AbrB is a regulator of the sigma(W) regulon in Bacillus subtilis

The Bacillus subtilis global regulator AbrB was found to negatively control expression of sigW and genes of the sigma(W) regulon. AbrB bound to DNA regions in the autoregulatory sigW promoter and to some, but not all, of the other sigma(W)-dependent promoters in B. subtilis. Defects in antibiotic resistance properties caused by spo0A mutations are at least partially correlated with AbrB repression of the sigma(W) regulon.     

The single extracytoplasmic-function sigma factor of Xylella fastidiosa is involved in the heat shock response and presents an unusual regulatory mechanism

Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA, predicted to encode an extracytoplasmic function (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa sigma(E) regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 sigma(E)-dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative sigma(E)-dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like other ECF sigma factors, rpoE and rseA were shown to comprise an operon in X. fastidiosa, together with a third open reading frame (XF2241). However, upon heat shock, rpoE expression was not induced, while rseA and XF2241 were highly induced at a newly identified sigma(E)-dependent promoter internal to the operon. Therefore, unlike many other ECF sigma factors, rpoE is not autoregulated but instead positively regulates the gene encoding its putative anti-sigma factor.     

Identification of Bacillus subtilis sigma-dependent genes that provide intrinsic resistance to antimicrobial compounds produced by Bacilli

Bacillus subtilis produces many antibiotics of varying structures and specificity. Here we identify a prominent role for sigma(W), an extracytoplasmic function (ECF) sigma factor, in providing intrinsic resistance to antimicrobial compounds produced by other Bacilli. By using a panel of B. subtilis mutants disrupted for each of the 30 known sigma(W)-dependent operons we identified resistance genes for at least three different antimicrobial compounds. The ydbST and fosB genes contribute to resistance to antimicrobial compound(s) produced by B. amyloliquefaciens FZB42, the yqeZyqfAB operon provides resistance to the SPbeta prophage-encoded bacteriocin sublancin, and the yknWXYZ operon and yfhL provide resistance to the antimicrobial peptide SdpC. YfhL encodes a paralogue of SdpI, a membrane protein that provides immunity to SdpC. In competition experiments, we identify sigma(W) as a key factor in allowing B. subtilis to resist antibiotic killing and encroachment by competing strains. Together with the previous observation that sigma(W) provides inducible resistance against the Streptomyces antibiotic fosfomycin, these studies support the notion that sigma(W) controls an antibiosis regulon important in the microbial ecology of soil bacteria.     

The Bacillus subtilis sigmaW anti-sigma factor RsiW is degraded by intramembrane proteolysis through YluC

The Bacillus subtilis sigma(W) regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis. The activity of the alternative, extracytoplasmic function (ECF) sigma factor sigma(W) is modulated by a specific anti-sigma factor (RsiW or YbbM) encoded by the rsiW (ybbM) gene located immediately downstream of sigW. The RsiW membrane topology was determined, and a specific reporter system for RsiW function was constructed. Experiments using the yeast two-hybrid system suggested a direct interaction of sigma(W) with the cytoplasmic part of RsiW. Analysis of truncated forms of the RsiW protein revealed that sigma(W) induction by alkaline shock is dependent on both the transmembrane and the extracytoplasmic domain of RsiW. Western blot and pulse-chase experiments demonstrated degradation of RsiW after an alkaline shock. A B. subtilis mutant strain deleted for the Escherichia coli yaeL orthologue yluC, encoding a transmembrane protease, was defective in inducing a sigma(W)-controlled promoter after alkaline shock and accumulated a membrane-bound truncated form of RsiW, suggesting that the activity of sigma(W) is controlled by the proteolysis of RsiW by at least two different proteolytic steps.     

S-adenosylmethionine activates adpA transcription and promotes streptomycin biosynthesis in Streptomyces griseus

Using a two-plasmid system, we recently identified sigma(E)-dependent promoters directing expression of the sigma(E) regulon genes in Salmonella enterica serovar Typhimurium (S. Typhimurium). Comparison of the promoters revealed a consensus sequence almost identical to the sigma(E)-dependent rpoEp3 promoter directing expression of rpoE. This two-plasmid system was previously optimized to identify nucleotides critical for the rpoEp3 promoter activity. However, two highly conserved nucleotides in the sigma(E) consensus sequence were not identified by this screening. In the present study, we have improved the two-plasmid screening system using a new optimized error-prone PCR mutagenesis. Together with site-directed mutagenesis, we further identified nucleotides critical for activity of the rpoEp3 promoter and quantified the effect of the particular mutation upon promoter activity. All the identified critical nucleotides of the rpoEp3 promoter (in capital) were located in the -35 (ggAACtt) and -10 (gTCtaA) regions and corresponded to the most conserved nucleotides in the sigma(E) consensus sequence. The expression of the wild-type and mutated rpoEp3 promoters was confirmed in S. Typhimurium and was found to exhibit a different pattern of sigma(E) activation compared with Escherichia coli, with a peak rpoEp3 promoter activity in early stationary phase followed by a decrease in late stationary phase.