Regulation of ς factor competition by the alarmone ppGpp

Abstract

Many regulons controlled by alternative ς factors, including ς S and ς 32 , 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 ς 70 , specific mutations in rpoD ( rpoD40 and rpoD35 ), or overproduction of Rsd (anti-ς 70 ) 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 ς 70 to compete with ς 32 for core binding and the mutant ς 70 proteins, encoded by rpoD40 and rpoD35 , compete less efficiently than wild-type ς 70 . Similarly, an in vivo competition assay showed that the ability of both ς 32 and ς S to compete with ς 70 is diminished in cells lacking ppGpp. Consistently, the fraction of ς S and ς 32 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.