SummaryBacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors. The s W regulon includes functions involved in detoxification and protection against antimicrobials, whereas s M is essential for growth at high salt concentrations. We now report that antibiotics that inhibit cell wall biosynthesis induce both s W and s M regulons as monitored using DNA microarrays. Induction of selected s W -dependent genes was confirmed using lacZ reporter fusions and Northern blot analysis. The ability of vancomycin to induce the s W regulon is dependent on both s W and the cognate anti-s, RsiW, but is independent of the transition state regulator AbrB. These results suggest that the membrane-localized RsiW anti-s W factor mediates the transcriptional response to cell wall stress. Our findings are consistent with the idea that one function of ECF s factors is to coordinate antibiosis stress responses and cell envelope homeostasis.

No abstract

Bacitracin resistance is normally conferred by either of two major mechanisms, the BcrABC transporter, which pumps out bacitracin, or BacA, an undecaprenol kinase that provides C 55 -isoprenyl phosphate by de novo synthesis. We demonstrate that the Bacillus subtilis bcrC (ywoA) gene, encoding a putative bacitracin transport permease, is an important bacitracin resistance determinant. A bcrC mutant strain had an eightfoldhigher sensitivity to bacitracin. Expression of bcrC initiated from a single promoter site that could be recognized by either of two extracytoplasmic function (ECF) factors, X or M . Bacitracin induced expression of bcrC, and this induction was dependent on M but not on X . Under inducing conditions, expression was primarily dependent on M . As a consequence, a sigM mutant was fourfold more sensitive to bacitracin, while the sigX mutant was only slightly sensitive. A sigX sigM double mutant was similar to a bcrC mutant in sensitivity. These results support the suggestion that one function of B. subtilis ECF factors is to coordinate antibiotic stress responses.

SummaryThe Bacillus subtilis sigW gene encodes an extracytoplasmic function (ECF) factor that is expressed in early stationary phase from a sigW-dependent autoregulatory promoter, P W . Using a consensus-based search procedure, we have identified 15 operons preceded by promoters similar in sequence to P W . At least 14 of these promoters are dependent on W both in vivo and in vitro as judged by lacZ reporter fusions, run-off transcription assays and nucleotide resolution start site mapping. We conclude that W controls a regulon of more than 30 genes, many of which encode membrane proteins of unknown function. The W regulon includes a penicillin binding protein (PBP4*) and a co-transcribed amino acid racemase (RacX), homologues of signal peptide peptidase (YteI), flotillin (YuaG), ABC transporters (YknXYZ), non-haem bromoperoxidase (YdjP), epoxide hydrolase (YfhM) and three small peptides with structural similarities to bacteriocin precursor polypeptides. We suggest that W activates a large stationary-phase regulon that functions in detoxification, production of antimicrobial compounds or both.

Listeria monocytogenes is an intracellular bacterial pathogen whose virulence depends on the regulated expression of numerous secreted bacterial factors. As for other gram-positive bacteria, many proteins secreted by L. monocytogenes are translocated across the bacterial membrane in an unfolded state to the compartment existing between the membrane and the cell wall. This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH. We recently identified PrsA2 as a gene product required for L. monocytogenes virulence. PrsA2 was identified based on its increased secretion by strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression. The prsA2 gene product is one of at least two predicted peptidyl-prolyl cis/trans-isomerases encoded by L. monocytogenes; these proteins function as posttranslocation protein chaperones and/or foldases. In this study, we demonstrate that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis by promoting the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and a broad-specificity phospholipase. Loss of PrsA2 activity severely attenuated virulence in mice and impaired bacterial cell-to-cell spread in host cells. In contrast, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth and cell-to-cell spread as well as virulence in mice. PrsA2 is thus distinct from PrsA1 in its unique requirement for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection.

