Mutational analysis of the Rhizobium meliloti nifA promoter

Abstract

The nifA gene of Rhizobium meliloti, the bacterial endosymbiont of alfalfa, is a regulatory nitrogen fixation gene required for the induction of several key nif and fix genes. Transcription of nifA is strongly induced in planta and under microaerobic conditions ex planta. Induction of nifA, in turn, is positively controlled by the fixL and fixJ genes of R. meliloti, the sensor and regulator, respectively, of a two-component system responsible for oxygen sensing by this bacterium. This system is also responsible for the positive induction of fixK. Here, we report that chemical and oligonucleotide site-directed mutageneses of the nifA promoter (nifAp) were conducted to identify nucleotides essential for induction. Nineteen mutants, including 14 single-point mutants, were analyzed for microaerobic induction of nifAp in R. meliloti. Critical residues were identified in an upstream region between base pairs -54 and -39 relative to the transcription start site. Attempts at separating the upstream and downstream regions of the nifA promoter so as to maintain fixJ-dependent activity were unsuccessful. A 5' deletion of the fixK promoter (fixKp) to -67 indicates that sequences upstream of this position are not required for microaerobic induction. A sequence comparison of the -54 to -39 region of nifAp with the upstream sequences of fixKp does not reveal a block of identical nucleotides that could account for the fixJ-dependent microaerobic induction of both promoters. Many of the defective nifAp mutants in this region, however, are in residues with identity to fixKp in an alignment of the promoters according to their transcription start sites. Therefore, it is possible that there is a common sequence motif in the -54 to -39 region of the two promoters that is required for fixLJ-dependent microaerobic induction.