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Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti.

ABSTRACT: The complete nos region essential for dissimilatory nitrous oxide reduction by the endosymbiotic diazotroph Rhizobium meliloti was identified in a cosmid (pYC7) carrying a 10.1-kb EcoRI fragment of the nod megaplasmid. This gene region was localized by Southern hybridization and Tn5 mutagenesis to within 8 kb downstream from the fixGHIS cluster. Nucleotide sequence determination of a 4.6-kb DNA segment including the structural gene nosZ and its flanking regions showed sequence homology and similarity in genetic organization with the nosRZDFY genes of Pseudomonas stutzeri Zobell. The genes were arranged in three complementation groups, comprising the nosZ structural gene, the nosR regulatory gene, and the nosDFY copper-processing genes. The derived amino acid sequence of the R. meliloti nosZ product (a multi-copper nitrous oxide reductase) was more similar to those of the analogous gene products of Paracoccus and Pseudomonas species than to that of Alcaligenes eutrophus. The nosZ gene was preceded by nosR, which encodes a regulatory protein containing C-terminal cysteine clusters similar to those present in the 4Fe-4S binding region of bacterial ferredoxins, The nosDFY genes, located downstream from nosZ, were identified as copper-processing genes encoding a periplasmic protein, an ATP/GTP-binding protein, and a membrane protein presumably forming a copper-processing system. A consensus sequence for an Anr- or Fnr-binding site similar to that in the upstream sequence of nosZ in Paracoccus denitrificans or P. stutzeri was absent in R. meliloti. No rpoN-binding site preceding the nos genes was detected, and none of the Tn5 insertions in the nos gene region affected symbiotic N2-fixing ability.

SUBMITTER: Holloway P

PROVIDER: S-EPMC177832 | biostudies-other | 1996 Mar

REPOSITORIES: biostudies-other

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Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti.

Holloway P P McCormick W W Watson R J RJ Chan Y K YK

Journal of bacteriology 19960301 6

The complete nos region essential for dissimilatory nitrous oxide reduction by the endosymbiotic diazotroph Rhizobium meliloti was identified in a cosmid (pYC7) carrying a 10.1-kb EcoRI fragment of the nod megaplasmid. This gene region was localized by Southern hybridization and Tn5 mutagenesis to within 8 kb downstream from the fixGHIS cluster. Nucleotide sequence determination of a 4.6-kb DNA segment including the structural gene nosZ and its flanking regions showed sequence homology and simil ...[more]

PMID: 8626275

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Project description:Transposon Tn5-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could be complemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938) were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in the structural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17, RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogen fixation. These mutants were mutated in regulatory dct genes which do not play an essential role in the symbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class II mutants were analyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone, whereas nodules induced by class II mutants only differed in an enhanced content of starch granules compared with wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SalI-HindIII subfragment of cosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame, which was designated dctA because Tn5 insertion sites of the class I mutants mapped within this coding region. The dctA gene was preceded by a nif consensus promoter and an upstream NifA-binding element. Upstream of the dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of the N-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R. leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATG start codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicity plot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences of the R. meliloti and the R. leguminosarum DctA proteins were highly conserved (82%).

Dataset Information

Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti.

Publications

Identification and analysis of the dissimilatory nitrous oxide reduction genes, nosRZDFY, of Rhizobium meliloti.

Similar Datasets

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