Regulation of σ factor competition by the alarmone ppGpp

Abstract

Many regulons controlled by alternative σ factors, including σs and σ32, are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of σs-dependent promoters because underproduction of σ70, specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-σ70) restored expression from σs-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type σ70 to compete with σ32 for core binding and the mutant σ70 proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type σ70. Similarly, an in vivo competition assay showed that the ability of both σ32 and σs to compete with σ70 is diminished in cells lacking ppGpp. Consistently, the fraction of σs and σ32 bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of σ factors in accordance with cellular demands during physiological stress.