Genetic method to identify regulons controlled by nonessential elements: isolation of a gene dependent on alternate...

We describe a general, in vivo method for identifying Bacillus subtilis genes controlled by specific, nonessential regulatory factors. We establish the use of this approach by identifying, isolating, and characterizing a gene dependent on sigma B, an alternate transcription factor which is found early in stationary phase but which is not essential for sporulation. The method relies on two features: (i) a plate transformation technique to introduce a null mutation into the regulatory gene of interest and (ii) random transcriptional fusions to a reporter gene to monitor gene expression in the presence and absence of a functional regulatory product. We applied this genetic approach to isolate genes comprising the sigma B regulon. We screened a random Tn917lacZ library for fusions that required an intact sigma B structural gene (sigB) for greatest expression, converting the library strains from wild-type sigB+ to sigB delta::cat directly on plates selective for chloramphenicol resistance. We isolated one such fusion, csbA::Tn917lacZ (csb for controlled by sigma B), which mapped between hisA and degSU on the B. subtilis chromosome. We cloned the region surrounding the insertion, identified the csbA reading frame containing the transposon, and found that this frame encoded a predicted 76-residue product which was extremely hydrophobic and highly basic. Primer extension and promoter activity experiments identified a sigma B-dependent promoter 83 bp upstream of the csbA coding sequence. A weaker, tandem, sigma A-like promoter was likewise identified 28 bp upstream of csbA. The csbA fusion was maximally expressed during early stationary phase in cells grown in Luria broth containing 5% glucose and 0.2% glutamine. This timing of expression and medium dependence were very similar to those for ctc, the only other recognized gene dependent on sigma B.