Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Sep;173(17):5457–5469. doi: 10.1128/jb.173.17.5457-5469.1991

Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase.

D J Evans 1, R Jones 1, P R Woodley 1, J R Wilborn 1, R L Robson 1
PMCID: PMC208258  PMID: 1885524

Abstract

Nucleotide sequence was obtained for a region of 7,099 bp spanning the nifU, nifS, nifV, nifW, nifZ, and nifM genes from Azotobacter chroococcum. Chromosomal mutations constructed at several sites within the locus confirmed a requirement for this region for expression of the molybdenum nitrogenase in this organism. The genes are tightly clustered and ordered as in Klebsiella pneumoniae except for two additional open reading frames (ORFs) between nifV and nifW. The arrangement of genes in A. chroococcum closely matches that described for Azotobacter vinelandii. The polypeptide encoded by ORF4 immediately downstream from nifV is 41% identical over 186 amino acids to the product of the cysE gene from Escherichia coli, which encodes serine acetyltransferase (SAT), a key enzyme in cysteine biosynthesis. Plasmids which potentially express ORF4 complemented E. coli JM39, a cysteine auxotroph which lacks SAT. SAT activity was detected in crude extracts of one such complemented strain. A strain of A. chroococcum carrying a chromosomal disruption of ORF4 grew normally with ammonium as the N source but more slowly than the parental strain when N2 was the sole N source. These data suggest that ORF4 encodes a nif-specific SAT required for optimizing expression of nitrogenase activity. ORF4 was assigned the name nifP. nifP may be required to boost rates of synthesis or intracellular concentrations of cysteine or methionine. Sequence identity between nifV and leuA gene products suggests that nifV may catalyze a condensation reaction analogous to that carried out by isopropylmalate synthase (LEUA) but in which acetyl coenzyme and alpha-ketoglutarate are substrates for the formation of homocitrate, the proposed product of NIFV activity.

Full text

PDF
5460

Selected References

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

  1. Ambler R. P., Bartsch R. G., Daniel M., Kamen M. D., McLellan L., Meyer T. E., Van Beeumen J. Amino acid sequences of bacterial cytochromes c' and c-556. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6854–6857. doi: 10.1073/pnas.78.11.6854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnold W., Rump A., Klipp W., Priefer U. B., Pühler A. Nucleotide sequence of a 24,206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J Mol Biol. 1988 Oct 5;203(3):715–738. doi: 10.1016/0022-2836(88)90205-7. [DOI] [PubMed] [Google Scholar]
  3. Beltzer J. P., Chang L. F., Hinkkanen A. E., Kohlhaw G. B. Structure of yeast LEU4. The 5' flanking region contains features that predict two modes of control and two productive translation starts. J Biol Chem. 1986 Apr 15;261(11):5160–5167. [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. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Byrne C. R., Monroe R. S., Ward K. A., Kredich N. M. DNA sequences of the cysK regions of Salmonella typhimurium and Escherichia coli and linkage of the cysK regions to ptsH. J Bacteriol. 1988 Jul;170(7):3150–3157. doi: 10.1128/jb.170.7.3150-3157.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Denk D., Böck A. L-cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant. J Gen Microbiol. 1987 Mar;133(3):515–525. doi: 10.1099/00221287-133-3-515. [DOI] [PubMed] [Google Scholar]
  9. Dente L., Cesareni G., Cortese R. pEMBL: a new family of single stranded plasmids. Nucleic Acids Res. 1983 Mar 25;11(6):1645–1655. doi: 10.1093/nar/11.6.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Dixon R. A. The genetic complexity of nitrogen fixation. The ninth Fleming lecture. J Gen Microbiol. 1984 Nov;130(11):2745–2755. doi: 10.1099/00221287-130-11-2745. [DOI] [PubMed] [Google Scholar]
  12. Dixon R., Kennedy C., Kondorosi A., Krishnapillai V., Merrick M. Complementation analysis of Klebsiella pneumoniae mutants defective in nitrogen fixation. Mol Gen Genet. 1977 Nov 29;157(2):189–198. doi: 10.1007/BF00267397. [DOI] [PubMed] [Google Scholar]
  13. Downie J. A. The nodL gene from Rhizobium leguminosarum is homologous to the acetyl transferases encoded by lacA and cysE. Mol Microbiol. 1989 Nov;3(11):1649–1651. doi: 10.1111/j.1365-2958.1989.tb00150.x. [DOI] [PubMed] [Google Scholar]
  14. Earl C. D., Ronson C. W., Ausubel F. M. Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes. J Bacteriol. 1987 Mar;169(3):1127–1136. doi: 10.1128/jb.169.3.1127-1136.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Evans D., Jones R., Woodley P., Robson R. Further analysis of nitrogen fixation (nif) genes in Azotobacter chroococcum: identification and expression in Klebsiella pneumoniae of nifS, nifV, nifM, and nifB genes and localization of nifE/N-, nifU-, nifA- and fixABC-like genes. J Gen Microbiol. 1988 Apr;134(4):931–942. doi: 10.1099/00221287-134-4-931. [DOI] [PubMed] [Google Scholar]
  16. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [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. Hediger M. A., Johnson D. F., Nierlich D. P., Zabin I. DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6414–6418. doi: 10.1073/pnas.82.19.6414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hoover T. R., Imperial J., Ludden P. W., Shah V. K. Homocitrate is a component of the iron-molybdenum cofactor of nitrogenase. Biochemistry. 1989 Apr 4;28(7):2768–2771. doi: 10.1021/bi00433a004. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Howard K. S., McLean P. A., Hansen F. B., Lemley P. V., Koblan K. S., Orme-Johnson W. H. Klebsiella pneumoniae nifM gene product is required for stabilization and activation of nitrogenase iron protein in Escherichia coli. J Biol Chem. 1986 Jan 15;261(2):772–778. [PubMed] [Google Scholar]
  22. Hryniewicz M., Sirko A., Pałucha A., Böck A., Hulanicka D. Sulfate and thiosulfate transport in Escherichia coli K-12: identification of a gene encoding a novel protein involved in thiosulfate binding. J Bacteriol. 1990 Jun;172(6):3358–3366. doi: 10.1128/jb.172.6.3358-3366.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jacobson M. R., Brigle K. E., Bennett L. T., Setterquist R. A., Wilson M. S., Cash V. L., Beynon J., Newton W. E., Dean D. R. Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J Bacteriol. 1989 Feb;171(2):1017–1027. doi: 10.1128/jb.171.2.1017-1027.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jacobson M. R., Cash V. L., Weiss M. C., Laird N. F., Newton W. E., Dean D. R. Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii. Mol Gen Genet. 1989 Oct;219(1-2):49–57. doi: 10.1007/BF00261156. [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. Jones-Mortimer M. C. Positive control of sulphate reduction in Escherichia coli. Isolation, characterization and mapping oc cysteineless mutants of E. coli K12. Biochem J. 1968 Dec;110(3):589–595. doi: 10.1042/bj1100589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Kaminski P. A., Norel F., Desnoues N., Kush A., Salzano G., Elmerich C. Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene. Mol Gen Genet. 1988 Nov;214(3):496–502. doi: 10.1007/BF00330486. [DOI] [PubMed] [Google Scholar]
  29. Kredich N. M., Tomkins G. M. The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. J Biol Chem. 1966 Nov 10;241(21):4955–4965. [PubMed] [Google Scholar]
  30. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  31. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Levy S., Danchin A. Phylogeny of metabolic pathways: O-acetylserine sulphydrylase A is homologous to the tryptophan synthase beta subunit. