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The prevalence of methicillin-resitant Staphylococcus aureus (MRSA) in hospitals and the community poses an increasing health burden, which requires the discovery of alternative antimicrobials. Allicin (diallyl thiosulfinate) from garlic exhibits broad-spectrum antimicrobial activity against many multidrug resistant bacteria. The thiol-reactive mode of action of allicin involves its S-thioallylations of low molecular weight (LMW) thiols and protein thiols. To investigate the mode of action and stress response caused by allicin in S. aureus, we analyzed the transcriptome signature, the targets for S-thioallylation in the proteome and the changes in the bacillithiol (BSH) redox potential (E BSH ) under allicin stress. Allicin caused a strong thiol-specific oxidative and sulfur stress response and protein damage as revealed by the induction of the PerR, HypR, QsrR, MhqR, CstR, CtsR, HrcA and CymR regulons in the RNA-seq transcriptome. Allicin also interfered with metal and cell wall homeostasis and caused induction of the Zur, CsoR and GraRS regulons. Brx-roGFP2 biosensor measurements revealed a strongly increased E BSH under allicin stress. In the proteome, 57 proteins were identified with S-thioallylations under allicin treatment, including translation factors (EF-Tu, EF-Ts), metabolic and redox enzymes (AldA, GuaB, Tpx, KatA, BrxA, MsrB) as well as redox-sensitive MarR/SarA-family regulators (MgrA, SarA, SarH1, SarS). Phenotype and biochemical analyses revealed that BSH and the HypR-controlled disulfide reductase MerA are involved in allicin detoxification in S. aureus. The reversal of protein S-thioallylation was catalyzed by the Brx/BSH/YpdA pathway. Finally, the BSSB reductase YpdA was shown to use S-allylmercaptobacillithiol (BSSA) as substrate to regenerate BSH in S. aureus. In conclusion, allicin results in an oxidative shift of E BSH and protein S-thioallylation, which can be reversed by YpdA and the Brx/BSH/YpdA electron pathways in S. aureus to regenerate thiol homeostasis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (E MSH ) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial E MSH throughout the growth curve with basal E MSH levels of ~- 296 mV. Consistent with its H 2 O 2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H 2 O 2 , but is rapidly oxidized by low doses of NaOCl. We further monitored basal E MSH changes and the H 2 O 2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal E MSH was observed in the sigH mutant. The catalase KatA was confirmed as major H 2 O 2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on E MSH and H 2 O 2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic E MSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level E MSH and the role of OxyR and KatA for efficient H 2 O 2 detoxification under oxidative stress., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections, which requires efficient protection mechanisms to avoid destruction. Here, we have investigated the changes in the RNA-seq transcriptome by the strong oxidant sodium hypochlorite (NaOCl) in S. aureus USA300 to identify novel redox-sensing mechanisms that provide protection under infection conditions., Results: NaOCl stress caused an oxidative stress response in S. aureus as indicated by the induction of the PerR, QsrR, HrcA, and SigmaB regulons in the RNA-seq transcriptome. The hypR-merA (USA300HOU_0588-87) operon was most strongly upregulated under NaOCl stress, which encodes for the Rrf2-family regulator HypR and the pyridine nucleotide disulfide reductase MerA. We have characterized HypR as a novel redox-sensitive repressor that controls MerA expression and directly senses and responds to NaOCl and diamide stress via a thiol-based mechanism in S. aureus. Mutational analysis identified Cys33 and the conserved Cys99 as essential for NaOCl sensing, while Cys99 is also important for repressor activity of HypR in vivo. The redox-sensing mechanism of HypR involves Cys33-Cys99 intersubunit disulfide formation by NaOCl stress both in vitro and in vivo. Moreover, the HypR-controlled flavin disulfide reductase MerA was shown to protect S. aureus against NaOCl stress and increased survival in J774A.1 macrophage infection assays. Conclusion and Innovation: Here, we identified a new member of the widespread Rrf2 family as redox sensor of NaOCl stress in S. aureus that uses a thiol/disulfide switch to regulate defense mechanisms against the oxidative burst under infections in S. aureus. Antioxid. Redox Signal. 29, 615-636.

