulus is conducive to characteristic behavior.

Authors: , Sree N Sreenath, Radina P Soebiyanto, Jayant Avva, Kwang-Hyun Cho,

Date Published: 17th Jan 2007

Publication Type: Not specified

Integrated Sampling Procedure for Metabolome Analysis

Abstract

Not specified

Authors: Jochen Schaub, Carola Schiesling, , Michael Dauner

Date Published: 2006

Publication Type: Not specified

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