We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires W , and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn 2؉ -dependent glutathione transferases but with a preference for Mg 2؉ and L-cysteine as cofactors.Sequencing of the Bacillus subtilis genome revealed the presence of seven new factors, all members of the extracytoplasmic function subfamily (12, 13). We have begun to investigate their functions by mutation of each gene and the identification of target operons (8)(9)(10)(11). In this work, we demonstrate that yndN encodes a fosfomycin resistance (Fos r ) protein that depends on W for expression. We have renamed yndN as fosB, based on its similarity to the fosB gene identified on a Staphylococcus epidermidis plasmid (Fig. 1B).Transcription of fosB requires W . Previously, 15 W -dependent operons were identified by searching the genome for sequences matching the W autoregulatory site, P w : TGAAAC N 16 CGTA (10). Additional candidate promoters, including one for fosB (Fig. 1A), were identified with 17-bp spacer regions (10).To confirm the role of this predicted W -dependent promoter, we generated a P fosB -cat-lacZ operon fusion inserted ectopically in the SP␤ prophage (HB8083; Table 1) and transduced the reporter fusion into wild-type, sigW, and rsiW mutant backgrounds. Promoter activity as determined in early-stationary-phase cells yielded 18.4 Miller units of ␤-galactosidase in the wild-type strain (HB0052), and this was reduced to background levels (ϳ1 unit) in the sigW mutant (HB0023). In the rsiW (anti-W ) mutant (HB0012), expression was elevated approximately twofold (30.5 units). This pattern is precisely that expected for a W -dependent promoter. We used reverse transcriptase primer extension mapping to identify the transcriptional start site for fosB as a G residue 10 bases downstream from the Ϫ10 region CGTA motif (Fig. 2). There were no other start sites visible in the primer extension experiment, which is consistent with the idea that W is largely, if not exclusively, responsible for fosB transcription. The fosB gene is apparently monocistronic, as it is flanked on either side by genes transcribed from the complementary strand of the genome (Fig. 1A).fosB and sigW mutants are sensitive to fosfomycin. Both fosB (HB0008) and sigW (HB0020) mutants are fosfomycin sensitive: an MIC of 50 g/ml for the mutants compared to 800 g/ml for the wild type in liquid culture. Similarly, the sigW and fosB mutants have a much greater zone of growth inhibition in disk diffusion assays (ϳ25-mm zone for wild type versus Ͼ50 mm for the mutants). The fosB and sigW mutant strains did not display altered sensitivity to several other antibiotics, including vancomycin, cephalosporin C, penicillin G, D-cycloserine, tunicamycin, nisin, and bacitracin. Induction of fosB from a xylose-inducible promoter completely restores Fos r to either the sigW mutant (HB0081) or, as expected, t...

Bacillus subtilis contains seven extracytoplasmic-function factors that activate partially overlapping regulons. We here identify four additional members of the X regulon, pbpX (penicillin-binding protein), ywnJ, the dlt operon (D-alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis). Modification of teichoic acids by esterification with D-alanine and incorporation of phosphatidylethanolamine into the cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the cell envelope. The resulting reduction in the net negative charge of the cell envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides. Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type cells. We conclude that activation of the X regulon serves to alter cell surface properties to provide protection against antimicrobial peptides.Bacillus subtilis encodes seven extracytoplasmic-function (ECF) factors. Most studies to date have focused on three:X , W , and M (reviewed in reference 19). sigX and its downstream gene rsiX (encoding the anti-X factor) were originally observed to be homologous (but not orthologous) to Escherichia coli fecI and fecR, which are involved in expression of ferric citrate transport genes (37). Although expression of sigX in E. coli can complement a fecI mutant (4), the B. subtilis sigX mutant is not affected in any known ferri-siderophore uptake systems (20).To understand the function of X , we identified several X -regulated genes using a consensus promoter search method (22). In these initial studies, we characterized six genes that are preceded by promoters recognized by X : sigX, abh (an AbrB homolog), csbB (a membrane-bound glucosyl transferase) (2), divIC (a membrane-bound cell-division initiation protein), lytR (a negative regulator of autolysin) (30), and rapD (a response regulator aspartate phosphatase) (43). These results suggested that X modulates aspects of cell envelope metabolism. Interestingly, most X -controlled genes are also transcribed by other forms of holoenzyme. For example, csbB has an additional B -dependent promoter, lytR and rapD both have additional A -dependent promoters, and sigX itself is preferentially transcribed from an upstream A -dependent site in addition to the X -dependent autoregulatory promoter (20,22). Moreover, in some cases (e.g., abh and divIC) the promoter activated by the E X holoenzyme can also be recognized by the E W holoenzyme at least in vitro (21). Similarly, the recently defined bcrC gene (a bacitracin resistance gene) is transcribed from a promoter that is recognized by either X or M (7, 38). The unknown function of many X -controlled genes makes it difficult to predict a phenotype for the sigX mutant. This challenge is exacerbated by the fact that many of the genes are expressed from multiple promoters or by multiple holoenzyme forms activating the same promoter. The latte...

Background Streptococcus suis serotype 2 (S. suis 2, SS2) has evolved into a highly infectious entity, which caused the two recent large-scale outbreaks of human SS2 epidemic in China, and is characterized by a toxic shock-like syndrome. However, the molecular pathogenesis of this new emerging pathogen is still poorly understood.Methodology/Principal Findings89K is a newly predicted pathogenicity island (PAI) which is specific to Chinese epidemic strains isolated from these two SS2 outbreaks. Further bioinformatics analysis revealed a unique two-component signal transduction system (TCSTS) located in the candidate 89K PAI, which is orthologous to the SalK/SalR regulatory system of Streptococcus salivarius. Knockout of salKR eliminated the lethality of SS2 in experimental infection of piglets. Functional complementation of salKR into the isogenic mutant ΔsalKR restored its soaring pathogenicity. Colonization experiments showed that the ΔsalKR mutant could not colonize any susceptible tissue of piglets when administered alone. Bactericidal assays demonstrated that resistance of the mutant to polymorphonuclear leukocyte (PMN)-mediated killing was greatly decreased. Expression microarray analysis exhibited a transcription profile alteration of 26 various genes down-regulated in the ΔsalKR mutant.Conclusions/SignificanceThese findings suggest that SalK/SalR is requisite for the full virulence of ethnic Chinese isolates of highly pathogenic SS2, thus providing experimental evidence for the validity of this bioinformatically predicted PAI.