Aims: Bacillithiol (BSH) is the major low-molecular-weight thiol of the human pathogen Staphylococcus aureus. In this study, we used OxICAT and Voronoi redox treemaps to quantify hypochlorite-sensitive protein thiols in S. aureus USA300 and analyzed the role of BSH in protein S-bacillithiolation., Results: The OxICAT analyses enabled the quantification of 228 Cys residues in the redox proteome of S. aureus USA300. Hypochlorite stress resulted in >10% increased oxidation of 58 Cys residues (25.4%) in the thiol redox proteome. Among the highly oxidized sodium hypochlorite (NaOCl)-sensitive proteins are five S-bacillithiolated proteins (Gap, AldA, GuaB, RpmJ, and PpaC). The glyceraldehyde-3-phosphate (G3P) dehydrogenase Gap represents the most abundant S-bacillithiolated protein contributing 4% to the total Cys proteome. The active site Cys151 of Gap was very sensitive to overoxidation and irreversible inactivation by hydrogen peroxide (H 2 O 2 ) or NaOCl in vitro. Treatment with H 2 O 2 or NaOCl in the presence of BSH resulted in reversible Gap inactivation due to S-bacillithiolation, which could be regenerated by the bacilliredoxin Brx (SAUSA300_1321) in vitro. Molecular docking was used to model the S-bacillithiolated Gap active site, suggesting that formation of the BSH mixed disulfide does not require major structural changes. Conclusion and Innovation: Using OxICAT analyses, we identified 58 novel NaOCl-sensitive proteins in the pathogen S. aureus that could play protective roles against the host immune defense and include the glycolytic Gap as major target for S-bacillithiolation. S-bacillithiolation of Gap did not require structural changes, but efficiently functions in redox regulation and protection of the active site against irreversible overoxidation in S. aureus. Antioxid. Redox Signal. 28, 410-430.

Thiol-redox proteomics methods are rapidly developing tools in redox biology. These are applied to identify and quantify proteins with reversible thiol oxidations that are formed under normal growth and oxidative stress conditions inside cells. The proteins with reversible thiol oxidations are usually prepared by alkylation of reduced thiols, subsequent reduction of disulfide bonds followed by a second differential alkylation of newly released thiols. Here, we describe two methods for detection of protein S-thiolations in Gram-positive bacteria using the direct shotgun approach and the fluorescent-label thiol-redox proteomics method that have been successfully applied in our previous work.

Aims: Bacillithiol (BSH) is utilized as a major thiol-redox buffer in the human pathogen Staphylococcus aureus. Under oxidative stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolation, which can be reversed by bacilliredoxins (Brx). In eukaryotes, glutaredoxin-fused roGFP2 biosensors have been applied for dynamic live imaging of the glutathione redox potential. Here, we have constructed a genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus., Results: The Brx-roGFP2 biosensor showed a specific and rapid response to low levels of bacillithiol disulfide (BSSB) in vitro that required the active-site Cys of Brx. Dynamic live imaging in two methicillin-resistant S. aureus (MRSA) USA300 and COL strains revealed fast and dynamic responses of the Brx-roGFP2 biosensor under hypochlorite and hydrogen peroxide (H 2 O 2 ) stress and constitutive oxidation of the probe in different BSH-deficient mutants. Furthermore, we found that the Brx-roGFP2 expression level and the dynamic range are higher in S. aureus COL compared with the USA300 strain. In phagocytosis assays with THP-1 macrophages, the biosensor was 87% oxidized in S. aureus COL. However, no changes in the BSH redox potential were measured after treatment with different antibiotics classes, indicating that antibiotics do not cause oxidative stress in S. aureus. Conclusion and Innovation: This Brx-roGFP2 biosensor catalyzes specific equilibration between the BSH and roGFP2 redox couples and can be applied for dynamic live imaging of redox changes in S. aureus and other BSH-producing Firmicutes. Antioxid. Redox Signal. 26, 835-848.

Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA anti-sigma factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols.

The opportunistic pathogen Pseudomonas aeruginosa uses secretion systems to deliver exoproteins into the environment. These exoproteins contribute to bacterial survival, adaptation, and virulence. The Twin arginine translocation (Tat) export system enables the export of folded proteins into the periplasm, some of which can then be further secreted outside the cell. However, the full range of proteins that are conveyed by Tat is unknown, despite the importance of Tat for the adaptability and full virulence of P. aeruginosa. In this work, we explored the P. aeruginosa Tat-dependent exoproteome under phosphate starvation by two-dimensional gel analysis. We identified the major secreted proteins and new Tat-dependent exoproteins. These exoproteins were further analyzed by a combination of in silico analysis, regulation studies, and protein localization. Altogether we reveal that the absence of the Tat system significantly affects the composition of the exoproteome by impairing protein export and affecting gene expression. Notably we discovered three new Tat exoproteins and one novel type II secretion substrate. Our data also allowed the identification of two new start codons highlighting the importance of protein annotation for subcellular predictions. The new exoproteins that we identify may play a significant role in P. aeruginosa pathogenesis, host interaction and niche adaptation.

