GENETIC-ANALYSIS OF THE RHIZOBIUM-MELILOTI NIFH PROMOTER, USING THE P22 CHALLENGE PHAGE SYSTEM

In several genera of bacteria, the sigma(54)-RNA polymerase holoenzyme (E sigma(54)) is a minor form of RNA polymerase that is responsible f or transcribing genes whose products are involved in diverse metabolic processes. E sigma(54) binds to the promoters of these genes to form a closed promoter complex. An activator protein is required for the tr ansition of this closed promoter complex to an open complex that is tr anscriptionally competent. In this study, the P22-based challenge phag e system was used to investigate interactions between E sigma(54) and the Rhizobium meliloti nifH promoter. Challenge phages were constructe d 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 Salmon ella typhimurium strain that overexpressed sigma(54) was infected with these challenge phages, E sigma(54) bound to the nifH promoter and re pressed transcription of the ant gene as seen by the increased frequen cy 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 sigma(54) were isolated . These phages had mutations within the nifH promoter that decreased t he affinity of the promoter for E sigma(54). The mutations were cluste red in seven highly conserved residues within the -12 and -24 regions of the nifH promoter.