Molecular genetic analysis of the Rhizobium meliloti fixK promoter: identification of sequences involved in positive and negative regulation

Abstract

Transcription of the Rhizobium meliloti fixK gene is induced in symbiotic and microaerobic growth conditions by the FixL/FixJ modulator/effector pair. Transcription of fixK is also negatively autoregulated. By 5' deletion analysis, the involvement in negative regulation of a DNA region between -514 and -450 with respect to the transcription start was demonstrated. Site-directed mutagenesis allowed us to show that a sequence homologous to the binding site of the Escherichia coli Fnr protein, centred at position -487, participates in this effect. However, deletion or mutagenesis of this Fnr-like sequence does not completely eliminate FixK-dependent repression, which suggests that either an additional DNA region is involved in negative regulation or that it is mediated at the level of fixLJ transcription. Deletion analysis also allowed the definition of a DNA region involved in FixJ-mediated activation of the fixK promoter, between -79 and -42. Different point mutations in the -60, -45 and -35 regions were shown to affect promoter activity. In some cases, the activity of mutant promoters could be partly or fully restored by increasing the expression of the fixLJ regulatory genes, in an E. coli strain harbouring a plasmid with fixLJ under the control of an inducible (p-tac) promoter.