Unlabelled: The PhoPR two-component signal transduction system controls one of three responses activated by Bacillus subtilis to adapt to phosphate-limiting conditions (PHO response). The response involves the production of enzymes and transporters that scavenge for phosphate in the environment and assimilate it into the cell. However, in B. subtilis and some other Firmicutes bacteria, cell wall metabolism is also part of the PHO response due to the high phosphate content of the teichoic acids attached either to peptidoglycan (wall teichoic acid) or to the cytoplasmic membrane (lipoteichoic acid). Prompted by our observation that the phosphorylated WalR (WalR∼P) response regulator binds to more chromosomal loci than are revealed by transcriptome analysis, we established the PhoP∼P bindome in phosphate-limited cells. Here, we show that PhoP∼P binds to the chromosome at 25 loci: 12 are within the promoters of previously identified PhoPR regulon genes, while 13 are newly identified. We extend the role of PhoPR in cell wall metabolism showing that PhoP∼P binds to the promoters of four cell wall-associated operons (ggaAB, yqgS, wapA, and dacA), although none show PhoPR-dependent expression under the conditions of this study. We also show that positive autoregulation of phoPR expression and full induction of the PHO response upon phosphate limitation require PhoP∼P binding to the 3' end of the phoPR operon., Importance: The PhoPR two-component system controls one of three responses mounted by B. subtilis to adapt to phosphate limitation (PHO response). Here, establishment of the phosphorylated PhoP (PhoP∼P) bindome enhances our understanding of the PHO response in two important ways. First, PhoPR plays a more extensive role in adaptation to phosphate-limiting conditions than was deduced from transcriptome analyses. Among 13 newly identified binding sites, 4 are cell wall associated (ggaAB, yqgS, wapA, and dacA), revealing that PhoPR has an extended involvement in cell wall metabolism. Second, amplification of the PHO response must occur by a novel mechanism since positive autoregulation of phoPR expression requires PhoP∼P binding to the 3' end of the operon., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Aims: In bacillithiol (BSH)-utilizing organisms, protein S-bacillithiolation functions as a redox switch in response to oxidative stress and protects critical Cys residues against overoxidation. In Bacillus subtilis, both the redox-sensing repressor OhrR and the methionine synthase MetE are redox controlled by S-bacillithiolation in vivo. Here, we identify pathways of protein de-bacillithiolation and test the hypothesis that YphP(BrxA) and YqiW(BrxB) act as bacilliredoxins (Brx) to remove BSH from OhrR and MetE mixed disulfides., Results: We present evidence that the BrxA and BrxB paralogs have de-bacillithiolation activity. This Brx activity results from attack of the amino-terminal Cys residue in a CGC motif on protein BSH-mixed disulfides. B. subtilis OhrR DNA-binding activity is eliminated by S-thiolation on its sole Cys residue. Both the BrxA and BrxB bacilliredoxins mediate de-bacillithiolation of OhrR accompanied by the transfer of BSH to the amino-terminal cysteine of their CGC active site motif. In vitro studies demonstrate that BrxB can restore DNA-binding activity to OhrR which is S-bacillithiolated, but not to OhrR that is S-cysteinylated. MetE is most strongly S-bacillithiolated at Cys719 in vitro and can be efficiently de-bacillithiolated by both BrxA and BrxB., Innovation and Conclusion: We demonstrate that BrxA and BrxB function in the reduction of BSH mixed protein disulfides with two natural substrates (MetE, OhrR). These results provide biochemical evidence for a new class of bacterial redox-regulatory proteins, the bacilliredoxins, which function analogously to glutaredoxins. Bacilliredoxins function in concert with other thiol-disulfide oxidoreductases to maintain redox homeostasis in response to disulfide stress conditions.

Aims: Protein S-bacillithiolation was recently discovered as important thiol protection and redox-switch mechanism in response to hypochlorite stress in Firmicutes bacteria. Here we used transcriptomics to analyze the NaOCl stress response in the mycothiol (MSH)-producing Corynebacterium glutamicum. We further applied thiol-redox proteomics and mass spectrometry (MS) to identify protein S-mycothiolation., Results: Transcriptomics revealed the strong upregulation of the disulfide stress σ(H) regulon by NaOCl stress in C. glutamicum, including genes for the anti sigma factor (rshA), the thioredoxin and MSH pathways (trxB1, trxC, cg1375, trxB, mshC, mca, mtr) that maintain the redox balance. We identified 25 S-mycothiolated proteins in NaOCl-treated cells by liquid chromatography-tandem mass spectrometry (LC-MS/MS), including 16 proteins that are reversibly oxidized by NaOCl in the thiol-redox proteome. The S-mycothiolome includes the methionine synthase (MetE), the maltodextrin phosphorylase (MalP), the myoinositol-1-phosphate synthase (Ino1), enzymes for the biosynthesis of nucleotides (GuaB1, GuaB2, PurL, NadC), and thiamine (ThiD), translation proteins (TufA, PheT, RpsF, RplM, RpsM, RpsC), and antioxidant enzymes (Tpx, Gpx, MsrA). We further show that S-mycothiolation of the thiol peroxidase (Tpx) affects its peroxiredoxin activity in vitro that can be restored by mycoredoxin1. LC-MS/MS analysis further identified 8 proteins with S-cysteinylations in the mshC mutant suggesting that cysteine can be used for S-thiolations in the absence of MSH., Innovation and Conclusion: We identified widespread protein S-mycothiolations in the MSH-producing C. glutamicum and demonstrate that S-mycothiolation reversibly affects the peroxidase activity of Tpx. Interestingly, many targets are conserved S-thiolated across bacillithiol- and MSH-producing bacteria, which could become future drug targets in related pathogenic Gram-positives.

