Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Feb;171(2):1017–1027. doi: 10.1128/jb.171.2.1017-1027.1989

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

M R Jacobson 1, K E Brigle 1, L T Bennett 1, R A Setterquist 1, M S Wilson 1, V L Cash 1, J Beynon 1, W E Newton 1, D R Dean 1
PMCID: PMC209696  PMID: 2644218

Abstract

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.

Full text

PDF
1017

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baughman G., Nomura M. Localization of the target site for translational regulation of the L11 operon and direct evidence for translational coupling in Escherichia coli. Cell. 1983 Oct;34(3):979–988. doi: 10.1016/0092-8674(83)90555-x. [DOI] [PubMed] [Google Scholar]
  2. Bennett L. T., Cannon F., Dean D. R. Nucleotide sequence and mutagenesis of the nifA gene from Azotobacter vinelandii. Mol Microbiol. 1988 May;2(3):315–321. doi: 10.1111/j.1365-2958.1988.tb00034.x. [DOI] [PubMed] [Google Scholar]
  3. Bennett L. T., Jacobson M. R., Dean D. R. Isolation, sequencing, and mutagenesis of the nifF gene encoding flavodoxin from Azotobacter vinelandii. J Biol Chem. 1988 Jan 25;263(3):1364–1369. [PubMed] [Google Scholar]
  4. Beynon J., Ally A., Cannon M., Cannon F., Jacobson M., Cash V., Dean D. Comparative organization of nitrogen fixation-specific genes from Azotobacter vinelandii and Klebsiella pneumoniae: DNA sequence of the nifUSV genes. J Bacteriol. 1987 Sep;169(9):4024–4029. doi: 10.1128/jb.169.9.4024-4029.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  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., Dean D. R. Revised nucleotide sequence of the Azotobacter vinelandii nifE gene. Nucleic Acids Res. 1988 Jun 10;16(11):5214–5214. doi: 10.1093/nar/16.11.5214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Brigle K. E., Setterquist R. A., Dean D. R., Cantwell J. S., Weiss M. C., Newton W. E. Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7066–7069. doi: 10.1073/pnas.84.20.7066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brigle K. E., Weiss M. C., Newton W. E., Dean D. R. Products of the iron-molybdenum cofactor-specific biosynthetic genes, nifE and nifN, are structurally homologous to the products of the nitrogenase molybdenum-iron protein genes, nifD and nifK. J Bacteriol. 1987 Apr;169(4):1547–1553. doi: 10.1128/jb.169.4.1547-1553.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Buikema W. J., Klingensmith J. A., Gibbons S. L., Ausubel F. M. Conservation of structure and location of Rhizobium meliloti and Klebsiella pneumoniae nifB genes. J Bacteriol. 1987 Mar;169(3):1120–1126. doi: 10.1128/jb.169.3.1120-1126.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Buikema W. J., Szeto W. W., Lemley P. V., Orme-Johnson W. H., Ausubel F. M. Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae. Nucleic Acids Res. 1985 Jun 25;13(12):4539–4555. doi: 10.1093/nar/13.12.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Dean D. R., Brigle K. E. Azotobacter vinelandii nifD- and nifE-encoded polypeptides share structural homology. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5720–5723. doi: 10.1073/pnas.82.17.5720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Drummond M. H. The base sequence of the nifF gene of Klebsiella pneumoniae and homology of the predicted amino acid sequence of its protein product to other flavodoxins. Biochem J. 1985 Dec 15;232(3):891–896. doi: 10.1042/bj2320891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Drummond M. H., Wootton J. C. Sequence of nifL from Klebsiella pneumoniae: mode of action and relationship to two families of regulatory proteins. Mol Microbiol. 1987 Jul;1(1):37–44. doi: 10.1111/j.1365-2958.1987.tb00524.x. [DOI] [PubMed] [Google Scholar]
