Transcription from the Pseudomonas-derived σ54-dependent Po promoter of the dmp operon is mediated by the aromatic-responsive regulator DmpR. However, physiological control is superimposed on this regulatory system causing silencing of the DmpR-mediated transcriptional response in rich media until the transition between exponential and stationary phase is reached. Here, the positive role of the nutritional alarmone (p)ppGpp in DmpR regulation of the Po promoter has been identified and investigated in vivo. Overproduction of (p)ppGpp in a Pseudomonas reporter system was found to allow an immediate transcriptional response under normally non-permissive conditions. Conversely (p)ppGpp-deficient Escherichia coli strains were found to be severely defective in DmpR-mediated transcription, demonstrating the requirement for this metabolic signal. A subset of mutations in the β, β′ and σ70 subunits of RNA polymerase, which confer prototrophy on ppGpp0E. coli, was also found to restore specific DmpR-mediated transcription from Po, suggesting that the metabolic signal is mediated directly through the σ54-RNA polymerase. These data provide a direct mechanistic link between the physiological status of the cell and expression from σ54 promoters.