THE HYPBFCDE OPERON FROM RHIZOBIUM-LEGUMINOSARUM BIOVAR VICIAE IS EXPRESSED FROM AN FNR-TYPE PROMOTER THAT ESCAPES MUTAGENESIS OF THE FNRN GENE

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum by. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H-2 arising from the nitrogen fixation process in root nodules. Synthesis of the active enzyme requires the products of the structural genes hupSL and an array of accessory proteins from at least 15 additional genes, including the gene cluster hypABFCDE, likely involved in nickel metabolism. Unlike the hupSL genes, which are expressed only in symbiosis, the hypBFCDE operon was also activated in vegetative cells in response to low pO(2) in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P-5b, with a transcription initiation site located 190 bp upstream of the ATG start codon of hypB, within the coding sequence of hypA. Transcription start site 5b was preceded by an Fnr box (anaerobox), 5'-TTGAgccatgTCAA-3', centered at position -39.5. Expression of the P-5b promoter in the heterologous Rhizobium meliloti bacterial host was dependent on the presence of an active fixK gene. A 2.6-kb EcoRI fragment was isolated from an R. leguminosarum by. viciae UPM791 gene bank by complementing an R. meliloti FixK(-) mutant. Sequencing of this DNA fragment identified an fnrN gene, and cassette insertion mutagenesis demonstrated that R. leguminosarum by. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum by. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN(-) mutant of R. leguminosarum by. viciae exhibited wild-type levels of hydrogenase activity. Microaerobic expression of P-5b, was reduced to ca. 50% of the wild-type level in the FnrN(-) mutant. These results indicate that hyp gene expression escapes mutagenesis of the fnrN gene and suggest the existence of a second fnr-like gene in R. leguminosarum bv. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.