Transcription Activity of IndividualrrnOperons inBacillus subtilisMutants Deficient in (p)ppGpp Synthetase Genes,relA,yjbM, andywaC

Abstract

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.

Towards an Elucidation of the Roles of the Ribosome during Different Growth Phases inBacillus subtilis

“…The first is RelA, a bi-functional (p)ppGpp synthetase and hydrolase, and the other two are small (p)ppGpp synthetases, YjbM and YwaC, both of which contain only the (p)ppGpp synthetase domain. 38) We also found that the transcription of all rrn operons from their P1 promoters is drastically reduced in the relA mutant, whereas this transcription is almost completely normal in the relA yjbM ywaC triple mutant, 39) but the contribution of the two small (p)ppGpp synthetases to the transcription of rRNA remains unclear. Elucidation of the biological significance of these two small (p)ppGpp synthetases can help in characterizing the transcriptional regulation of rRNA during sporulation in the near future.…”

“…Using this system, we successfully identified the transcriptional start sites of the various rrn operon promoters, which had been only partly determined previously. 39) In addition, this system was valuable for analyzing the effects of simultaneous deletion of the three genes that encode ppGpp synthetases on the transcription of each rRNA operon. 39) Although further investigations are required, we believe that by using these systems, it should be possible to elucidate the mechanism of transcription of each rRNA operon during sporulation completely in the near future.…”

“…To elucidate the mechanism of transcription of rRNA operons during sporulation, we inserted a truncated chloramphenicol resistance gene (catpt1), which lacked both its promoter and a rho-independent transcriptional terminator, downstream of the known P2 or P3 promoter regions in seven rrn operons (rrnO, -A, -J, -I, -E, -D, and -B) and upstream of sites where the primary transcript is cleaved during the post-transcriptional processing events that generate mature 16S, 23S, and 5S rRNAs. 39) Then we performed primer extension analyses on the RNA isolated from these strains using a labeled reverse primer that was complementary to the 5 0 region of the inserted cat gene. Using this system, we successfully identified the transcriptional start sites of the various rrn operon promoters, which had been only partly determined previously.…”

From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

“…There is emerging evidence that SASs make important contributions to bacterial physiology despite the dominant role of Rel in SR activation (23). For example, SASs were shown to be required for fully efficient SR induction, GTP homeostasis, growth rate control, tolerance to cell wall antibiotics, and virulence (10,21,(24)(25)(26)(27)(28)(29). Moreover, the stable genomic coexistence of the bifunctional Rel with one or two SASs in a large number of bacteria indicates an evolutionary pressure that favors retention of the Rel-SAS (p)ppGpp synthetase redundancy.…”

Many Means to a Common End: the Intricacies of (p)ppGpp Metabolism and Its Control of Bacterial Homeostasis

“…The link between (p)ppGpp and CodY in Firmicutes has been recently reviewed (23) and will be only briefly discussed in this article. Col-lectively, these results indicate that (p)ppGpp indirectly controls the action of RNAP in Firmicutes through modulation of intracellular purine concentrations (19,24,25).…”