Fig 10. Effects of Rho over-expression in B. subtilis cells.

(A) Over-expression of Rho reinforces Rho-dependent termination within kinB gene. The analysis of chloramphenicol-resistance of the BSB1 WT cells containing pKinB-L or pKinB-S plasmids described in Fig 8C was reproduced in the presence of Rho over-producing plasmid pRho or the control p148 vector; the applied concentration of Cm 3μg/ml. The experiment was repeated three times with nine biological replicas of each strain. (B) Over-expression of Rho suppresses synthesis of KinB in B. subtilis BSB1 WT cells. B. subtilis cells containing kinB-SPA translational fusion at natural chromosomal loci and pRho or p148 plasmids were grown in sporulation-inducing DS medium at 37°C to indicated OD₆₀₀ and analyzed for KinB-SPA protein as described in Materials and Methods. To control equilibrium between the samples, total protein extracts were analyzed for Mbl protein using anti-Mbl specific antibodies. The experiment was reproduced twice. (C and D) Over-expression of Rho reduces sporulation. (C) Kinetics of luciferase expression from spoIIA-luc fusion in BSB1 WT (blue squares) and RM (red circles) cells in the presence of pRho (filled-in symbols) or p148 (opened symbols) plasmids. The experiments were performed as in Fig 6 three times. The results from the representative experiment are shown. (D) Analysis of the sporulation efficiency of B. subtilis BSB1 WT, BSB1 kinA and BSB1 kinB mutant cells containing pRho or p148 plasmids was performed as in Fig 7C after 20 hours of incubation in DS medium. Average values and standard deviations from three independent experiments are multiplied by 100 and plotted in a log10 scale. In total, twelve biological replicas of each strain were analyzed. (E) Colony biofilm formation by B. subtilis NCIB 3610 WT and RM cells containing pRho plasmid or p148 vector. Relevant genotypes are indicated on the side of each image. Each icon represents image of individual colonies grown on MSgg agar medium for 72h at 30°C. The experiment was reproduced at least five times. The results from the representative experiment are presented. (F and G) Over-expression of Rho reinforces cells motility. (F) Swarming motility of NCIB 3610 WT and NCIB 3610 RM containing pRho plasmid or p148 vector was assayed as in Fig 3 using LB 0.7% agar plates. The images were acquired after 20 hours of incubation at 37°C. The experiment was performed five times. The results from the representative experiment are shown. (G) Quantitative swarming assay of the NCIB 3610 (blue) and NCIB 3610 RM (red) strains containing pRho plasmid (solid lines; filled-in triangles) or p148 vector (dotted lines; empty triangles). Values represent the mean of five experiments including two replicas for each strain.