The respiratory pathogen Streptococcus pneumoniae has evolved efficient mechanisms to resist oxidative stress conditions and to displace other bacteria in the nasopharynx. Here we characterize at physiological, functional and structural levels two novel surface-exposed thioredoxin-family lipoproteins, Etrx1 and Etrx2. The impact of both Etrx proteins and their redox partner methionine sulfoxide reductase SpMsrAB2 on pneumococcal pathogenesis was assessed in mouse virulence studies and phagocytosis assays. The results demonstrate that loss of function of either both Etrx proteins or SpMsrAB2 dramatically attenuated pneumococcal virulence in the acute mouse pneumonia model and that Etrx proteins compensate each other. The deficiency of Etrx proteins or SpMsrAB2 further enhanced bacterial uptake by macrophages, and accelerated pneumococcal killing by H2 O2 or free methionine sulfoxides (MetSO). Moreover, the absence of both Etrx redox pathways provokes an accumulation of oxidized SpMsrAB2 in vivo. Taken together our results reveal insights into the role of two extracellular electron pathways required for reduction of SpMsrAB2 and surface-exposed MetSO. Identification of this system and its target proteins paves the way for the design of novel antimicrobials., (© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Aims: Protein S-bacillithiolations are mixed disulfides between protein thiols and the bacillithiol (BSH) redox buffer that occur in response to NaOCl in Bacillus subtilis. We used BSH-specific immunoblots, shotgun liquid chromatography (LC)-tandem mass spectrometry (MS/MS) analysis and redox proteomics to characterize the S-bacillithiolomes of B. subtilis, B. megaterium, B. pumilus, B. amyloliquefaciens, and Staphylococcus carnosus and also measured the BSH/oxidized bacillithiol disulfide (BSSB) redox ratio after NaOCl stress., Results: In total, 54 proteins with characteristic S-bacillithiolation (SSB) sites were identified, including 29 unique proteins and eight proteins conserved in two or more of these bacteria. The methionine synthase MetE is the most abundant S-bacillithiolated protein in Bacillus species after NaOCl exposure. Further, S-bacillithiolated proteins include the translation elongation factor EF-Tu and aminoacyl-tRNA synthetases (ThrS), the DnaK and GrpE chaperones, the two-Cys peroxiredoxin YkuU, the ferredoxin-NADP(+) oxidoreductase YumC, the inorganic pyrophosphatase PpaC, the inosine-5'-monophosphate dehydrogenase GuaB, proteins involved in thiamine biosynthesis (ThiG and ThiM), queuosine biosynthesis (QueF), biosynthesis of aromatic amino acids (AroA and AroE), serine (SerA), branched-chain amino acids (YwaA), and homocysteine (LuxS and MetI). The thioredoxin-like proteins, YphP and YtxJ, are S-bacillithiolated at their active sites, suggesting a function in the de-bacillithiolation process. S-bacillithiolation is accompanied by a two-fold increase in the BSSB level and a decrease in the BSH/BSSB redox ratio in B. subtilis., Innovation: Many essential and conserved proteins, including the dominant MetE, were identified in the S-bacillithiolome of different Bacillus species and S. carnosus using shotgun-LC-MS/MS analyses., Conclusion: S-bacillithiolation is a widespread redox control mechanism among Firmicutes bacteria that protects conserved metabolic enzymes and essential proteins against overoxidation.

The ability of bacteria to adapt to environmental changes has allowed these organisms to thrive in all parts of the globe. By monitoring their extracellular and intracellular environments, bacteria assure their most appropriate response for each environment. Post-translational modification of proteins is one mechanism by which cells respond to their changing environments. Here, we report that two post-translational modifications regulate transcription of the extracytoplasmic stress-responsive promoter cpxP: (i) acetyl phosphate-dependent phosphorylation of the response regulator CpxR and (ii) acetyl coenzyme A-dependent acetylation of the α subunit of RNA polymerase. Together, these two post-translational modifications fine-tune cpxP transcription in response to changes in the intracellular environment.

Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In Bacillus subtilis the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation. Here we have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid in B. subtilis. The expression profile of NaOCl stress is indicative of disulfide stress as shown by the induction of the thiol- and oxidative stress-specific Spx, CtsR, and PerR regulons. Thiol redox proteomics identified only few cytoplasmic proteins with reversible thiol-oxidations in response to NaOCl stress that include GapA and MetE. Shotgun-liquid chromatography-tandem MS analyses revealed that GapA, Spx, and PerR are oxidized to intramolecular disulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, the 3-D-phosphoglycerate dehydrogenase SerA, and the putative bacilliredoxin YphP. S-bacillithiolation of the OhrR repressor leads to up-regulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE, YxjG, PpaC and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. The mechanism of S-glutathionylation of MetE has been described in Escherichia coli also leading to enzyme inactivation and methionine auxotrophy. In summary, our studies discover an important role of the bacillithiol redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of four enzymes of the methionine biosynthesis pathway.

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

The redox-sensing MarR/DUF24-type repressor YodB controls expression of the azoreductase AzoR1 and the nitroreductase YodC that are involved in detoxification of quinones and diamide in Bacillus subtilis. In the present paper, we identified YodB and its paralog YvaP (CatR) as repressors of the yfiDE (catDE) operon encoding a catechol-2,3-dioxygenase that also contributes to quinone resistance. Inactivation of both CatR and YodB is required for full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis studies showed that CatR protects two inverted repeats with the consensus sequence TTAC-N(5)-GTAA overlapping the -35 promoter region (BS1) and the transcriptional start site (TSS) (BS2). The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue, which is required for redox sensing of CatR in vivo as shown by Cys-to-Ser mutagenesis. Our data suggest that CatR is modified by intermolecular disulfide formation in response to diamide and quinones in vitro and in vivo. Redox regulation of CatR occurs independently of YodB, and no protein interaction was detected between CatR and YodB in vivo using protein cross-linking and mass spectrometry.

The MarR/DUF24-type repressor YodB controls the azoreductase AzoR1, the nitroreductase YodC and the redox-sensing regulator Spx in response to quinones and diamide in Bacillus subtilis. Previously, we showed using a yodBCys6-Ala mutant that the conserved Cys6 apparently contributes to the DNA-binding activity of YodB in vivo. Here, we present data that mutation of Cys6 to Ser led to a form of the protein that was reduced in redox-sensing in response to diamide and 2-methylhydroquinone (MHQ) in vivo. DNA-binding experiments indicate that YodB is regulated by a reversible thiol-modification in response to diamide and MHQ in vitro. Redox-regulation of YodB involves Cys6-Cys101' intermolecular disulfide formation by diamide and quinones in vitro. Diagonal Western blot analyses confirm the formation of intersubunit disulfides in YodB in vivo that require the conserved Cys6 and either of the C-terminal Cys101' or Cys108' residues. This study reveals a thiol-disulfide switch model of redox-regulation for the YodB repressor to sense electrophilic compounds in vivo.

The gram-positive pathogen Staphylococcus aureus secretes various proteins into its extracellular milieu. Bioinformatics analyses have indicated that most of these proteins are directed to the canonical Sec pathway, which consists of the translocation motor SecA and a membrane-embedded channel composed of the SecY, SecE, and SecG proteins. In addition, S. aureus contains an accessory Sec2 pathway involving the SecA2 and SecY2 proteins. Here, we have addressed the roles of the nonessential channel components SecG and SecY2 in the biogenesis of the extracellular proteome of S. aureus. The results show that SecG is of major importance for protein secretion by S. aureus. Specifically, the extracellular accumulation of nine abundant exoproteins and seven cell wall-bound proteins was significantly affected in an secG mutant. No secretion defects were detected for strains with a secY2 single mutation. However, deletion of secY2 exacerbated the secretion defects of secG mutants, affecting the extracellular accumulation of one additional exoprotein and one cell wall protein. Furthermore, an secG secY2 double mutant displayed a synthetic growth defect. This might relate to a slightly elevated expression of sraP, encoding the only known substrate for the Sec2 pathway, in cells lacking SecG. Additionally, the results suggest that SecY2 can interact with the Sec1 channel, which would be consistent with the presence of a single set of secE and secG genes in S. aureus.

Summary Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and DeltatatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The DeltatatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild-type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild-type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.

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.

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.

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.

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.

The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can secrete numerous proteins into the surrounding medium enabling them to use high-molecular-weight substances, which are abundant in soils, as nutrient sources. The availability of complete genome sequences allows for the prediction of the proteins containing signals for secretion into the extracellular milieu and also of the proteins which form the secretion machinery needed for protein translocation through the cytoplasmic membrane. To confirm the predicted subcellular localization of proteins, proteomics is the best choice. The extracellular proteomes of B. subtilis and B. licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species., (Copyright (c) 2008 S. Karger AG, Basel.)

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.

