Analysis of Promoter Recognition In Vivo Directed by ς F of Bacillus subtilis by Using Random-Sequence Oligonucleotides

Abstract

ABSTRACT 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 ς factors. Of these, ς F is the first to become active. Few genes have been identified that are transcribed by RNA polymerase containing ς F (E-ς F ), and only two genes of known function are exclusively under the control of E-ς F , spoIIR and spoIIQ . In order to investigate the features of promoters that are recognized by E-ς 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-ς F in vivo. The results indicate a weak ς F -specific −10 consensus, GG/tNNANNNT, of which the ANNNT portion is common to all sporulation-associated ς factors, as well as to ς A . There was a rather stronger −35 consensus, GTATA/T, of which GNATA is also recognized by other sporulation-associated ς factors. The looseness of the ς F promoter requirement contrasts with the strict requirement for ς A -directed promoters of B. subtilis . It suggests that additional, unknown, parameters may help determine the specificity of promoter recognition by E-ς F in vivo.