Genetic composition of the Bacillus subtilis SOS system

Abstract

The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5'-CGAACATATGTTCG-3'. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.

FIG. 1.

Sequence requirements for LexA binding. The preferred half site sequence based on a thermodynamic analysis of LexA binding to recA operator mutants. Base substitutions labeled as destabilizing abolish LexA binding to the recA operator (Groban et al., submitted).

FIG. 2.

Binding of B. subtilis LexA to potential SOS promoters. Mobility shift assays were conducted with purified LexA, radiolabeled recA promoter DNA (5 to 10 nM), and a 5- to 50-fold molar excess of the indicated promoter DNA as described in Materials and Methods. The lower and upper bands correspond to unbound and LexA-bound recA promoter DNA, respectively. Lanes with no LexA protein or competitor DNA added are indicated.

FIG. 3.

Binding of B. subtilis LexA to the recA promoter. Graphical analyses of mobility shift titration of ³²P-labeled recA promoter (10 nM) incubated with increasing concentrations of LexA as described in Materials and Methods.

FIG. 4.

Binding of B. subtilis LexA to the lexA and yqjW promoters. Mobility shift assays were conducted with purified LexA (0 to 96 nM) and radiolabeled lexA (12 nM) or yqjW (12 nM) promoter DNA as described in Materials and Methods.

FIG. 5.

Genetic map locations of B. subtilis SOS genes. Primary (black) and secondary (gray) SOS genes are indicated, with arrows depicting the direction of transcription.