Suppression of ctc promoter mutations in Bacillus subtilis

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Abstract
Transcription from the Bacillus subtilis ctc promoter is induced as cells enter stationary phase under conditions in which the enzymes of the tricarboxylic acid cycle are repressed. This transcription requires the presence of a secondary form of RNA polymerase, E sigma B, that is found in exponentially growing cells and in early-stationary-phase cells. Starting with a defective ctc promoter that had either a base substitution at position -15 or a base substitution at position -36, we were able to identify four independent second-site mutations within these mutated promoters that suppressed the effect of the original mutations and thereby restored function to the ctc promoter. Three of these mutated promoters had an additional base substitution(s) at positions -5, -9, or both -5 and -9 that enhanced their utilization in vivo by E sigma B, whereas one of the promoters had a single-base-pair deletion in the -15 region that placed it under a completely different form of regulation than that of the wild-type ctc promoter. In addition to mutations in the ctc promoter region, we also isolated three classes of mutants that exhibited increased ctc expression. The effects of the mutations in these strains were not allele specific, since they increased expression from both mutant and wild-type ctc promoters. One class of mutants which affected expression from the ctc promoter carried mutations that blocked the activity of the tricarboxylic acid cycle. A second class of mutations mapped near cysA and was unable to sporulate. Three-factor transformation crosses and complementation analysis indicated that one of these mutations was an allele of spo0H. The third class of mutations is closely linked to dal and may define a regulatory gene for sigB, the sigma B structural gene.