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. 1989 Feb;171(2):1075–1086. doi: 10.1128/jb.171.2.1075-1086.1989

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii.

R D Joerger 1, M R Jacobson 1, R Premakumar 1, E D Wolfinger 1, P E Bishop 1
PMCID: PMC209704  PMID: 2644222

Abstract

The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

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Selected References

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  1. Beynon J., Cannon M., Buchanan-Wollaston V., Cannon F. The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell. 1983 Sep;34(2):665–671. doi: 10.1016/0092-8674(83)90399-9. [DOI] [PubMed] [Google Scholar]
  2. Bishop P. E., Hawkins M. E., Eady R. R. Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem J. 1986 Sep 1;238(2):437–442. doi: 10.1042/bj2380437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishop P. E., Jarlenski D. M., Hetherington D. R. Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7342–7346. doi: 10.1073/pnas.77.12.7342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bishop P. E., Jarlenski D. M., Hetherington D. R. Expression of an alternative nitrogen fixation system in Azotobacter vinelandii. J Bacteriol. 1982 Jun;150(3):1244–1251. doi: 10.1128/jb.150.3.1244-1251.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bishop P. E., Premakumar R., Dean D. R., Jacobson M. R., Chisnell J. R., Rizzo T. M., Kopczynski J. Nitrogen Fixation by Azotobacter vinelandii Strains Having Deletions in Structural Genes for Nitrogenase. Science. 1986 Apr 4;232(4746):92–94. doi: 10.1126/science.232.4746.92. [DOI] [PubMed] [Google Scholar]
  6. Bishop P. E., Rizzo T. M., Bott K. F. Molecular cloning of nif DNA from Azotobacter vinelandii. J Bacteriol. 1985 Apr;162(1):21–28. doi: 10.1128/jb.162.1.21-28.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brigle K. E., Newton W. E., Dean D. R. Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster. Gene. 1985;37(1-3):37–44. doi: 10.1016/0378-1119(85)90255-0. [DOI] [PubMed] [Google Scholar]
  8. Bulen W. A., LeComte J. R. The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and ATP hydrolysis. Proc Natl Acad Sci U S A. 1966 Sep;56(3):979–986. doi: 10.1073/pnas.56.3.979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carlomagno M. S., Riccio A., Bruni C. B. Convergently functional, Rho-independent terminator in Salmonella typhimurium. J Bacteriol. 1985 Jul;163(1):362–368. doi: 10.1128/jb.163.1.362-368.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chisnell J. R., Premakumar R., Bishop P. E. Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii. J Bacteriol. 1988 Jan;170(1):27–33. doi: 10.1128/jb.170.1.27-33.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DE WITT C. W., ROWE J. A. N,O-Diacetylneuraminic acid and N-acetylneuraminic acid in Escherichia coli. Nature. 1959 Aug 1;184(Suppl 6):381–382. doi: 10.1038/184381b0. [DOI] [PubMed] [Google Scholar]
  12. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eady R. R., Robson R. L., Richardson T. H., Miller R. W., Hawkins M. The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein. Biochem J. 1987 May 15;244(1):197–207. doi: 10.1042/bj2440197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fickett J. W. Recognition of protein coding regions in DNA sequences. Nucleic Acids Res. 1982 Sep 11;10(17):5303–5318. doi: 10.1093/nar/10.17.5303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  16. Gillum W. O., Mortenson L. E., Chen J. S., Holm R. H. Quantitative extrusions of the Fe4S4 cores of the active sites of ferredoxins and the hydrogenase of Clostridium pasteurianum. J Am Chem Soc. 1977 Jan 19;99(2):584–595. doi: 10.1021/ja00444a044. [DOI] [PubMed] [Google Scholar]
  17. Gribskov M., Devereux J., Burgess R. R. The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):539–549. doi: 10.1093/nar/12.1part2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hales B. J., Case E. E., Morningstar J. E., Dzeda M. F., Mauterer L. A. Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii. Biochemistry. 1986 Nov 18;25(23):7251–7255. doi: 10.1021/bi00371a001. [DOI] [PubMed] [Google Scholar]
  19. Hales B. J., Langosch D. J., Case E. E. Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii. J Biol Chem. 1986 Nov 15;261(32):15301–15306. [PubMed] [Google Scholar]
  20. Hausinger R. P., Howard J. B. Thiol reactivity of the nitrogenase Fe-protein from Azotobacter vinelandii. J Biol Chem. 1983 Nov 25;258(22):13486–13492. [PubMed] [Google Scholar]
  21. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  22. Holmes W. M., Platt T., Rosenberg M. Termination of transcription in E. coli. Cell. 1983 Apr;32(4):1029–1032. doi: 10.1016/0092-8674(83)90287-8. [DOI] [PubMed] [Google Scholar]
  23. Jacobson M. R., Premakumar R., Bishop P. E. Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii. J Bacteriol. 1986 Aug;167(2):480–486. doi: 10.1128/jb.167.2.480-486.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Joerger R. D., Bishop P. E. Bacterial alternative nitrogen fixation systems. Crit Rev Microbiol. 1988;16(1):1–14. doi: 10.3109/10408418809104465. [DOI] [PubMed] [Google Scholar]
