<i>B</i>. <i>subtilis</i> Rho⁺ exhibits competence–negative phenotype.

(A) Schematics of PcomK regulation in B. subtilis cells. The promoter of the comK gene (PcomK) encoding the master regulator of competence is regulated by five transcription factors: Spo0A, Rok, AbrB, CodY, and DegU, and by ComK itself. The gradual increase in Spo0A∼P first induces the PcomK promoter activity directly (topmost arrow) and by antagonizing the transcriptional repressors AbrB and Rok, but represses it when concentration of Spo0A~P reaches a high threshold level (bottommost bar-headed line). Along with action on the PcomK, AbrB represses the promoter of rok. ComK activates its own expression by direct binding to PcomK assisted by the response regulator DegU. Eventually, an increase of ComK leads to the activation of the expression of DNA-binding, -uptake and -recombination genes that determines development of the competence state. Arrows and bar-headed lines represent positive and negative effects, respectively. (B) Kinetics of competence development in B. subtilis WT (blue line), Rho+ (red line) and Rho+Q146Stop (red double-line) strains. Cells grown in a defined rich medium SpC to stationary phase were transferred to competence medium (T0) and tested for transformation by homologous genomic DNA over three hours as described in Materials and Methods. The experiment included three biological replicas of each strain and was reproduced three times. Plotted are the mean values and SD from a representative experiment. (C) Kinetics of luciferase expression from the promoter of comK gene in B. subtilis WT (blue line) and Rho+ (red line) cells. Cells bearing the PcomK-luc transcription fusion were grown in competence medium and analyzed for luciferase activity as described in Materials and Methods. Plotted are the mean values of luminescence readings corrected for OD from four independent cultures of each strain analyzed simultaneously. The double-lined curves depict characteristic growth kinetics of cells measured by OD600. (D) Kinetics of luciferase expression in B. subtilis mutant strains: abrB rok PcomK-luc (purple line) and Rho+ abrB rok PcomK-luc (purple double-line). The mutants were analyzed in parallel with the parental strains WT PcomK-luc (blue line) and Rho+ PcomK-luc (red line). For each strain, data acquisition and processing were performed as in (C). Each strain was analyzed at least three times. The results of representative experiments are shown. (E) Inactivation of the known repressors of comK does not rescue competence–negative phenotype of Rho+ cells. B. subtilis WT and Rho+ strains and their respective derivatives carrying single mutations in the abrB, rok, codY genes or double abrB rok mutation were transformed by donor genomic DNA after two hours of growth in competence medium as described in Materials and Methods. Shown are the mean values with SD (in parentheses) from two independent experiments each incorporating three biological replicas of each strain. (F and G) De-repression of comK leads to the activation of the late competence genes in Rho+ cells. (F) The WT PcomGA-gfp, Rho+ PcomGA-gfp strains and their abrB rok mutant derivatives were grown in competence medium up to stationary phase (OD600 1.5). Cells were sampled two hours after entering stationary phase and analyzed by fluorescence microscopy for the activity of PcomGA-gfp reporter in two replicas. (G) The proportion of GFP-expressing cells was determined by manual counting of a minimum 600 cells per strain and per replica. The experiment was reproduced twice. Plotted are the mean values and SD from a representative experiment. Statistical significance was estimated with a two-tailed t-test. The displayed p-values are as follows: ***, p ≤ 0.001; **, p ≤ 0.01; *, 0.01 ≤ p ≤ 0.05.