Expression from the nifB promoter of Azotobacter vinelandii can be activated by NifA, VnfA, or AnfA transcriptional activators

Abstract

In Azotobacter vinelandii, nifB is required for the activity of all three nitrogenases. Expression of a nifB-lacZ fusion was examined to determine which regulatory gene products are important for nifB expression and how its transcription is regulated in response to metals. In all conditions, expression in A. vinelandii was eliminated by an rpoN mutation, confirming the absolute requirement for sigma N. In the wild type, nifB-lacZ expression was approximately twofold higher in cells grown with Mo than without. Expression was negligible in a nifA mutant grown with Mo but was much higher in Mo-free medium, suggesting that in these conditions, another sigma N-dependent activator was responsible for nifB expression, possibly VnfA, AnfA, or NtrC. Although expression of the nifB-lacZ fusion in A. vinelandii vnfA, anfA, and ntrC mutants was little different from that in the wild type, nifB transcription could be activated by NifA, VnfA, or a truncated form of AnfA in Escherichia coli. The two potential NifA binding sites centered at -87 and -129 bp upstream of the transcription start site each overlapped a VnfA recognition sequence, motifs also found in Azotobacter chroococcum in two exactly conserved regions. Deletion analysis showed that both regions are important for nifB expression. Activation of the full-length promoter by AnfA was impaired by overexpressing the DNA-binding domain of NifA, suggesting that binding of NifA and AnfA can be competitive.