Elicitation of High (p)ppGpp Levels in Streptococcus equisimilis without Imposing Nutritional Deprivation

Abstract

In Escherichia coli, the intracellular levels of guanosine polyphosphates [(p)ppGpp] rise following numerous environmental stresses and have wide-reaching effects on gene expression and, consequently, cellular physiology and metabolism¹. To contribute to understanding how the pathogenic streptococci respond to environmental cues during the infectious process, we have analyzed the function of the rel _Seq gene found in the streptokinase gene region of S. equisimilis H46A⁴. This strain accumulates (p)ppGpp during amino acid starvation and carbon source exhaustion, a response that is accompanied by the coupled cessation of net protein and RNA synthesis³. As judged by the relaxed response of insertion mutants, rel _Seq determines (p)ppGpp accumulation during amino acid deprivation and can thus be operationally defined as encoding the stringent factor of S. equisimilis ³. However, unlike the relA gene of E. coli, rel _Seq is bifunctional, encoding, like E. coli spoT, also a strong (p)ppGpp hydrolytic activity². Literally nothing is known about the pleiotropic effects of the stringent response in the streptococci, which is particularly true of possible regulatory functions of (p)ppGpp in the expression control of virulence genes. To approach this problem, we describe here a plasmid that specifies elevated (p)ppGpp levels in S. equisimilis. Such a construct should circumvent the need of provoking the stringent response by transient nutrient starvation protocols, which may complicate interpretations of effects due solely to (p)ppGpp.