Transcriptional analysis of the ylaABCD operon of Bacillus subtilis encoding a sigma factor of extracytoplasmic function family

The ylaABCD operon of Bacillus subtilis contains four predicted ORFs in the order ylaA, ylaB, ylaC and ylaD, where ylaC is assumed to code for a sigma factor of the extracytoplasmic function (ECF) family. Predicted YlaD may function as the anti-YlaC factor as it has an oxidative stress sensing domain similar to that of the RsrA, which is the anti-sigma factor of SigR, an ECF sigma of Streptomyces coelicolor. Northern blot analysis of the ylaABCD operon revealed two transcriptional products resulting from a distal promoter upstream of ylaA and from an internal promoter located at the first codon of ylaC. Both transcription start sites were determine by primer extension and 5'-RACE PCR. The transcription from the distal. promoter was initiated by over-expression of YlaC on a multi-copy plasmid and depended on YlaC. DNA sequences of the -35 and -10 regions were similar to those recognized by other ECF sigmas of B. subtilis. On the other hand the transcription from the internal promoter was induced by oxidative stress and depended on Spx, which is an oxidative stress responding factor interacting with the alpha subunit of RNA polymerase core enzyme. The latter transcription depended possibly on SigA. We could not detect translation of YlaC from this transcript. Experiments with ylaD-disruption or co-overexpression of ylaD with ylaC suggested that YlaD functions as the anti-YlaC factor. Although YlaD has an oxidative stress sensing domain, oxidative stress did not induce the whole ylaABCD operon.