  25. Joerger R. D., Bishop P. E. Nucleotide sequence and genetic analysis of the nifB-nifQ region from Azotobacter vinelandii. J Bacteriol. 1988 Apr;170(4):1475–1487. doi: 10.1128/jb.170.4.1475-1487.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Joerger R. D., Premakumar R., Bishop P. E. Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions. J Bacteriol. 1986 Nov;168(2):673–682. doi: 10.1128/jb.168.2.673-682.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Johnston R. E., Mackenzie J. M., Jr, Dougherty W. G. Assembly of overlapping DNA sequences by a program written in BASIC for 64K CP/M and MS-DOS IBM-compatible microcomputers. Nucleic Acids Res. 1986 Jan 10;14(1):517–527. doi: 10.1093/nar/14.1.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jones R., Woodley P., Robson R. Cloning and organisation of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumoniae. Mol Gen Genet. 1984;197(2):318–327. doi: 10.1007/BF00330980. [DOI] [PubMed] [Google Scholar]
  29. Kennedy C., Eady R. R., Kondorosi E., Rekosh D. K. The molybdenum--iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide 'mapping'. Biochem J. 1976 May 1;155(2):383–389. doi: 10.1042/bj1550383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
  31. Marinus M. G., Morris N. R. Pleiotropic effects of a DNA adenine methylation mutation (dam-3) in Escherichia coli K12. Mutat Res. 1975 Apr;28(1):15–26. doi: 10.1016/0027-5107(75)90309-7. [DOI] [PubMed] [Google Scholar]
  32. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  34. Misra T. K. A new strategy to create ordered deletions for rapid nucleotide sequencing. Gene. 1985;34(2-3):263–268. doi: 10.1016/0378-1119(85)90135-0. [DOI] [PubMed] [Google Scholar]
  35. Mount D. W., Conrad B. Microcomputer programs for graphic analysis of nucleic acid and protein sequences. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):811–817. doi: 10.1093/nar/12.1part2.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nebert D. W., Gonzalez F. J. P450 genes: structure, evolution, and regulation. Annu Rev Biochem. 1987;56:945–993. doi: 10.1146/annurev.bi.56.070187.004501. [DOI] [PubMed] [Google Scholar]
  37. Norgard M. V. Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase. Anal Biochem. 1981 May 1;113(1):34–42. doi: 10.1016/0003-2697(81)90040-3. [DOI] [PubMed] [Google Scholar]
  38. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  39. Orme-Johnson W. H. Molecular basis of biological nitrogen fixation. Annu Rev Biophys Biophys Chem. 1985;14:419–459. doi: 10.1146/annurev.bb.14.060185.002223. [DOI] [PubMed] [Google Scholar]
  40. Page W. J., von Tigerstrom M. Optimal conditions for transformation of Azotobacter vinelandii. J Bacteriol. 1979 Sep;139(3):1058–1061. doi: 10.1128/jb.139.3.1058-1061.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Phillips I. R., Shephard E. A., Ashworth A., Rabin B. R. Isolation and sequence of a human cytochrome P-450 cDNA clone. Proc Natl Acad Sci U S A. 1985 Feb;82(4):983–987. doi: 10.1073/pnas.82.4.983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Robinson A. C., Dean D. R., Burgess B. K. Iron-molybdenum cofactor biosynthesis in Azotobacter vinelandii requires the iron protein of nitrogenase. J Biol Chem. 1987 Oct 15;262(29):14327–14332. [PubMed] [Google Scholar]
  43. Robson R., Woodley P., Jones R. Second gene (nifH*) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein. EMBO J. 1986 Jun;5(6):1159–1163. doi: 10.1002/j.1460-2075.1986.tb04341.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shah V. K., Brill W. J. Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3249–3253. doi: 10.1073/pnas.74.8.3249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Smith B. E., Eady R. R., Lowe D. J., Gormal C. The vanadium-iron protein of vanadium nitrogenase from Azotobacter chroococcum contains an iron-vanadium cofactor. Biochem J. 1988 Feb 15;250(1):299–302. doi: 10.1042/bj2500299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Souillard N., Magot M., Possot O., Sibold L. Nucleotide sequence of regions homologous to nifH (nitrogenase Fe protein) from the nitrogen-fixing archaebacteria Methanococcus thermolithotrophicus and Methanobacterium ivanovii: evolutionary implications. J Mol Evol. 1988;27(1):65–76. doi: 10.1007/BF02099731. [DOI] [PubMed] [Google Scholar]
  48. Sternberg M. J., Taylor W. R. Modelling the ATP-binding site of oncogene products, the epidermal growth factor receptor and related proteins. FEBS Lett. 1984 Oct 1;175(2):387–392. doi: 10.1016/0014-5793(84)80774-7. [DOI] [PubMed] [Google Scholar]
  49. Unger B. P., Gunsalus I. C., Sligar S. G. Nucleotide sequence of the Pseudomonas putida cytochrome P-450cam gene and its expression in Escherichia coli. J Biol Chem. 1986 Jan 25;261(3):1158–1163. [PubMed] [Google Scholar]
  50. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  51. Wang S. Z., Chen J. S., Johnson J. L. The presence of five nifH-like sequences in Clostridium pasteurianum: sequence divergence and transcription properties. Nucleic Acids Res. 1988 Jan 25;16(2):439–454. doi: 10.1093/nar/16.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  53. Yates M. G., Planqué K. Nitrogenase from Azotobacter chroococcum. Purification and properties of the component proteins. Eur J Biochem. 1975 Dec 15;60(2):467–476. doi: 10.1111/j.1432-1033.1975.tb21025.x. [DOI] [PubMed] [Google Scholar]

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