Analysis of promoter recognition in vivo directed by σF of Bacillus subtilis by using random-sequence oligonucleotides

Formation of spores from vegetative bacteria by Bacillus subtilis is a primitive system of cell differentiation. Critical to spore formation is the action of a series of sporulation-specific RNA polymerase a factors. Of these, sigma (F) is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing sigma (F) (E-sigma (F)), and only two genes of known function are exclusively under the control of E-sigma (F), spoIIR and spoIIQ. In order to investigate the features of promoters that are recognized by E-sigma (F), We studied the effects of randomizing sequences for the -10 and -35 regions of the promoter for spoIIQ. The randomized promoter regions were cloned in front of a promoterless copy of lacZ in a vector designed for insertion by double crossover of single copies of the promoter-lacZ fusions into the amyE region of the B. subtilis chromosome. This system made it possible to test for transcription of lacZ by E-sigma (F) in vivo. The results indicate a weak sigma (F)-specific -10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated a factors, as well as to sigma (A). There was a rather stronger -35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated a factors. The looseness of the sigma (F) promoter requirement contrasts with the strict requirement for sigma (A)-directed promoters of B. subtilis. It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-sigma (F) in vivo.