FNRN CONTROLS SYMBIOTIC NITROGEN-FIXATION AND HYDROGENASE ACTIVITIES IN RHIZOBIUM-LEGUMINOSARUM BIOVAR VICIAE UPM791

Rhizobium leguminosarum by. viciae UPM791 contains a second copy of th e fnrN gene, which encodes a redox-sensitive transcriptional activator functionally homologous to Escherichia coli Fnr, This second copy (fn rN2) is located in the symbiotic plasmid, while fnrN1 is in the chromo some, Isolation and sequencing of the fnrN2 gene revealed that the ded uced amino acid sequence of FnrN2 is 87.5% identical to the sequence o f FnrN1, including a conserved cysteine-rich motif characteristic of F nr-like proteins, Individual R. leguminosarum fnrN1 and fnrN2 mutants exhibited a Fix(+) phenotype and near wild-type levels of nitrogenase and hydrogenase activities in pea (Pisum sativum L.) nodules, In contr ast, an fnrN1 fnrN2 double mutant formed ineffective nodules lacking b oth nitrogenase and hydrogenase activities, Unlike the wild-type strai n and single fnrN1 or fnrN2 mutants, the fnrN1 fnrN2 double mutant was unable to induce micro-oxic or bacteroid activation of the hypBFCDEX operon, which encodes proteins essential for hydrogenase synthesis. In the search for symbiotic genes that could be controlled by FnrN, a fi xNOQP operon, putatively encoding a micro-oxically induced; bacteroid- specific cbb(3)-type terminal cytochrome oxidase, was isolated from st rain UPM791 and partially sequenced. The fixNOQP operon was present in a single copy located in the symbiotic plasmid, and an anaerobox was identified in the firN promoter region. Consistent with this, a fixNOQ P'-lacZ fusion was shown to be highly induced in micro-oxic cells of t he wild-type strain. A high level of micro-oxic induction was also obs erved in single fnrN1 and fnrN2 mutants, but no detectable induction,v as observed in the fnrN1 fnrN2 double mutant. The lack of expression o f fixNOQP in the fnrN1 fnrN2 double mutant is likely to cause the obse rved Fix-phenotype. These data demonstrate that, contrary to the situa tion in other rhizobia, FnrN controls both hydrogenase and nitrogenase activities of R. leguminosarum by. viciae UPM791 in the nodule and su ggest that this strain lacks a functional fixK gene.