Cloning and characterization of nitrogenase genes from Anabaena variabilis

Three cloned Klebsiella pneumoniae nitrogenase gene probes were used to perform genomic Southern hybridization analysis and to screen an Anabaena variabilis-Charon 30 genetic library for Anabaena nitrogenase genes. One of ten phage clones that were isolated using a Klebsiella nifHD gene probe was studied in detail. This clone (ABN 10) contains a 9 kb Eco R1 fragment which was observed in genomic Southerns. ABN 10 does not hybridize with a Klebsiella nifK gene probe. A 2.8 kb Hind III fragment that lies within the 9 kb Eco R1 fragment contains the linked Anabaena nifH and nifD genes; the nifK gene lies at least 4 kb away. Partial sequencing of the nifH and nifD genes confirmed their identity and showed strong conservation of amino acid sequence in comparison to Anabaena 7120, Klebsiella pneumoniae, Azotobacter vinlandii, and Clostridium pasteurianum. When the cloned Anabaena nif genes were put into Klebsiella cells on plasmids, they were not expressed, suggesting that the Anabaena promoters are not recognized by Klebsiella transcription factors.