Abstract
NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria. In Rhizobium leguminosarum bv. viciae UPM791, the nifA gene is part of a gene cluster (orf71 orf79 fixW orf5 fixABCX nifAB) separated by 896 bp from an upstream and divergent truncated duplication of nifH (DeltanifH). Symbiotic expression analysis of genomic nifA::lacZ fusions revealed that in strain UPM791 nifA is expressed mainly from a sigma54-dependent promoter (P(nifA1)) located upstream of orf71. This promoter contains canonical NifA upstream activating sequences located 91 bp from the transcription initiation site. The transcript initiated in P(nifA1) spans 5.1 kb and includes nifA and nifB genes. NifA from Klebsiella pneumoniae was able to activate transcription from P(nifA1) in a heterologous Escherichia coli system. In R. leguminosarum, the P(nifA1) promoter is essential for effective nitrogen fixation in symbiosis with peas. In its absence, partially efficient nitrogen-fixing nodules were produced, and the corresponding bacteroids exhibited only low levels of nifA gene expression. The basal level of nifA expression resulted from a promoter activity originating upstream of the fixX-nifA intergenic region and probably from an incomplete duplication of P(nifA1) located immediately upstream of fixA.