The Rho⁺ strain exhibits sporulation-negative phenotype.

(A) Sporulation efficiency of B. subtilis WT, Δrho and Rho+ cells. Cells inoculated at OD600 0.025 were grown in DS medium at 37° for 20 hours and analyzed for heat-resistant spores as described in Materials and Methods. Sporulation efficiency was estimated as proportion of viable cells in the heated and unheated cultures. Plotted are the mean values from four independent experiments with three biological replicas for each strain with standard deviation SD (bar-headed lines). (B) Schematics of the multicomponent Spo0A phosphorelay. Only the key elements relevant to this study are shown. Phosphoryl groups are transferred from sensor protein kinases (KinA-E) to Spo0F, Spo0B, and ultimately to Spo0A. Sporulation is triggered when the level of Spo0A∼P reaches a high threshold level. The bar-headed line indicates negative Rho-mediated regulation of KinB expression. (C) Kinetics of luciferase expression from the promoter of early sporulation gene spoIIAA in B. subtilis WT (blue line) and Rho+ (red line) cells induced for sporulation. (D) Kinetics of luciferase expression from the promoters of spo0A gene in WT (blue line), Rho+ (red line) and sigH mutant (green line) cells induced for sporulation. In C and D, cells bearing transcriptional fusions spoIIAA-luc and spo0A-luc, respectively, were grown in DS medium and analyzed for luciferase activity at five-minute intervals in a multimode microplate reader as described in Materials and Methods. For each strain, plotted are the mean values of luminescence readings corrected for OD600 from four independent cultures analyzed simultaneously (solid lines with symbols) and characteristic growth curves (double-lined) measured by OD600. The experiments were reproduced at least three times. The results from the representative experiments are shown. (E and F) Synthetic over-production of sensor histidine kinases KinA or KinC does not rescue sporulation-negative phenotype of Rho+ cells. Sporulation efficiency of B. subtilis WT and Rho+ strains and their respective derivatives expressing kinA (E) and kinC (F) genes under control of the IPTG-inducible promoter. Cells were inoculated at OD600 0.025 in DS medium containing IPTG at the indicated concentrations and grown at 37° during 20 hours; sporulation efficiency was analyzed as described above (Fig 2A) and in Materials and Methods. Plotted are the mean values from three independent experiments with three biological replicas of each strain with standard deviation SD (bar-headed lines). Statistical significance was estimated with a two-tailed t-test. The displayed p-values are as follows: ****, p ≤ 0.0001; ***, p ≤ 0.001; **, p ≤ 0.01.