Single mutations introduced in the essential ribosomal proteins L3 and S10 cause a sporulation defect in Bacillus subtilis.

We introduced single mutations into the rplC and rpsJ genes, which encode the essential ribosomal proteins L3 (RplC) and S10 (RpsJ), respectively, and are located in the S10 gene cluster of the gram-positive, endospore-forming bacterium Bacillus subtilis, and examined whether these mutations affected their growth rate, sporulation, competence development and 70S ribosome formation. Mutant cells harboring the G52D mutation in the L3 ribosomal protein, which is located at the peptidyl transferase center of 50S, accumulated 30S subunit at 45°C, probably due to a defect in 50S formation, and exhibited a reduction in the sporulation frequency at high temperature. On the other hand, mutant cells harboring the H56R mutation in the S10 protein, which is located near the aminoacyl-tRNA site of 30S, showed severe growth defect and deficiency in spore formation, and also exhibited significant delay in competence development.

Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp(0) mutant of Bacillus subtilis triggers YvyD-dependent dimerization of ribosome.

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-ß-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the ΔrelA ΔyjbM ΔywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.