sigma54-promoter discrimination and regulation by ppGpp and DksA.

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 sigma(54)-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 sigma(54)-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 sigma(54)-dependent transcription in combination with a series of sigma(54)-promoters with graded affinities for sigma(54)-RNA polymerase. For in vitro analysis we used a P. putida-based reconstituted sigma(54)-transcription assay system in conjunction with DNA-binding plasmon resonance analysis of native and heterologous sigma(54)-RNA polymerase holoenzymes. In comparison with E. coli, ppGpp/DksA responsive sigma(54)-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 sigma(54)-promoters that is conferred by the strong DNA binding properties of the P. putida sigma(54)-RNA polymerase holoenzyme.

sigma54-RNA polymerase controls sigma70-dependent transcription from a non-overlapping divergent promoter.

Divergent transcription of a regulatory gene and a cognate promoter under its control is a common theme in bacterial regulatory circuits. This genetic organization is found for the dmpR gene that encodes the substrate-responsive specific regulator of the sigma(54)-dependent Po promoter, which controls (methyl)phenol catabolism. Here we identify the Pr promoter of dmpR as a sigma(70)-dependent promoter that is regulated by a novel mechanism in which sigma(54)-RNA polymerase occupancy of the non-overlapping sigma(54)-Po promoter stimulates sigma(70)-Pr output. In addition, we show that DmpR stimulates its own production through Po activity both in vivo and in vitro. Hence, the demonstrated regulatory circuit reveals a novel role for sigma(54)-RNA polymerase, namely regulation of a sigma(70)-dependent promoter, and a new mechanism that places a single promoter under dual control of two alternative forms of RNA polymerase. We present a model in which guanosine tetra-phosphate plays a major role in the interplay between sigma(54)- and sigma(70)-dependent transcription to ensure metabolic integration to couple sigma(70)-Pr output to both low-energy conditions and the presence of substrate.

Properties of RNA polymerase bypass mutants: implications for the role of ppGpp and its co-factor DksA in controlling transcription dependent on sigma54.

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain sigma70-dependent promoters. A number of promoters that are dependent on alternative sigma-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived sigma54-dependent Po promoter and several other sigma54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro sigma54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the beta- and beta'-subunits of core RNA polymerase that allow high levels of transcription from the sigma54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with sigma-factors to favor sigma54-holoenzyme formation over that with sigma70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent sigma70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of sigma54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent sigma70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative sigma-factors is controlled to meet cellular requirements.

The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of sigma-dependent transcription.

The RNA polymerase-binding protein DksA is a cofactor required for guanosine tetraphosphate (ppGpp)-responsive control of transcription from sigma70 promoters. Here we present evidence: (i) that both DksA and ppGpp are required for in vivo sigma54 transcription even though they do not have any major direct effects on sigma54 transcription in reconstituted in vitro transcription and sigma-factor competition assays, (ii) that previously defined mutations rendering the housekeeping sigma70 less effective at competing with sigma54 for limiting amounts of core RNA polymerase similarly suppress the requirement for DksA and ppGpp in vivo and (iii) that the extent to which ppGpp and DksA affect transcription from sigma54 promoters in vivo reflects the innate affinity of the promoters for sigma54-RNA polymerase holoenzyme in vitro. Based on these findings, we propose a passive model for ppGpp/DksA regulation of sigma54-dependent transcription that depends on the potent negative effects of these regulatory molecules on transcription from powerful stringently regulated sigma70 promoters.