Analysis of a suppressor mutation ssb (kinC) of sur0B20 (spo0A) mutation in Bacillus subtilis reveals that kinC encodes a histidine protein kinase

Abstract

sur0B20 is a mutation that suppresses the effects of spo0B delta B or spo0F221 mutations in Bacillus subtilis, sur0B20 is an allele of the spo0A gene (Glu-14 to Val-14 conversion) and restores the sporulation of spo0B or spo0F mutants to the wild-type level. Here, we report the isolation of suppressor mutations of sur0B20 (ssb). One of these mutations, ssb-12, severely impairs the suppressor activity of sur0B20. A 2.5-kbp MboI fragment which complements the ssb-12 mutation was cloned by the prophage transformation method using phi CM as a vector. Nucleotide sequencing of the fragment revealed two open reading frames (orf1 and orf2). Gene disruption and complementation experiments showed that orf2 is the ssb gene. ssb was shown to encode a protein with a molecular weight of 48,846 (428 amino acid residues) showing strong similarity to transmitter kinases, especially KinA, of two-component regulatory systems. Therefore, ssb was renamed kinC. Deletion of kinC had no observable effect on sporulation. kinC transcription was induced at the onset of sporulation, probably from a sigma A-dependent promoter, and its expression was shut off at T3. DNase I protection experiments showed that the Spo0A protein binds to two adjoining sites in the kinC promoter region with different affinities. These results suggest that kinC expression might be regulated by Spo0A.