Regulation of ς factor competition by the alarmone ppGpp

Abstract

Many regulons controlled by alternative ς factors, including ς^S and ς³², are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of ς^S-dependent promoters because underproduction of ς⁷⁰, specific mutations inrpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-ς⁷⁰) restored expression from ς^S-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 ς⁷⁰proteins, 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 ς^S to compete with ς⁷⁰ is diminished in cells lacking ppGpp. Consistently, the fraction of ς^S 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.