Genetic analysis of the Rhizobium meliloti nifH promoter, using the P22 challenge phage system

In several genera of bacteria, the sigma54-RNA polymerase holoenzyme (E sigma54) is a minor form of RNA polymerase that is responsible for transcribing genes whose products are involved in diverse metabolic processes. Esigma54 binds to the promoters of these genes to form a closed promoter complex. An activator protein is required for the transition of this closed promoter complex to an open complex that is transcriptionally competent. In this study, the P22-based challenge phage system was used to investigate interactions between Esigma54 and the Rhizobium meliloti nifH promoter. Challenge phages were constructed in which the R. meliloti nifH promoter replaced the binding site for the Mnt protein, a repressor of the phage P22 ant gene. When a Salmonella typhimurium strain that overexpressed sigma54 was infected with these challenge phages, E sigma54 bound to the nifH promoter and repressed transcription of the ant gene as seen by the increased frequency of lysogeny. Following mutagenesis of challenge phages that carried the R. meliloti nifH promoter, mutant phages that could form plaques on an S. typhimurium strain that overexpressed sigma54 were isolated. These phages had mutations within the nifH promoter that decreased the affinity of the promoter for Esigma54. The mutations were clustered in seven highly conserved residues within the -12 and-24 regions of the nifH promoter.