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.

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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.

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...