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 inrpoD (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 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 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.