Regulation of bacterial iron homeostasis is often controlled by the iron-sensing ferric uptake repressor (Fur). The Bacillus subtilis Fur protein acts as an iron-dependent repressor for siderophore biosynthesis and iron transport proteins. Here, we demonstrate that Fur also coordinates an iron-sparing response that acts to repress the expression of iron-rich proteins when iron is limiting. When Fur is inactive, numerous iron-containing proteins are down-regulated, including succinate dehydrogenase, aconitase, cytochromes, and biosynthetic enzymes for heme, cysteine, and branched chain amino acids. As a result, a fur mutant grows slowly in a variety of nutrient conditions. Depending on the growth medium, rapid growth can be restored by mutations in one or more of the molecular effectors of the iron-sparing response. These effectors include the products of three Fur-regulated operons that encode a small RNA (FsrA) and three small, basic proteins (FbpA, FbpB, and FbpC). Extensive complementarity between FsrA and the leader region of the succinate dehydrogenase operon is consistent with an RNA-mediated translational repression mechanism for this target. Thus, iron deprivation in B. subtilis activates pathways to remodel the proteome to preserve iron for the most critical cellular functions.

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.

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

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

Disulphide bond formation catalysed by thiol-disulphide oxidoreductases (TDORs) is a universally conserved mechanism for stabilizing extracytoplasmic proteins. In Escherichia coli, disulphide bond formation requires a concerted action of distinct TDORs in thiol oxidation and subsequent quinone reduction. TDOR function in other bacteria has remained largely unexplored. Here we focus on TDORs of low-GC Gram-positive bacteria, in particular DsbA of Staphylococcus aureus and BdbA-D of Bacillus subtilis. Phylogenetic analyses reveal that the homologues DsbA and BdbD cluster in distinct groups typical for Staphylococcus and Bacillus species respectively. To compare the function of these TDORs, DsbA was produced in various bdb mutants of B. subtilis. Next, we assessed the ability of DsbA to sustain different TDOR-dependent processes, including heterologous secretion of E. coli PhoA, competence development and bacteriocin (sublancin 168) production. The results show that DsbA can function in all three processes. While BdbD needs a quinone oxidoreductase for activity, DsbA activity appears to depend on redox-active medium components. Unexpectedly, both quinone oxidoreductases of B. subtilis are sufficient to sustain production of sublancin. Moreover, DsbA can functionally replace these quinone oxidoreductases in sublancin production. Taken together, our unprecedented findings imply that TDOR systems of low-GC Gram-positive bacteria have a modular composition.

Bacillus subtilis is exposed to a variety of antimicrobial compounds in the soil. In this paper, we report on the response of B. subtilis to the fungal-related antimicrobials 6-brom-2-vinyl-chroman-4-on (chromanon) and 2-methylhydroquinone (2-MHQ) using proteome and transcriptome analyses. Chromanon, a derivative of aposphaerins from Aposphaeria species caused predominant protein damage in B. subtilis as indicated by the induction of the HrcA, CtsR, and Spx regulons. The expression profile of the ganomycin-related substance 2-MHQ was similar to that of catechol as reflected by the common induction of the thiol-specific oxidative stress response. Several putative ring-cleavage dioxygenases and oxidoreductases were differentially up-regulated by 2-MHQ, catechol, and chromanon including yfiDE, ydfNOP, yodED, ycnDE, yodC, and ykcA. The nitroreductase encoding yodC gene is induced in response to catechol, 2-MHQ, and chromanon, which depend on the MarR-type repressor YodB. The yfiDE (catDE) operon encodes a catechol-2,3-dioxygenase which is most strongly induced by catechol. The yodED (mhqED), ydfNOP (mhqNOP) operons, and ykcA (mhqA) respond most strongly to 2-MHQ and encode putative hydroquinone-specific extradiol dioxygenases. The ycnDE operon was most strongly induced by chromanon. Mutational analyses revealed that the putative hydroquinone-specific dioxygenases MhqO and MhqA confer resistance to 2-MHQ in B. subtilis.

With the emergence of mass spectrometry in protein science and the availability of complete genome sequences, proteomics has gone through a rapid development. The soil bacterium Bacillus subtilis, as one of the first DNA sequenced species, represents a model for Gram-positive bacteria and its proteome was extensively studied throughout the years. Having the final goal to elucidate how life really functions, one basic requirement is to know the entirety of cellular proteins. This review presents how far we have got in unraveling the proteome of B. subtilis. The application of gel-based and gel-free technologies, the analyses of different subcellular proteome fractions, and the pursuance of various physiological strategies resulted in a coverage of more than one-third of B. subtilis theoretical proteome.

