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46 Publications visible to you, out of a total of 46
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

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

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

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

Proteomic signatures uncover thiol-specific electrophile resistance mechanisms in Bacillus subtilis

Abstract (Expand)

Proteomic and transcriptomics signatures are powerful tools for visualizing global changes in gene expression in bacterial cells after exposure to stress, starvation or toxic compounds. Based on the … global expression profile and the dissection into specific regulons, this knowledge can be used to predict the mode of action for novel antimicrobial compounds. This review summarizes our recent progress of proteomic signatures in the model bacterium for low-GC Gram-positive bacteria Bacillus subtilis in response to the antimicrobial compounds phenol, catechol, salicylic acid, 2-methylhydroquinone (2-MHQ) and 6-brom-2-vinyl-chroman-4-on (chromanon). Catechol, 2-MHQ and diamide displayed a common mode of action, as revealed by the induction of the thiol-specific oxidative stress response. In addition, multiple dioxygenases/glyoxalases, azoreductases and nitroreductases were induced by thiol-reactive compounds that are regulated by two novel thiol-specific regulators, YodB and MhqR (YkvE), both of which contribute to electrophile resistance in B. subtilis. These novel thiol-stress-responsive mechanisms are highly conserved among Gram-positive bacteria and are thought to have evolved to detoxify quinone-like electrophiles.

Authors: Haike Antelmann, , Peter Zuber

Date Published: 20th Feb 2008

Publication Type: Not specified

Regulation of quinone detoxification by the thiol stress sensing DUF24/MarR-like repressor, YodB in Bacillus subtilis

Abstract (Expand)

Recently, we showed that the MarR-type repressor YkvE (MhqR) regulates multiple dioxygenases/glyoxalases, oxidoreductases and the azoreductase encoding yvaB (azoR2) gene in response to thiol-specific … stress conditions, such as diamide, catechol and 2-methylhydroquinone (MHQ). Here we report on the regulation of the yocJ (azoR1) gene encoding another azoreductase by the novel DUF24/MarR-type repressor, YodB after exposure to thiol-reactive compounds. DNA binding activity of YodB is directly inhibited by thiol-reactive compounds in vitro. Mass spectrometry identified YodB-Cys-S-adducts that are formed upon exposure of YodB to MHQ and catechol in vitro. This confirms that catechol and MHQ are auto-oxidized to toxic ortho- and para-benzoquinones which act like diamide as thiol-reactive electrophiles. Mutational analyses further showed that the conserved Cys6 residue of YodB is required for optimal repression in vivo and in vitro while substitution of all three Cys residues of YodB affects induction of azoR1 transcription. Finally, phenotype analyses revealed that both azoreductases, AzoR1 and AzoR2 confer resistance to catechol, MHQ, 1,4-benzoquinone and diamide. Thus, both azoreductases that are controlled by different regulatory mechanisms have common functions in quinone and azo-compound reduction to protect cells against the thiol reactivity of electrophiles.

Authors: Montira Leelakriangsak, Nguyen Thi Thu Huyen, Stefanie Töwe, Nguyen van Duy, Dörte Becher, , Haike Antelmann, Peter Zuber

Date Published: 16th Jan 2008

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

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

The MarR-type repressor MhqR (YkvE) regulates multiple dioxygenases/glyoxalases and an azoreductase which confer resistance to 2-methylhydroquinone and catechol in Bacillus subtilis

Abstract (Expand)

Catechol and 2-methylhydroquinone (2-MHQ) cause the induction of the thiol-specific stress response and four dioxygenases/glyoxalases in Bacillus subtilis. Using transcription factor arrays, the MarR-type … regulator YkvE was identified as a repressor of the dioxygenase/glyoxalase-encoding mhqE gene. Transcriptional and proteome analyses of the DeltaykvE mutant revealed the upregulation of ykcA (mhqA), ydfNOP (mhqNOP), yodED (mhqED) and yvaB (azoR2) encoding multiple dioxygenases/glyoxalases, oxidoreductases and an azoreductase. Primer extension experiments identified sigma(A)-type promoter sequences upstream of mhqA, mhqNOP, mhqED and azoR2 from which transcription is elevated after thiol stress. DNase I footprinting analysis showed that YkvE protects a primary imperfect inverted repeat with the consensus sequence of tATCTcgaAtTCgAGATaaaa in the azoR2, mhqE and mhqN promoter regions. Analysis of mhqE-promoter-bgaB fusions confirmed the significance of YkvE binding to this operator in vivo. Adjacent secondary repeats were protected by YkvE in the azoR2 and mhqN promoter regions consistent with multiple DNA-protein binding complexes. DNA-binding activity of YkvE was not directly affected by thiol-reactive compounds in vitro. Mutational analyses showed that MhqA, MhqO and AzoR2 confer resistance to 2-MHQ. Moreover, the DeltaykvE mutant displayed a 2-MHQ and catechol resistant phenotype. YkvE was renamed as MhqR controlling a 2-MHQ and catechol-resistance regulon of B. subtilis.

Authors: Stefanie Töwe, Montira Leelakriangsak, Kazuo Kobayashi, Nguyen Van Duy, , Peter Zuber, Haike Antelmann

Date Published: 27th Aug 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

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

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