Identification and DNA sequence of fixZ, a nifB-like gene from Rhizobium leguminosarum

Abstract

Previously, several mutants which nodulated peas but which failed to fix nitrogen were isolated following Tn5 mutagenesis of pRL 1JI, a symbiotic plasmid of Rhizobium leguminosarum. Two of these alleles, fix52::Tn5 and fix137::Tn5 were in a region of pRL 1JI which hybridized to a probe that contained the nifA gene and the amino-terminal region of the nifB gene of Klebsiella pneumoniae. The nitrogen fixation defect of the fix52::Tn5 mutant strain was corrected by a 2.0kb fragment of the corresponding wild-type DNA cloned in a wide host-range plasmid. The DNA sequence of this region revealed an open reading frame corresponding to the gene within which the fix52::Tn5 allele was located. The polypeptide corresponding to this open reading frame had a deduced molecular weight of 39,936 and the gene was termed fixZ. The deduced amino acid sequence of the fixZ gene product contained two clusters of cysteine residues, suggesting that the protein may contain an iron-sulphur cluster. The sequence of the fixZ polypeptide was very similar to the sequence of the K. pneumoniae nifB gene (provided by W. Arnold and A. Pühler) which is required for the synthesis of the FeMo-cofactor of nitrogenase. It was shown that the previously observed hybridization was due to homology between the amino terminal regions of fixZ and nifB. Upstream from fixZ was found another open reading frame whose 5' terminus was not established, but within which was located the fix137::Tn5 allele. This gene was termed fixY. The deduced amino acid sequence of the sequenced part of fixY showed similarity to that of the regulatory nifA gene of K. pneumoniae (provided by W. J. Buikema and F. M. Ausubel). Thus in R. leguminoarum the fix genes that correspond to the nifA and nifB genes are in the same relative orientation as in K. pneumoniae.