SummaryThe Bacillus subtilis s s s s W regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis.

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The chromosome of Bacillus subtilis codes for seven extracytoplasmic function sigma factors the activity of which is modulated normally by a cognate anti-sigma factor. While inducing factors and genes for four of them (sigma(M), sigma(W), sigma(X), and sigma(Y)) have been identified, those of the remaining three sigma factors including sigma(V) remain elusive. The objective of the present study was the unequivocal identification of its anti-sigma factor and of genes controlled by sigma(V). In many cases reported so far the gene coding for the anti-sigma factor is located immediately downstream of the gene coding for the sigma factor, and both form a bicistronic operon. We could show by two different experimental approaches that this is also the case for sigV and rsiV. Under conditions of overproduction of sigma(V), 13 genes could be identified being induced several-fold by the DNA macroarray technique. Induction of three of them was confirmed by Northern blots, and the potential promoter of sigV was identified by primer extension. This led to the deduction of a consensus sequence recognized by sigma(V).

FtsH is a membrane-bound and energy-dependent metalloprotease in bacteria which is involved in the posttranslational control of the activity of a variety of important transcription factors and in the degradation of uncomplexed integral membrane proteins. For Bacillus subtilis, little is known about the target proteins of FtsH protease. Its gene is not essential, but knockout strains display a pleiotropic phenotype including sensitivity toward salt and heat stress, defects in sporulation and competence, and largely filamentous growth. Comparison of the intracellular proteomes of wild-type and ftsH knockout strains revealed that at least nine proteins accumulated in the absence of ftsH, four of which could be identified. Two of these proteins turned out to be members of the W regulon. Accumulation of one of these W -controlled proteins, the penicillin-binding protein PBP4*, was analyzed in more detail. We could show that PBP4* is not a proteolytic substrate of FtsH and that its overproduction is due to the enhanced transcription of its gene (pbpE) in ftsH null mutants. The filamentous growth phenotype of ⌬ftsH strains was abolished in a ⌬ftsH ⌬pbpE double knockout. In ftsH wild-type strains with the pbpE gene under regulatable control, pbpE overexpression caused filamentation of the cells. DNA macroarray analysis revealed that most genes of the W regulon are transcribed at elevated levels in an ftsH mutant. The influence of FtsH on W -controlled genes is discussed.FtsH belongs to the AAA (ATPases associated with diverse cellular activities) protein superfamily, and members of this family are involved in a wide variety of cellular processes, such as vesicle-mediated protein transport, cell cycle control, control of gene expression, and proteolysis (for recent reviews, see references 19 and 35). The AAA proteins constitute a subfamily of the Walker-type nucleoside triphosphatases and contain conserved ATPase domains, typically spanning 200 to 250 residues, referred to as AAA modules (6,20). FtsH is a member of the AAA proteases, which occur in eubacteria and eukaryotic organelles such as mitochondria and chloroplasts, but not in archaea, and contain an AAA module and a zinc metalloprotease domain in a single polypeptide chain. Bacterial FtsH proteins are anchored via two transmembrane segments in the cytoplasmic membrane, with the short N-and the long Cterminal parts facing the cytoplasm. These metalloproteases are involved in the quality control of membrane-bound proteins and in the degradation of short-lived cytoplasmic regulatory proteins (for recent reviews, see references 16 and 26).While several target proteins of the Escherichia coli FtsH metalloprotease have already been identified, they remained elusive in Bacillus subtilis. Therefore, we reasoned that comparing the proteomes of B. subtilis wild-type and ftsH knockout cells should reveal target proteins accumulating in the absence of the protease. It should be mentioned that, in contrast to that of E. coli, the B. subtilis ftsH gene is dispensable, though i...