EXPRESSION FROM THE NIFB PROMOTER OF AZOTOBACTER-VINELANDII CAN BE ACTIVATED BY NIFA, VNFA, OR ANFA TRANSCRIPTIONAL ACTIVATORS

In Azotobacter vinelandii, nifB is required for the activity of all th ree nitrogenases. Expression of a nifB-lacZ fusion was examined to det ermine which regulatory gene products are important for nifB expressio n and how its transcription is regulated in response to metals. In all conditions, expression in A. vinelandii was eliminated by an rpoN mut ation, confirming the absolute requirement for sigma(N). In the wild t ype, nifB-lacZ expression was approximately twofold higher in cells gr own with Mo than without. Expression was negligible in a nifA mutant g rown with Mo but was much higher in Mo-free medium, suggesting that in these conditions, another sigma(N)-dependent activator was responsibl e for nifB expression, possibly VnfA, AnfA, or NtrC. Although expressi on of the nifB-lacZ fusion in A. vinelandii vnfA, anfA, and ntrC mutan ts was little different from that in the wild type, nifB transcription could be activated by NifA, VnfA, or a truncated form of AnfA in Esch erichia coli. The two potential NifA binding sites centered at -87 and -129 bp upstream of the transcription start site each overlapped a Vn fA recognition sequence, motifs also found in Azotobacter chroococcum in two exactly conserved regions. Deletion analysis showed that both r egions are important for nifB expression. Activation of the full-lengt h promoter by AnfA was impaired by overexpressing the DNA-binding doma in of NifA, suggesting that binding of NifA and AnfA fan be competitiv e.