Enhanced expression of Bacillus subtilis yaaA can restore both the growth and the sporulation defects caused by mutation of rplB, encoding ribosomal protein L2.

A temperature-sensitive mutation in rplB, designated rplB142, encodes a missense mutation at position 142 [His (CAT) to Leu (CTT)] of Bacillus subtilis ribosomal protein L2. The strain carrying the mutation grew more slowly than the wild-type, even at low temperatures, probably due to the formation of defective 70S ribosomes and the accumulation of incomplete 50S subunits (50S* subunits). Gel analysis indicated that amounts of L2 protein and also of L16 protein were reduced in ribosomes prepared from the rplB142 mutant 90 min after increasing the growth temperature to 45 °C. These results suggest that the assembly of the L16 protein into the 50S subunit requires the native L2 protein. The H142L mutation in the defective L2 protein affected sporulation as well as growth, even at the permissive temperature. A suppressor mutation that restored both growth and sporulation of the rplB142 mutant at low temperature was identified as a single base deletion located immediately upstream of the yaaA gene that resulted in an increase in its transcription. Furthermore, genetic analysis showed that enhanced synthesis of YaaA restores the functionality of L2 (H142L) by facilitating its assembly into 50S subunits.

Transcription activity of individual rrn operons in Bacillus subtilis mutants deficient in (p)ppGpp synthetase genes, relA, yjbM, and ywaC.

In Bacillus subtilis a null mutation of the relA gene, whose gene product is involved in the synthesis and/or hydrolysis of (p)ppGpp, causes a growth defect that can be suppressed by mutation(s) of yjbM and/or ywaC coding for small (p)ppGpp synthetases. All 35 suppressor mutations newly isolated were classified into two groups, either yjbM or ywaC, by mapping and sequencing their mutations, suggesting that there are no (p)ppGpp synthetases other than RelA, YjbM, and YwaC in B. subtilis. In order to understand better the relation between RelA and rRNA synthesis, we studied in the relA mutant the transcriptional regulation of seven rRNA operons (rrnO, -A, -J, -I, -E, -D, or -B) individually after integration of a promoter- and terminatorless cat gene. We identified the transcriptional start sites of each rrn operon (a G) and found that transcription of all rrn operons from their P1 promoters was drastically reduced in the relA mutant while this was almost completely restored in the relA yjbM ywaC triple mutant. Taken together with previous results showing that the intracellular GTP concentration was reduced in the relA mutant while it was restored in the triple mutant, it seems likely that continuous (p)ppGpp synthesis by YjbM and/or YwaC at a basal level causes a decrease in the amounts of intracellular GTP.