  17. Drummond M., Whitty P., Wootton J. Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J. 1986 Feb;5(2):441–447. doi: 10.1002/j.1460-2075.1986.tb04230.x. [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. Hiratsuka K., Roy K. L. Sequence of a 1.4 kb Eco RI fragment of Azotobacter vinelandii nif DNA. Nucleic Acids Res. 1988 Feb 11;16(3):1207–1207. doi: 10.1093/nar/16.3.1207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Holland D., Zilberstein A., Zamir A., Sussman J. L. A quantitative approach to sequence comparisons of nitrogenase MoFe protein alpha- and beta-subunits including the newly sequenced nifK gene from Klebsiella pneumoniae. Biochem J. 1987 Oct 15;247(2):277–285. doi: 10.1042/bj2470277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hoover T. R., Robertson A. D., Cerny R. L., Hayes R. N., Imperial J., Shah V. K., Ludden P. W. Identification of the V factor needed for synthesis of the iron-molybdenum cofactor of nitrogenase as homocitrate. 1987 Oct 29-Nov 4Nature. 329(6142):855–857. doi: 10.1038/329855a0. [DOI] [PubMed] [Google Scholar]
  22. Ioannidis I., Buck M. Nucleotide sequence of the Klebsiella pneumoniae nifD gene and predicted amino acid sequence of the alpha-subunit of nitrogenase MoFe protein. Biochem J. 1987 Oct 15;247(2):287–291. doi: 10.1042/bj2470287. [DOI] [PMC free article] [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. 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]
  25. Merrick M., Filser M., Dixon R., Elmerich C., Sibold L., Houmard J. The use of translocatable genetic elements to construct a fine-structure map of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster. J Gen Microbiol. 1980 Apr;117(2):509–520. doi: 10.1099/00221287-117-2-509. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Nieva-Gómez D., Roberts G. P., Klevickis S., Brill W. J. Electron transport to nitrogenase in Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1980 May;77(5):2555–2558. doi: 10.1073/pnas.77.5.2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Oppenheim D. S., Yanofsky C. Translational coupling during expression of the tryptophan operon of Escherichia coli. Genetics. 1980 Aug;95(4):785–795. doi: 10.1093/genetics/95.4.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Paul W., Merrick M. The nucleotide sequence of the nifM gene of Klebsiella pneumoniae and identification of a new nif gene: nifZ. Eur J Biochem. 1987 Dec 30;170(1-2):259–265. doi: 10.1111/j.1432-1033.1987.tb13694.x. [DOI] [PubMed] [Google Scholar]
  31. Roberts G. P., Brill W. J. Gene-product relationships of the nif regulon of Klebsiella pneumoniae. J Bacteriol. 1980 Oct;144(1):210–216. doi: 10.1128/jb.144.1.210-216.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Robinson A. C., Burgess B. K., Dean D. R. Activity, reconstitution, and accumulation of nitrogenase components in Azotobacter vinelandii mutant strains containing defined deletions within the nitrogenase structural gene cluster. J Bacteriol. 1986 Apr;166(1):180–186. doi: 10.1128/jb.166.1.180-186.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Scott K. F., Rolfe B. G., Shine J. Biological nitrogen fixation: primary structure of the Klebsiella pneumoniae nifH and nifD genes. J Mol Appl Genet. 1981;1(1):71–81. [PubMed] [Google Scholar]
  36. Setterquist R., Brigle K. E., Beynon J., Cannon M., Ally A., Cannon F., Dean D. R. Nucleotide sequence of the nifE and nifN genes from Klebsiella pneumoniae. Nucleic Acids Res. 1988 Jun 10;16(11):5215–5215. doi: 10.1093/nar/16.11.5215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shah V. K., Stacey G., Brill W. J. Electron transport to nitrogenase. Purification and characterization of pyruvate:flavodoxin oxidoreductase. The nifJ gene product. J Biol Chem. 1983 Oct 10;258(19):12064–12068. [PubMed] [Google Scholar]
  38. Strandberg G. W., Wilson P. W. Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can J Microbiol. 1968 Jan;14(1):25–31. doi: 10.1139/m68-005. [DOI] [PubMed] [Google Scholar]
  39. Sundaresan V., Ausubel F. M. Nucleotide sequence of the gene coding for the nitrogenase iron protein from Klebsiella pneumoniae. J Biol Chem. 1981 Mar 25;256(6):2808–2812. [PubMed] [Google Scholar]
  40. Toukdarian A., Kennedy C. Regulation of nitrogen metabolism in Azotobacter vinelandii: isolation of ntr and glnA genes and construction of ntr mutants. EMBO J. 1986 Feb;5(2):399–407. doi: 10.1002/j.1460-2075.1986.tb04225.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES