Changes in conserved region 3 of Escherichia coli sigma 70 mediate ppGpp-dependent functions in vivo

In Escherichia coli, deletion of relA and spoT results 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(P504L) and rpoD(S506F), able to confer complete prototrophy without the reappearance of ppGpp, were localized to that portion of rpoD coding for conserved region 3.1 of sigma 70. Characterization of these mutants revealed distinct physiological effects. Both mutations cause growth rate defects exacerbated in the presence of ppGpp and paralleled by reductions in rrnB P1-lacZ reporter gene expression, as if growth of these mutants is limited by rrn P1 promoter activity. Levels of ppGpp, as a function of growth rate, are lowered by a constant fraction (75%) in the rpoD(P504L) strain and by a decreasing fraction at lower growth rates in the rpoD(S506F) strain. Comparisons of rrnB P1-lacZ expression at different ppGpp levels is interpreted for the rpoD(P504L) mutant as resulting from a hypersensitivity to ppGpp. For the rpoD(S506F) mutant there is a normal sensitivity to ppGpp but the action of ppGpp is functionally mimicked; that is, a low intrinsic rrnB P1 promoter activity is manifested even in the absence of ppGpp. In addition to effects on rrnB P1 promoters, the accumulation of the stationary phase-specific sigma factor (sigma s), which is normally ppGpp-dependent, was assayed in the rpoD mutants 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 sigma 70 associated with holoenzyme appears reduced by both rpoD mutations as judged by comparison with wild-type and ppGpp-deficient strains. Interestingly, the amount of holoenzyme-associated sigma 70 was higher in the ppGpp-deficient than in the wild-type strain, possibly indicating that sigma 70-core RNA polymerase interactions are decreased by ppGpp.