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21 Publications visible to you, out of a total of 21
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

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

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 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

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

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

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

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

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

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

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

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

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

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

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

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