Cloning and Characterization of a Cluster of Linked Bacillus subtilis Late Competence Mutations

Abstract

We characterized a segment of chromosomal DNA from Bacillus subtilis that was required for the development of genetic competence. The chromosomal DNA was cloned from a group of genetically linked and phenotypically similar Tn9171ac insertion mutants deficient in competence. This cluster of mutations defined the comG locus. Chromosomal DNA flanking each of the six insertions was cloned. Restriction maps of the cloned plasmids revealed that their chromosomal inserts consisted of overlapping fragments. These data, together with Southern blots of chromosomal DNA from the comG mutants, showed that the six Tn9171ac comG insertions occurred in the following order: comG12, comG39, comG412, comG107, comG56, and comG210. Expression of the comG Tn9171ac insertions was from a promoter located upstream from the first insertion, comG12. This was determined genetically and by low-resolution S1 nuclease mapping of the 3 ' terminus. The comG region spanned about 5 kilobase pairs, based on low-resolution S1 nuclease mapping of the transcription terminator and Northern blotting. The comG12 mutation had a partial epistatic effect on the expression of one other com locus, comE, but none of the other comG mutations affected expression of this or any other com gene tested. Based on these conclusions, and on its size and phenotype, the comG locus must be organized as a polycistronic operon that is subject to competence-specific regulation. Genetic competence in Bacillus subtilis is an example of