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The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks

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Tn5 was introduced into Azotobacter vinelandii on a suicide vector, pGS9. Three Nif- mutants were found to carry Tn5 in nifH (MV6), in nifN (MV22), and in or near nifM (MV21), from the results of hybridisation experiments. For MV21 and MV22 this was also shown by complementation with the nif genes of Klebsiella pneumoniae on pRD1. MV6 failed to synthesis the nifH, D and K gene products. MV6 and MV22 fixed nitrogen in the absence of supplied molybdenum while mutant MV21 did not, suggesting that the nifM gene product may be required for the alternative nitrogenase system synthesised in azotobacteria under conditions of molybdenum deprivation. Reconstitution experiments with mutant extracts showed that MV22 (nifN -) lacked the FeMo cofactor and that MV21 (NifM-) synthesised inactive Fe protein. These biochemical phenotypes are identical to those of the K. pneumoniae nifN and nifM mutants, respectively, demonstrating that these genes have the same function in both K. pneumoniae and A. vinelandii. Complementation of the A. vinelandii mutants with pLAFR1 gene banks of A. vinelandii or a. chroococcum yielded three cosmids of interest. pLV10 complemented UW91, a nifH mutant, and corrected the defect in MV6 after recombination with the mutant genome. It also carried nifD (but not nifK) and about 18 kb of DNA upstream from nifH. pLV1 from the A. vinelandii gene bank complemented both MV21 and MV22 as did pLC11, isolated from the A. chroococcum gene bank. Both pLV1 and pLC11 carried part of the nif cluster downstream of nifHDK which also includes nifEN and nifMVS on about 22 kb of DNA.

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Author notes

  1. Rawia Gamal

    Present address: Department of Microbiology, Faculty of Agriculture, Ain Shams University, Cairo, Egypt

  2. Juan Ramos

    Present address: Department of Biochemical Medicine, University of Geneva, Geneva, Switzerland

  3. Kevin Brigle & Dennis Dean

    Present address: Department of Anaerobic Microbiology, Virginia Polytechnic Institute, Blacksburg, Virginia, USA

Authors and Affiliations

  1. AFRC Unit of Nitrogen Fixation, University of Sussex, BN1 9RQ, Brighton, East Sussex, UK

    Christina Kennedy, Rawia Gamal, Richard Humphrey & Juan Ramos

  2. Battelle-C.F. Kettering Laboratories, 150 East South College St., 45387, Yellow Springs, OH, USA

    Kevin Brigle & Dennis Dean

Authors
  1. Christina Kennedy
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  2. Rawia Gamal
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  3. Richard Humphrey
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  4. Juan Ramos
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  5. Kevin Brigle
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  6. Dennis Dean
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Communicated by J. Schell

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Kennedy, C., Gamal, R., Humphrey, R. et al. The nifH, nifM and nifN genes of Azotobacter vinelandii: Characterisation by Tn5 mutagenesis and isolation from pLAFR1 gene banks. Molec. Gen. Genet. 205, 318–325 (1986). https://doi.org/10.1007/BF00430445

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  • DOI: https://doi.org/10.1007/BF00430445

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