Transcriptional regulation of comC: evidence for a competence-specific transcription factor in Bacillus subtilis

Abstract

comC specifies a protein product that is required for genetic competence in Bacillus subtilis. The probable transcriptional start site of comC has been localized by high-resolution primer extension analysis and shown to be preceded by an appropriately positioned sequence that resembles the consensus promoter for the sigma A form of RNA polymerase. Low-resolution S1 nuclease transcription mapping was used to identify the comC terminator, which is located near a palindromic element recognizable in the DNA sequence. Deletion analysis of the sequence upstream from the likely promoter identified a region required in cis for the expression of comC. An overlapping, and possibly identical, sequence was shown to inhibit the expression of competence and of several late competence genes, when present in multiple copies. This was interpreted as due to the titration of a positively acting competence transcription factor (CTF) by multiple copies of the promoter-bearing fragment. In crude lysates of B. subtilis grown to competence, a DNA-binding activity that appeared to be specific for the comC promoter fragment was detected by gel retardation assays. This activity, postulated to be due to CTF, was detected only following growth in competence medium, only in the stationary phase of growth, and was dependent on the expression of ComA, a known competence-regulatory factor. In the presence of the mecA42 mutation, the ComA requirement for CTF activity was bypassed, and CTF activity could be detected in lysates prepared from a strain grown in complex medium. This behavior suggested that either the expression or the activation of CTF was regulated in a competence-specific manner. Comparison of the putative CTF-binding site defined by deletion analysis with a similarly positioned sequence upstream from the start site of the late competence gene comG revealed that both sequences contained palindromes, with 5 of 6 identical base pairs in each arm. It is suggested that these palindromic sequences comprise recognition elements for CTF binding and that CTF binding must occur for the appropriate expression of late competence genes.