Transcription Activity of Individual rrn Operons in Bacillus subtilis Mutants Deficient in (p)ppGpp Synthetase Genes, relA, yjbM, and ywaC

Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and guanosine 5′-triphosphate 3′-diphosphate (pppGpp), generally referred to as (p)ppGpp, are produced in cells of many bacteria and plants when they encounter adverse environmental conditions such as amino acid starvation (4, 5). As a global regulator, (p)ppGpp is known to control several cellular processes including transcription, translation, nucleotide metabolism, and DNA replication (1, 12, 22, 36). In Escherichia coli two homologous enzymes, RelA and SpoT, are involved in the regulation of intracellular (p)ppGpp levels. RelA is a ribosome-associated (p)ppGpp synthetase responding mainly to uncharged tRNAs that accumulate as a result of amino acid limitation (4). SpoT is a bifunctional (p)ppGpp synthetase and hydrolase and regulates (p)ppGpp levels in response to limitation of carbon source, fatty acid, or iron (3, 32, 35, 40). In contrast to E. coli, many other bacteria were assumed to possess only one gene encoding a RelA-SpoT homolog, Rel or RelA, considered to be a bifunctional (p)ppGpp synthetase and hydrolase (23, 38). We have recently found that two small RelA homologues, YjbM and YwaC, are capable of synthesizing (p)ppGpp in Bacillus subtilis, one of the best-characterized gram-positive bacteria (26). The putative homologues of RelA, YjbM and YwaC, are found in Streptococcus mutans and many gram-positive bacteria (20, 26), suggesting that intracellular (p)ppGpp levels in these bacteria are controlled by these three enzymes although the detailed regulatory mechanisms remain unclear. During the course of characterizing a relA null mutant of B. subtilis, we found that this mutant strain grew more slowly than wild-type cells in LB medium (26). This growth defect could be suppressed by introduction of the yjbM and/or ywaC null mutation(s) or by expression of relA(D264G), encoding a RelA protein with a D264G mutation that abolishes (p)ppGpp synthetase activity (26). As this mutant RelA protein has normal hydrolase activity (26), these results suggest that the slow-growth characteristics of the relA null mutant could result from a slightly enhanced basal level of (p)ppGpp, which, however, was below the level of detection by our high-performance liquid chromatography system (26). (p)ppGpp binds directly to RNA polymerase and thereby inhibits the transcription of rRNA (rrn) operons, resulting in growth arrest (1, 2, 5, 6, 9, 29). Furthermore, it has been shown that increased levels of ppGpp caused by the relA mutation likely lead to a decrease in GTP pools, which inhibits rRNA operon promoter activity due to the reduced availability of initiating GTP (17, 30). Therefore, we studied the regulation of transcription for each individual rRNA operon in the relA mutant. Seven novel strains were constructed, each carrying a promoter- and terminatorless cat gene within either of the rRNA operons rrnO, -A, -J, -I, -E, -D, or -B to monitor their transcription activity. Using these strains, we experimentally determined all transcription start sites from promoters of seven individual rRNA operons and assessed the effects of the relA gene disruption and its suppressor mutations on the transcription activity of these rrn operons in B. subtilis.