The Rok protein of Bacillus subtilis represses genes for cell surface and extracellular functions

Abstract

Rok is a repressor of the transcriptional activator ComK and is therefore an important regulator of competence in Bacillus subtilis (T. T. Hoa, P. Tortosa, M. Albano, and D. Dubnau, Mol. Microbiol. 43:15-26, 2002). To address the wider role of Rok in the physiology of B. subtilis, we have used a combination of transcriptional profiling, gel shift experiments, and the analysis of lacZ fusions. We demonstrate that Rok is a repressor of a family of genes that specify membrane-localized and secreted proteins, including a number of genes that encode products with antibiotic activity. We present evidence for the recent introduction of rok into the B. subtilis-Bacillus licheniformis-Bacilllus amyloliquefaciens group by horizontal transmission.

FIG. 1.

Genetic maps of rok-regulated gene clusters. Genes are indicated with arrows, indicating the direction of transcription. Black arrows indicate genes that meet the criteria for rok regulation, as described in the text; grey arrows indicate genes that nearly met these criteria but are classified as rok regulated, based on their proximity to regulated genes; white arrows indicate genes that are judged as not rok regulated, based on the transcription profiling experiments. yybN is in brackets, since the transcription profiling for this gene was inconclusive (see the text). Dotted arrows indicate putative rok-regulated transcription units. Hairpins indicate terminators as derived from the Subtilist database (http://genolist.pasteur.fr/SubtiList/). The sizes in the figure are not proportional to the lengths of the open reading frames or intergenic regions.

FIG. 2.

Validation of transcriptional profiling results using lacZ fusions. Strains carrying fusions of lacZ to candidate rok-regulated genes were grown in competence medium, and samples were withdrawn at the indicated times for measurement of β-galactosidase specific activities. T₀ refers to the time of departure from exponential growth. Each fusion was placed in both rok (▪) and rok⁺ (•) backgrounds for determination of β-galactosidase.

FIG. 3.

Gel retardations. DNA fragments from the indicated genes were prepared by PCR and end labeled with ³²P. In each panel the lane marked with an x corresponds to probe alone. The remaining lanes correspond to probe samples incubated with Rok-His₆ at concentrations ranging from 7 to 1,792 nM. Each successive lane, from left to right, corresponds to a doubling in the concentration of Rok-His₆. The arrows indicate the positions of the unshifted probe fragments. In each case, the unshifted band that migrated slower than the probe probably corresponds to single-stranded DNA, due to slightly unequal concentrations of PCR primers. This material is not shifted by Rok-His₆.

FIG. 4.

Relationships among group 1 bacilli (adapted from reference 2). Species in which a rok ortholog have been identified are boxed, and those in which it is known to be absent are underlined.