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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.sub.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. doi: 10.1128/JB.00409-10

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. doi: 10.1128/JB.01452-09

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. doi: 10.1128/JB.00937-09

YvrI is a recently identified alternative [sigma] factor in Bacillus subtilis that requires the coactivator YvrHa to activate transcription. Previously, a strain engineered to overproduce YvrI was found to overproduce oxalate decarboxylase (OxdC), and further analysis identified three YvrI-activated promoters preceding the yvrI-yvrHa, yvrJ, and oxdC-yvrL operons. Independently, proteome analyses identified OxdC as a highly abundant, cell wall-associated protein that accumulated under acidic growth conditions. We show here that the accumulation of OxdC in the cell wall proteome under acidic growth conditions is absolutely dependent on YvrI and is correlated with enhanced transcription of both the yvrl-yvrHa and the oxdC-yvrL operons. Conversely, OxdC accumulates to a high level even under nonacidic growth conditions in cells lacking YvrL, a negative regulator of YvrI/YvrHa-dependent transcription. These results indicate that YvrI and its associated coregulators YvrHa and YvrL are required for the regulation of OxdC expression by acid stress. The high-level accumulation of OxdC depends, in part, on a strong oxdC promoter. A regulatory sequence with similarity to an upstream promoter element (UP) was identified upstream of the oxdC promoter and is required for high-level promoter activity. Conservation of the YvrI/YvrHa/YvrL regulatory system among related species allowed us to deduce an expanded consensus sequence for the compositionally unusual promoters recognized by this new [sigma] factor.

The Dlt system modulates the density of negative charge in the cell wall of Gram-positive bacteria by substituting anionic polymers (wall and lipoteichoic acids) with D-alanine. The htrA and htrB genes, regulated by the CssRS two-component system (TCS) and encoding membrane-associated protein quality control proteases, were expressed at strongly decreased levels in a mutant with defective Dlt (dltD:: miniTn 10) as compared to the [dlt.sup.+] wild-type strain under a secretion stress condition (hypersecretion of AmyQ [alpha]-amylase). The level of HtrA protein in the extracellular proteome of the dltD mutant was decreased consistently. Expression from the promoter of the lialHGFSR (yvqlHGFEC) operon ([P.sub.lial]) is dependent on the LiaRS TCS. The Dlt defect increased the expression from [P.sub.lial] under two stress conditions, AmyQ hypersecretion and treatment with a cationic antimicrobial peptide (LL-37), but decreased the expression in vancomycin-treated cells. Furthermore, Dlt inactivation enhanced the expression of the YxdJK-regulated yxdL gene in LL-37-treated cells. The increased net negative charge of the cell wall seems to cause varied and opposite effects on the expression of CssRS-, LiaRS- and YxdJK-regulated genes under different stress conditions. The results suggest that TCSs which sense misfolded proteins or peptides are modulated by the density of negative charge in the cell wall. The density of negative charge on the outer surface of the cell membrane did not have a similar effect on TCSs.

An investigation on whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in Bacillus subtilis is presented. Results reveal that Eschericia coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis.

The toxin-antitoxin operon of pSM19035 encodes three proteins: the [omega] global regulator, the [epsilon] labile antitoxin and the stable [zeta] toxin. Accumulation of [zeta] toxin free of [epsilon] antitoxin induced loss of cell proliferation in both Bacillus subtilis and Escherichia coli cells. Induction of a [zeta] variant ([zeta]Y83C) triggered stasis, in which B. subtilis cells were viable but unable to proliferate, without selectively affecting protein translation. In E. coli cells, accumulation of free [zeta] toxin induced stasis, but this was fully reversed by expression of the [epsilon] antitoxin within a defined time window. The time window for reversion of [zeta] toxicity by expression of [epsilon] antitoxin was dependent on the initial cellular level of [zeta]. After 240 min of constitutive expression, or inducible expression of high levels of [zeta] toxin for 30 min, expression of [epsilon] failed to reverse the toxic effect exerted by [zeta] in cells growing in minimal medium. Under the latter conditions, [zeta] inhibited replication, transcription and translation and finally induced death in a fraction (~ 50 %) of the cell population. These results support the view that [zeta] interacts with its specific target and reversibly inhibits cell proliferation, but accumulation of [zeta] might lead to cell death due to pleiotropic effects.

