Characterization of the SOS regulon of Caulobacter crescentus

Abstract

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.

FIG. 1.

Phenotypic characterization of the C. crescentus lexA strain. (A and B) Growth curve of the wild-type NA1000 and lexA strains. Cells were grown in PYE medium at 30°C and monitored for up to 9 h (CFU counting) and 24 h (OD₆₀₀). OD₆₀₀ determination (A) and CFU determination (B). Error bars indicate standard errors. Solid lines, wild-type NA1000 strain; dotted lines, the lexA strain. (C to E) Light microscopy of C. crescentus strains using a 100× objective. Wild-type NA1000 strain (C), the lexA strain (D), and the lexA strain after genotypic complementation with a low-copy-number vector containing the wild-type allele of lexA (E).

FIG. 2.

Caulobacter crescentus LexA binding site model. The sequence logo was generated using the WebLogo program (14).

FIG. 3.

Site-directed mutagenesis of the imuA promoter and analysis of promoter activity in β-galactosidase assays. (A) The SOS box in the promoter of the imuA gene is shown, with the conserved, directed repeats shown in bold. The underlined bases were altered in PimuAO^c, eliminating the directed repeat. (B) The chart shows the average of three β-galactosidase assays performed with the wild-type (wt) and lexA strains with plasmids pP3213 and pP3213Oc, containing PimuA and PimuAO^c, respectively. Induction of the SOS response was achieved by the irradiation of cells with 45 J/m² of UVC.

FIG. 4.

Determination of the transcriptional start sites of imuA and CC_2272. The sequence around the predicted transcriptional start site is shown, with the coding sequence highlighted in bold. The transcriptional start sites are shown inside the black boxes and in italics. The SOS box is shown in gray shading, and the conserved −35 and −10 sequences are underlined. The consensus promoter for the vegetative sigma factor (34) is shown at the bottom.