Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes

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Abstract
The fixA, fixB, fixC, and fixX genes of Rhizobium meliloti 1021 constitute an operon and are required for nitrogen fixation in alfalfa nodules. DNA homologous to the R. meliloti fixABC genes is present in all other Rhizobium and Bradyrhizobium species examined, but fixABC-homologous sequences were found in only one free-living diazotroph, Azotobacter vinelandii. To determine whether the fixABCX genes share sequence homology with any of the 17 Klebsiella pneumoniae nif genes, we determined the entire nucleotide sequence of the fixA, fixB, fixC, and fixX genes and defined four open reading frames that code for polypeptides of molecular weights 31,146, 37,786, 47,288, and 10,937, respectively. Neither DNA nor amino acid sequence homology to the R. meliloti fixA, -B, -C, and -X genes was found in the K. pneumoniae nif operon. The fixX gene contains a cluster of cysteine residues characteristic of ferredoxins and is highly homologous to an Azotobacter ferredoxin which has been shown to donate electrons to nitrogenase. The fixABC operon contains a promoter region that is highly homologous to other nifA-activated promoters. We also found a duplication of the 5' end of the fixABCX operon; a 250-bp region located 520 bp upstream of the fixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.