The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding ~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 Iow 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.

The deletion of six AT-rich islands from the Bacillus subtilis genome is demonstrated that results in reduced amounts of extracellular LipA. The findings suggest that SkfA protects LipA, directly or indirectly, against proteolytic degradation.

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.

Results show that majority of the phosphate starvation-induced proteins are regulated by the sigma(sup)B protein. Data further indicate that phosphate starvation-induced proteins are regulated by different regulon proteins in wild type and mutants of Bacillus subtilis.

A study was conducted to identify and analyze two Bacillus subtilis genes, yklA and ykzA, which are homologous to osmC gene from Escherichia coli. Expression of ykzA was found to be non-medium dependent but increased when cells were exposed to stress and starvation. The stress-induced increase was dependent on the alternative sigma factor B which controls a large stationary-phase and stress regulon. The composite complex expression pattern of the two B. subtilis genes was similar to osmC in E. coli.

Research was conducted to characterize delta(super B) dependent stress proteins of Bacillus subtilis that are similar to the delta(super S)-dependent Dps protein from Escherichia coli. Cells were harvested in a synthetic medium under conditions of glucose starvation to establish oxidative-stress resistance. Results showed that growing cells are capable to responding to specific growth-restricting conditions by the induction of heat shock proteins to prevent cellular damage.

The effects of ethanol, heat shock and salt stress on the protein profile of Bacillus subtilis were analyzed by high resolution two-dimensional (2-D) polyacrylamide gel electrophoresis. High resolution 2-D polyacrylamide gel electrophoretic analysis of Bacillus subtilis stress proteins indicated the presence of stress-specific proteins that were triggered by ethanol, heat shock and salt stress. Furthermore, general stress proteins were also activated by general stress induction mechanisms.

The vegetative proteins of Bacillus subtilis were characterized by the construction of two-dimensional (2-D) protein gels from crude extracts of the microbe. The 2-D gel electrophoresis of Bacillus subtilis extracts indicated the role of osmotic stress and glucose starvation in modulating the synthesis of enzymes involved in glycolysis and amino acid metabolism. Furthermore, 2-D gel electrophoresis of Bacillus subtilis crude extracts permitted the identification and separation of 600 distinct protein spots.

The AhpC subunit of the Bacillus subtilis alkyl hydroperoxide reductase was identified as a general stress protein induced in response to heat or salt stress or after entry of the organism into the stationary phase. The ahp operon, encoding the two subunits AhpC and AhpF, was cloned and localized between the gntRKPZ operon and the bglA locus. Findings indicated an especially strong induction of AhpC and AhpF in cells subjected to oxidative stress. Stress induction occurred at a sigma-A-dependent promoter that overlaps with operator sites similar to the per box.

Two-dimensional polyacrylamide gel electrophoresis of general stress proteins (Gsps) in Bacillus subtilis showed that genes for Gsps are localized in regions controlled by sigmaB. These regions contain two striking spots, GsiB and GspA. N-terminal sequencing of GspA indicated that it is similar to a 33.5-kDa protein in B. subtilis. Northern blot analysis of the GspA-encoding gene gspA revealed a 0.95-kb transcript indicating a monocistronic transcriptional unit. Construction of an insertion mutation in gspA confirmed that gspA gene is not essential for growth in response to stress.

Secretory proteins perform a variety of important 'remote-control' functions for bacterial survival in the environment. The study concludes that proteomics has yielded a variety of novel leads for the analysis of protein traffic in B.subtilis and other gram-positive bacteria.

Research has been conducted on response of Bacillus subtilis sigma factor (super)B to environmental stress. Results indicate that sigma (super)B concentration after stress cannot compensate for its low affinity for core RNA polymerase.

Nine new members of the sigma(super B) regulon in Bacillus subtilis have been identified and subjected to transcriptional analysis. Putative functions of BmrU, Ycdf, YdaD, YdaP, YhdN and YocK indicate the possible protective role of sigma(super B)-dependent general stress proteins, whose induction makes Bacillus subtilis respond to different stimuli such as ethanol, heat and salt stress. The other new sigma(super B)-dependent genes encoding proteins with still unknown functions are ydaE, ydaG and yfkM.