A novel autoregulation mechanism of fnrN expression in Rhizobium leguminosarum bv viciae

The fnrN gene from Rhizobium leguminosarum UPM791 controls microaerobic expression of both nitrogen fixation and hydrogenase activities in symbiotic cells. Two copies of fnrN are present in this strain, one chromosomal (fnrN1) and the other located in the symbiotic plasmid (fnrN2). Their expression was studied by cloning the regulatory regions in lacZ promoter-probe vectors. The fnrN genes were found to be autoregulated: they are expressed only at basal levels under aerobic conditions; they are highly expressed under microaerobic conditions; and they are expressed at basal levels in the double mutant DG2 (fnrN1 fnrN2) under any condition. The promoters of both genes contain two FnrN-binding sequences (anaeroboxes), centred at positions -12.5 (proximal anaerobox) and -44.5 (distal anaerobox). Expression analysis and gel retardation experiments with fnrN1-derivative promoter mutants altered in key bases of the anaerobox sequences demonstrated that binding of FnrN1 to the distal anaerobox is necessary for microaerobic activation of transcription, and that binding of FnrN1 to the proximal anaerobox results in transcriptional repression. The apparent affinity of FnrN1 for the proximal anaerobox was fivefold lower than for the distal anaerobox, resulting in repression of transcription of fnrN1 only at high-FnrN1 concentrations. This positive and negative autoregulation mechanism ensures an equilibrated expression of fnrN in response to microaerobic conditions.