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The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene.

ABSTRACT: Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 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 pO2 in the culture medium. In microaerobic cells and in bacteroids, transcription of the hypBFCDE operon occurred from a promoter, P5b, 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 P5b 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 bv. 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 bv. viciae fnrN is able to replace the R. meliloti fixK gene for activation of both the R. leguminosarum bv. viciae hypBFCDE operon and the R. meliloti fix genes. However, bacteroids from a genomic FnrN- mutant of R. leguminosarum bv. 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 by. viciae. Southern blot analysis with an fnrN internal probe revealed the presence of a second genomic region with homology to fnrN.

SUBMITTER: Hernando Y

PROVIDER: S-EPMC177378 | biostudies-other | 1995 Oct

REPOSITORIES: biostudies-other

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The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene.

Hernando Y Y Palacios J M JM Imperial J J Ruiz-Argüeso T T

Journal of bacteriology 19951001 19

Pea (Pisum sativum L.) bacteroids produced by Rhizobium leguminosarum bv. viciae UPM791 synthesize a membrane-bound (NiFe) hydrogenase which oxidizes H2 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 symbio ...[more]

PMID: 7559356

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Project description:Rhizobium leguminosarum bv. viciae UPM791 contains a second copy of the fnrN gene, which encodes a redox-sensitive transcriptional activator functionally homologous to Escherichia coli Fnr. This second copy (fnrN2) is located in the symbiotic plasmid, while fnrN1 is in the chromosome. Isolation and sequencing of the fnrN2 gene revealed that the deduced amino acid sequence of FnrN2 is 87.5% identical to the sequence of FnrN1, including a conserved cysteine-rich motif characteristic of Fnr-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 contrast, an fnrN1 fnrN2 double mutant formed ineffective nodules lacking both nitrogenase and hydrogenase activities. Unlike the wild-type strain 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 fixNOQP operon, putatively encoding a micro-oxically induced, bacteroid-specific cbb3-type terminal cytochrome oxidase, was isolated from strain 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 fixN promoter region. Consistent with this, a fixNOQP'-lacZ fusion was shown to be highly induced in micro-oxic cells of the wild-type strain. A high level of micro-oxic induction was also observed in single fnrN1 and fnrN2 mutants, but no detectable induction was observed in the fnrN1 fnrN2 double mutant. The lack of expression of fixNOQP in the fnrN1 fnrN2 double mutant is likely to cause the observed Fix- phenotype. These data demonstrate that, contrary to the situation in other rhizobia, FnrN controls both hydrogenase and nitrogenase activities of R. leguminosarum bv. viciae UPM791 in the nodule and suggest that this strain lacks a functional fixK gene.

Project description:The rhi genes of Rhizobium leguminosarum biovar viciae are expressed in the rhizosphere and play a role in the interaction with legumes, such as the pea. Previously (K. M. Gray, J. P. Pearson, J. A. Downie, B. E. A. Boboye, and E. P. Greenberg, J. Bacteriol. 178:372-376, 1996) the rhiABC operon had been shown to be regulated by RhiR and to be induced by added N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone (3OH, C14:1-HSL). Mutagenesis of a cosmid carrying the rhiABC and rhiR gene region identified a gene (rhiI) that affects the level of rhiA expression. Mutation of rhiI slightly increased the number of nodules formed on the pea. The rhiI gene is (like rhiA) regulated by rhiR in a cell density-dependent manner. RhiI is similar to LuxI and other proteins involved in the synthesis of N-acyl-homoserine lactones (AHLs). Chemical analyses of spent culture supernatants demonstrated that RhiI produces N-(hexanoyl)-L-homoserine lactone (C6-HSL) and N-(octanoyl)-L-homoserine lactone (C8-HSL). Both of these AHLs induced rhiA-lacZ and rhiI-lacZ expression on plasmids introduced into an Agrobacterium strain that produces no AHLs, showing that rhiI is positively regulated by autoinduction. However, in this system no induction of rhiA or rhiI with 3OH,C14:1-HSL was observed. Analysis of the spent culture supernatant of the wild-type R. leguminosarum bv. viciae revealed that at least seven different AHLs are made. Mutation of rhiI decreased the amounts of C6-HSL and C8-HSL but did not block their formation, and in this background the rhiI mutation did not significantly affect the expression levels of the rhiI gene or rhiABC genes or the accumulation of RhiA protein. These observations suggest that there are additional loci involved in AHL production in R. leguminosarum bv. viciae and that they affect rhiI and rhiABC expression. We postulate that the previously observed induction of rhiA by 3OH,C14:1-HSL may be due to an indirect effect caused by induction of other AHL production loci.

Dataset Information

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene.

Publications

The hypBFCDE operon from Rhizobium leguminosarum biovar viciae is expressed from an Fnr-type promoter that escapes mutagenesis of the fnrN gene.

Similar Datasets

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