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228 Publications visible to you, out of a total of 228
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

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

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

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

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

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

Integrated Sampling Procedure for Metabolome Analysis

Abstract

Not specified

Authors: Jochen Schaub, Carola Schiesling, , Michael Dauner

Date Published: 2006

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

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

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

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

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

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

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