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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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