Phytate is the most abundant phosphorus source in plants. Since Bacillus subtilis is a soil-dwelling bacterium, the focus of this study was to investigate whether it can use phytate as a phosphorus source. The extracellular proteome analysis revealed that phytate is an alternative phosphorus source to overcome the phosphate starvation response in B. subtilis. However, the phytase was not induced neither under phosphate starvation conditions nor by phytate addition. Surprisingly, the proteome analyses demonstrated a re-distribution of the major cell wall protease WprA from the cell wall to the extracellular medium in phytate-supplemented medium. In contrast, several cell wall proteins such as autolysins and autolysin modifier proteins (e.g., LytB, -C, -D, -E, -F) are increased in the cell wall proteome in response to phytate which is not accompanied by increased transcription of the corresponding genes. These effects of phytate on the composition of the B. subtilis cell wall proteome do not depend on the acidic conditions, the increased sodium ion concentration, and the increased cell lysis. In addition, the previously predicted as cytoplasmic protein oxalate decarboxylase OxdC was identified as the most abundant cell wall protein which was induced at the transcriptional level due to the acidic conditions caused by phytate.

The global gene expression profile of Bacillus subtilis in response to ammonium and tryptophan starvation was analyzed using transcriptomics and proteomics which gained novel insights into these starvation responses. The results demonstrate that both starvation conditions induce specific, overlapping and general starvation responses. The TnrA regulon, the glutamine synthetase (glnA) as well as the sigma(L)-dependent bkd and roc operons were most strongly and specifically induced after ammonium starvation. These are involved in the uptake and utilization of ammonium and alternative nitrogen sources such as amino acids, gamma-aminobutyrate, nitrate/nitrite, uric acid/urea and oligopeptides. In addition, several carbon catabolite-controlled genes (e.g. acsA, citB), the alpha-acetolactate synthase/-decarboxylase alsSD operon and several aminotransferase genes were specifically induced after ammonium starvation. The induction of sigma(F)- and sigma(E)-dependent sporulation proteins at later time points in ammonium-starved cells was accompanied by an increased sporulation frequency. The specific response to tryptophan starvation includes the TRAP-regulated tryptophan biosynthesis genes, some RelA-dependent genes (e.g. adeC, ald) as well as spo0E. Furthermore, we recognized overlapping responses between ammonium and tryptophan starvation (e.g. dat, maeN) as well as the common induction of the CodY and sigma(H) general starvation regulons and the RelA-dependent stringent response. Many genes encoding proteins of so far unknown functions could be assigned to specifically or commonly induced genes., (Copyright (c) 2007 S. Karger AG, Basel.)

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for alpha-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.

In this paper we have defined proteome signatures of Bacillus subtilis in response to heat, salt, peroxide, and superoxide stress as well as after starvation for ammonium, tryptophan, glucose, and phosphate using the 2-D gel-based approach. In total, 79 stress-induced and 155 starvation-induced marker proteins were identified including 50% that are not expressed in the vegetative proteome. Fused proteome maps and a color coding approach have been used to define stress-specific regulons that are involved in specific adaptative functions (HrcA for heat, PerR and Fur for oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition, starvation-specific regulons are defined that are involved in the uptake or utilization of alternative nutrient sources (TnrA, sigmaL/BkdR for ammonium; tryptophan-activated RNA-binding attenuation protein for tryptophan; CcpA, CcpN, sigmaL/AcoR for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome signatures include the CtsR, Spx, sigmaL/RocR, sigmaB, sigmaH, CodY, sigmaF, and sigmaE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress-induced protein damages. Finally, a subset of sigmaH-dependent proteins (sporulation response regulator, YvyD, YtxH, YisK, YuxI, YpiB) and the CodY-dependent aspartyl phosphatase RapA were defined as general starvation proteins that indicate the transition to stationary phase caused by starvation.

The bacterial signal recognition particle (SRP)-dependent pathway is believed to be a major targeting route for membrane proteins, as well as for subsets of secretory proteins. The present studies were aimed at an assessment of the role of two key components of SRP, namely Ffh and FtsY, in protein secretion by the Gram-positive bacterium Bacillus subtilis. Our results show that both components are important for the extracellular accumulation of proteins containing known signal peptides. Remarkably, extracellular accumulation of individual proteins was affected to different extents by depletion of Ffh or FtsY, at least under the conditions tested. Moreover, the observed Ffh or FtsY dependence of certain secretory proteins did not seem to correlate with signal peptide length or hydrophobicity. Although it is presently difficult to distinguish between direct and indirect effects, these findings suggest that other, yet unidentified, determinants in secretory proteins are also important for their SRP dependence. High-level production of homologous and heterologous secretory proteins was shown to result in elevated cellular Ffh and FtsY levels. This phenomenon is, most likely, due to post-transcriptional regulation. In conclusion, the present proteomic dissection of SRP-dependent extracellular protein accumulation provides exciting leads to identify novel determinants for interactions between secretory proteins and SRP.

The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding approximately 40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT.

Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.

