Changes in Conserved Region 3 ofEscherichia coliσ70Mediate ppGpp-dependent FunctionsIn Vivo

Abstract

InEscherichia colideletion ofrelAandspoTresults in an inability to synthesize ppGpp, guanosine-3′, 5′-bis(pyrophosphate), and a loss in the ability to grow on amino acid-free minimal media. Two spontaneous missense suppressor alleles,rpoD(P505L)andrpoD(S506F)able to confer complete prototrophy without the reappearance of ppGpp, were localized to that portion ofrpoDcoding for conserved region 3.1 of σ70. Characterization of these mutants revealed distinct physiological effects. Both mutations cause growth rate defects exacerbated in the presence of ppGpp and paralleled by reductions inrrnBP1-lacZreporter gene expression, as if growth of these mutants is limited byrrnP1 promoter activity. Levels of ppGpp, as a function of growth rate, are lowered by a constant fraction (75%) in therpoD(P504L)strain and by a decreasing fraction at lower growth rates in therpoD(S506F)strain. Comparisons ofrrnBP1-lacZexpression at different ppGpp levels is interpreted for therpoD(P504L)mutant as resulting from a hypersensitivity to ppGpp. For therpoD(S506F)mutant there is a normal sensitivity to ppGpp but the action of ppGpp is functionally mimicked; that is, a low intrinsicrrnBP1 promoter activity is manifested even in the absence of ppGpp. In addition to effects onrrnBP1 promoters, the accumulation of the stationary phase-specific sigma factor (σs), which is normally ppGpp-dependent, was assayed in therpoDmutants and in one,rpoD(S506F)found to be restored in the absence of ppGpp. The behavior of these mutants thus seems consistent with a unitary effect of ppGpp on transcription resulting in both negative and positive regulation of gene expression. In addition, the cellular fraction of σ70associated with holoenzyme appears reduced by bothrpoDmutations as judged by comparison with wild-type and ppGpp-deficient strains. Interestingly, the amount of holo enzyme-associated σ70was higher in the ppGpp-deficient than in the wild-type strain, possibly indicating that σ70-core RNA polymerase interactions are decreased by ppGpp.