Occurrence of the regulatory nucleotides ppGpp and pppGpp following induction of the stringent response in staphylococci

Abstract

The stringent response in Escherichia coli and many other organisms is regulated by the nucleotides ppGpp and pppGpp. We show here for the first time that at least six staphylococcal species also synthesize ppGpp and pppGpp upon induction of the stringent response by mupirocin. Spots corresponding to ppGpp and pppGpp on thin-layer chromatograms suggest that pppGpp is the principal regulatory nucleotide synthesized by staphylococci in response to mupirocin, rather than ppGpp as in E. coli.Bacteria adapt to amino acid or carbon source insufficiency by a complex series of regulatory events known as the stringent response (reviewed in reference 4). In Escherichia coli, for example, amino acid starvation leads to an accumulation of uncharged cognate tRNA. When the ratio of charged to uncharged tRNA falls below a critical threshold (24), occupation of the vacant mRNA codon at the ribosomal A site by uncharged cognate tRNA leads to stalling of peptide chain elongation and synthesis of the nucleotides pppGpp and ppGpp from GTP and ATP in an idling reaction involving RelA (2, 9, 13). Intracellular concentrations of (p)ppGpp are controlled by the relA gene, encoding the ribosome-dependent (p)ppGpp synthetase I, or stringent factor (19), and the spoT gene, encoding the ribosome-independent ppGpp 3Ј-pyrophosphohydrolase/ppGpp synthetase II (14,33).Induction of the stringent response rapidly reduces the synthesis of mRNA because of inhibition of RNA polymerase by ppGpp (28,31). This coincides with the down-regulation of a wide range of energetically demanding cellular processes, including the synthesis of stable RNA, DNA, protein, and peptidoglycan. Close coordination between transcription and translation can be maintained through the effects of the stringent response on the regulation of individual ribosomal proteins; elongation factors G, Tu, and Ts; certain aminoacyl tRNA synthetases; and stationary-phase-specific sigma factor ( s ) (11). Ultimately, changes such as these enhance survival in a nutrient-poor environment.The antibiotic mupirocin (30) produces cellular effects similar to those of isoleucine starvation by preventing the charging of tRNA Ile due to inhibition of isoleucyl tRNA synthetase in E. coli (16,17), Staphylococcus aureus (5), and other organisms, thereby inhibiting protein synthesis. In the presence of mupirocin, RNA synthesis in E. coli and S. aureus is also inhibited as a consequence of the stringent response; however, this inhibition can be reversed by chloramphenicol (15), which in E. coli, binds close to the binding site for the terminal CCA of aminoacyl tRNA in the peptidyl transferase A site (23) and reduces the amount of RelA-dependent (p)ppGpp synthesis (8, 10). A stringent response mediated by (p)ppGpp has been detected in some gram-positive and gram-negative bacteria and higher organisms after amino acid starvation or induction by a variety of antibiotics (7,18,26). However, in halobacteria (25) and streptococci (21), stringency is not necessarily coupled with (p)ppGpp production. Thus, in S...

Characterization of the stringent and relaxed responses of Streptococcus equisimilis

“…In amino acid-starved E. coli cells, the pppGpp/ppGpp ratio is generally smaller than 1 but varies widely depending on the nature of the spoT or gpp alleles (reviewed in reference 3). In similarly starved S. equisimilis cells, we find a pppGpp/ppGpp ratio of Յ0.1, in contrast to the relative quantities of the two nucleotides in Bacillus subtilis (14) and Staphylococcus aureus (6), where pppGpp has been reported to be the more abundant nucleotide during amino acid starvation. Tetracycline efficiently blocks the net accumulation of (p)ppGpp (Fig.…”

Staphylococcus aureus HrtA Is an ATPase Required for Protection against Heme Toxicity and Prevention of a Transcriptional Heme Stress Response

“…Comparison of the global expression profile of heme-exposed S. aureus ⌬hrtA with the global expression profile of S. aureus upon cold shock, heat shock, SOS induction, stringent response induction, acid shock, alkaline shock, Mn 2ϩ starvation, or Zn 2ϩ starvation indicated that the stringent response most closely resembles the heme response of S. aureus ⌬hrtA (37.4% similarity) ( Table 2) (1, 7). The bacterial stringent response is induced under carbon or amino acid insufficiency through ribosome stalling, which leads to an increase in the intracellular concentration of (p)ppGpp (6). This occurs through the induction of the stringent response regulatory factor RelA (SACOL1689), the transcript of which is up-regulated upon treatment of S. aureus with the stringent response-inducing antimicrobial agent mupirocin and is increased 9.2-fold in S. aureus ⌬hrtA exposed to heme (see Table S1 in the supplemental material) (1).…”

Characterization of the Staphylococcus aureus Heat Shock, Cold Shock, Stringent, and SOS Responses and Their Effects on Log-Phase mRNA Turnover

“…Mupirocin is an antimicrobial agent that induces the staphylococcal stringent response by inhibiting isoleucyl tRNA synthetase, thereby increasing the cellular concentration of uncharged tRNA Ile molecules (8,11). Using realtime PCR, we determined that 30 min of mupirocin treatment (60 g ml Ϫ1 ) increased the transcript titers of the stringent response control factor relA and the ilv operon (3-and Ͼ1,000-fold, respectively), whereas rpsL transcript levels were decreased (5-fold); cell viability was marginally affected (data not shown).…”