Activity of the Tat machinery for protein transport across the inner membrane of Escherichia coli and the chloroplast thylakoidal membrane requires the presence of three membrane proteins: TatA, TatB and TatC. Here, we show that the Tat machinery of the Gram-positive bacterium Bacillus subtilis is very different because it contains at least two minimal Tat translocases, each composed of one specific TatA and one specific TatC component. A third, TatB-like component is apparently not required. This implies that TatA proteins of B. subtilis perform the functions of both TatA and TatB of E. coli and thylakoids. Notably, the two B. subtilis translocases named TatAdCd and TatAyCy both function as individual, substrate-specific translocases for the twin-arginine preproteins PhoD and YwbN, respectively. Importantly, these minimal TatAC translocases of B. subtilis are representative for the Tat machinery of the vast majority of Gram-positive bacteria, Streptomycetes being the only known exception with TatABC translocases.

Bacillus subtilis contains three proteins of the signal recognition particle-GTPase family known as Ffh, FtsY, and FlhF. Here we show that FlhF is dispensable for protein secretion, unlike Ffh and FtsY. Although flhF is located in the fla/che operon, B. subtilis 168 flhF mutant cells assemble flagella and are motile.

Using a combined quantitative proteomic and bioinformatic approach, we monitored the cytoplasmic proteome profile of the Gram-positive bacterium Bacillus subtilis during a fermentation process in complex medium. Proteome signatures were applied to elucidate the physiological changes occurring in the gene expression profile during growth. Furthermore, we determined the significance level of quantitative proteome changes, identified relative to the threshold of scatter in replicated samples and developed a statistically rigorous method that allowed us to determine significant fold-changes at 95% confidence between different proteomes. Different functional groups of proteins were clustered according to their pattern of significant expression changes. The largest group is induced by the exhaustion of glucose and the presence of alternative carbon and nitrogen sources. Furthermore, depletion of glucose caused the induction of the trichloroacetic acid (TCA) cycle enzymes and the downregulation of glycolytic enzymes. The onset of the transition phase may be provoked by amino acid starvation, resulting in the RelA-dependent repression of proteins involved in the translation process and in the induction of amino acid biosynthetic pathways. Comparisons between the parental strain and two subtilisin-hypersecreting strains revealed only small cytoplasmic differences in the main metabolic pathways. Instead, the overproduction of degradative enzymes in both of these mutants was reflected in the extracellular proteome.

Secretory proteins perform a variety of important "remote-control" functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which approximately 90 extracellular proteins were identified. Analysis of these proteins disclosed various "secrets of the secretome," such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only approximately 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

The accumulation of malfolded proteins in the cell envelope of the Gram-positive eubacterium Bacillus subtilis was previously shown to provoke a so-called secretion stress response. In the present studies, proteomic approaches were employed to identify changes in the extracellular proteome of B. subtilis in response to secretion stress. The data shows that, irrespective of the way in which secretion stress is imposed on the cells, the levels of only two extracellular proteins, HtrA and YqxI, display major variations in a parallel manner. Whereas the extracellular level of the HtrA protease is determined through transcriptional regulation, the level of YqxI in the growth medium is determined post-transcriptionally in an HtrA-dependent manner. In the absence of secretion stress, the extracellular levels of HtrA and YqxI are low because of extracytoplasmic proteolysis. Finally, the protease active site of HtrA is dispensable for post-transcriptional YqxI regulation. It is known that Escherichia coli HtrA has combined protease and chaperone-like activities. As this protein shares a high degree of similarity with B. subtilis HtrA, it can be hypothesized that both activities are conserved in B. subtilis HtrA. Thus, a chaperone-like activity of B. subtilis HtrA could be involved in the appearance of YqxI on the extracellular proteome.

In Bacillus subtilis, the alternative sigma factor sigma(B) is activated in response to environmental stress or energy depletion. The general stress regulon under the control of sigma(B) provides the cell with multiple stress resistance. Experiments were designed to determine how activated sigma(B) replaces sigma(A) as a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the sigma(A)-dependent stress gene clpE under sigma(B)-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type. The relative affinities of sigma(A) and sigma(B) for binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of sigma(B) for E was 60-fold lower than that of sigma(A). Western blot analyses with antibodies against sigma(A), sigma(B), and E showed that, after exposure to ethanol stress, the concentration of sigma(B) was only twofold higher than those of sigma(A) and E. Thus, the concentration of sigma(B) after stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of sigma(B) to E after stress.

Protein secretion from Bacillus species is a major industrial production tool with a market of over $1 billion per year. However, standard export technologies, based on the well-characterised general secretory (Sec) pathway, are frequently inapplicable for the production of proteins. The recently discovered twin-arginine translocation (Tat) pathway offers additional potential to transport proteins. Here we review the use of functional genomic and proteomic approaches to explore the Tat pathway of Bacillus subtilis. The properties of Tat pathway components and the twin-arginine signal peptides that direct proteins into this pathway are discussed. Where appropriate, a comparison is made with Tat systems from other organism, such as Escherichia coli. Recent findings with the latter organism in particular provide proof-of-principle that the Tat pathway can be exploited for the production of Sec-incompatible proteins.