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...