IDENTIFICATION AND CHARACTERIZATION OF THE NIFV-NIFZ-NIFT GENE REGIONFROM THE FILAMENTOUS CYANOBACTERIUM ANABENA SP STRAIN PCC-7120

The nifV and leuA gents, which encode homocitrate synthase and oc alph a-isopropylmalate synthase, respectively, were cloned from the filamen tous cyanobacterium Anabaena sp. strain PCC 7120 by a PCR-based strate gy. Since the N-terminal Darts of NifV and LeuA from other bacteria ar e highly similar to each other, a single pair of PCR primers was used to amplify internal fragments of both Anabaena strain 7120 genes, Sequ ence analysis of cloned PCR products confirmed the presence of two dif ferent nitV-like DNA fragments, which were subsequently used as nifV- and leuA-specific probes, respectively, to clone XbaI fragments of 2.1 kbp (pOST4) and 2.6 kbp (pOST2), Plasmid pOST4 carried the Anabaena s train 7120 nifV-nifZ-nifT genes, whereas pOST2 contained the leuA and dapF genes. The nifVZT genes were not located in close proximity to th e main nif gene cluster in Anabaena strain 7120, and therefore nifVZT forms a second nif gene cluster in this strain. Overlaps between the n ifV and nifZ genes and between the nifZ and nifT genes and the presenc e of a 1.8-kb transcript indicated that nifVZT might form one transcri ptional unit, Transcripts of nifV were Induced not only in a nitrogen- depleted culture but also by iron depletion irrespective of the nitrog en status, The nifV gene in Anabaena strain 7120 was interrupted by an interposon insertion (mutant strain BMB105) and by a plasmid integrat ion via a single crossover with a nifV internal fragment as a site for recombination (mutant strain BMB106). Both mutant strains were capabl e of diazotrophic growth, and their growth rates were only slightly im paired compared to that of the wild type. Heterologous complementation of the Rhodobacter capsulatus nifV mutant R229I by the Anabaena strai n 7120 nifV gene corroborated the assumption that Anabaena strain 7120 nifV also encodes a homocitrate synthase. In contrast, tile Anabaena strain 7120 leuA gene did not complement the nifV mutation of R229I ef ficiently.