Characterization of the stringent and relaxed responses of Streptococcus equisimilis

Abstract

The 739-codon rel Seq gene of Streptococcus equisimilis H46A is bifunctional, encoding a strong guanosine 3,5-bis(diphosphate) 3-pyrophosphohydrolase (ppGppase) and a weaker ribosome-independent ATP:GTP 3-pyrophosphoryltransferase [(p)ppGpp synthetase]. To analyze the function of this gene, (p)ppGpp accumulation patterns as well as protein and RNA synthesis were compared during amino acid deprivation and glucose exhaustion between the wild type and an insertion mutant carrying a rel Seq gene disrupted at codon 216. We found that under normal conditions, both strains contained basal levels of (p)ppGpp. Amino acid deprivation imposed by pseudomonic acid or isoleucine hydroxamate triggered a rel Seq -dependent stringent response characterized by rapid (p)ppGpp accumulation at the expense of GTP and abrupt cessation of net RNA accumulation in the wild type but not in the mutant. Tetracycline added to block (p)ppGpp synthesis caused the accumulated (p)ppGpp to degrade rapidly, with a concomitant increase of the GTP pool (decay constant of ppGpp, Ϸ0.7 min ؊1). Simultaneous addition of pseudomonic acid and tetracycline to mimic a relaxed response caused wild-type RNA synthesis to proceed at rates approximating those seen under either condition in the mutant. Glucose exhaustion provoked the (p)ppGpp accumulation response in both the wild type and the rel Seq insertion mutant, consistent with the block of net RNA accumulation in both strains. Although the source of (p)ppGpp synthesis during glucose exhaustion remains to be determined, these findings reinforce the idea entertained previously that rel Seq fulfils functions that reside separately in the paralogous relA and spoT genes of Escherichia coli. Analysis of (p)ppGpp accumulation patterns was complicated by finding an unknown phosphorylated compound that comigrated with ppGpp under two standard thin-layer chromatography conditions. Unlike ppGpp, this compound did not adsorb to charcoal and did not accumulate appreciably during isoleucine deprivation. Like ppGpp, the unknown compound did accumulate during energy source starvation.Bacteria have evolved elaborate strategies that enable them to adjust their macromolecular syntheses when their nutrient supply becomes limiting or other environmental stresses are imposed. Among the responses involving the modulation of gene expression is the well-known stringent response, the hallmarks of which include the restriction of the synthesis of stable RNAs, the stimulation of certain anabolic reaction pathways, and the induction of stationary-phase-specific genes as results of amino acid limitation or inhibition of tRNA amino acylation. This pleiotropic response is mediated by the accumulation of GDP and GTP derivatives that carry a pyrophosphate group on the 3Ј-hydroxyl position of ribose, i.e., ppGpp and pppGpp, respectively, hereafter together designated (p)ppGpp (see references 3 and 9 for recent reviews). Depending on the nature of the nutritional stress imposed, there are two different sources for (p)ppGpp accumulation in E...

Complete genome sequence of an M1 strain of Streptococcus pyogenes

“…The relA͞spoT proteins are key components of the bacterial stringent response. The genomic sequence revealed the presence of a gene (SPy1981, relA) encoding a bifunctional enzyme involved in the synthesis (Rel-like function) and hydrolysis (SpoT-like function) of (p)ppGpp during amino acid starvation (28). Thus rel fulfills functions that reside separately in the proteins encoded by the relA and spoT genes of E. coli.…”

Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation

“…Bacteria under the stress of starvation have developed efficient adaptive regulatory reactions to shift their metabolic balance away from biosynthesis and reproduction, toward the acquisition of energy for existing biological functions (Giard et al, 1997;Kim et al, 2001;Matin, 1990;Watson et al, 1998). Cultures of Escherichia coli, Streptococcus equisimilis, Bacillus subtilis and Streptococcus pyogenes that are deprived of amino acids, carbon, glucose or iron induce a stringent response dependent on the synthesis of guanosine nucleotides [(p)ppGpp], which favour activities that facilitate adaptation to the nutritional stress (Chatterji & Ojha, 2001; Mechold & Malke, 1997;Miethke et al, 2006;Steiner & Malke, 2000). Despite considerable speculation, information is scarce concerning starvation responses among oral bacteria, or whether such responses are stimulated in the oral environment (Bowden & Li, 1997;Bowden & Hamilton, 1998).…”

The role of the Corynebacterium glutamicum rel gene in (p)ppGpp metabolism

“…subtilis (Wendrich & Marahiel, 1997) and S. equisimilis (Mechold et al, 1996;Mechold & Malke, 1997), where most probably only one relA/spoT-like gene exists. Mutations affecting the SpoT (p)ppGpp hydrolase activity in E. coli, like the spoT203 allele, which was obtained by hydroxylamine mutagenesis, result in a slow-growth phenotype due to elevations of (p)ppGpp levels (Xiao et al, 1991).…”