σ54-promoter discrimination and regulation by ppGpp and DksA

Abstract

The & sigma;54-factor controls expression of a variety of genes in response to environmental cues. Much previous work has implicated the nucleotide alarmone ppGpp and its co-factor DksA in control of σ54-dependent transcription in the gut commensal Escherichia coli, which has evolved to live under very different environmental conditions than Pseudomonas putida. Here we compared ppGpp/DksA mediated control of σ54-dependent transcription in these two organisms. Our in vivo experiments employed P. putida mutants and manipulations of factors implicated in ppGpp/DksA mediated control of σ54-dependent transcription in combination with a series of σ54-promoters with graded affinities for σ54-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted σ54-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous σ54-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive σ54-transcription in the environmentally adaptable P. putida was found to be more robust under low energy conditions that occur upon nutrient depletion. The mechanism behind this difference can be traced to reduced promoter discrimination of low affinity σ54-promoters that is conferred by the strong DNA binding properties of the P. putida σ54-RNA polymerase holoenzyme. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.