Identification and characterization of the nifV-nifZ-nifT gene region from the filamentous cyanobacterium Anabaena sp. strain PCC 7120.

The nifV and leuA genes, which encode homocitrate synthase and alpha-isopropylmalate synthase, respectively, were cloned from the filamentous cyanobacterium Anabaena sp. strain PCC 7120 by a PCR-based strategy. Since the N-terminal parts of NifV and LeuA from other bacteria are highly similar to each other, a single pair of PCR primers was used to amplify internal fragments of both Anabaena strain 7120 genes. Sequence analysis of cloned PCR products confirmed the presence of two different nifV-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 strain 7120 nifV-nifZ-nifT genes, whereas pOST2 contained the leuA and dapF genes. The nifVZT genes were not located in close proximity to the main nif gene cluster in Anabaena strain 7120, and therefore nifVZT forms a second nif gene cluster in this strain. Overlaps between the nifV and nifZ genes and between the nifZ and nifT genes and the presence of a 1.8-kb transcript indicated that nifVZT might form one transcriptional unit. Transcripts of nifV were induced not only in a nitrogen-depleted culture but also by iron depletion irrespective of the nitrogen status. The nifV gene in Anabaena strain 7120 was interrupted by an interposon insertion (mutant strain BMB105) and by a plasmid integration via a single crossover with a nifV internal fragment as a site for recombination (mutant strain BMB106). Both mutant strains were capable of diazotrophic growth, and their growth rates were only slightly impaired compared to that of the wild type. Heterologous complementation of the Rhodobacter capsulatus nifV mutant R229I by the Anabaena strain 7120 nifV gene corroborated the assumption that Anabaena strain 7120 nifV also encodes a homocitrate synthase. In contrast, the Anabaena strain 7120 leuA gene did not complement the nifV mutation of R229I efficiently.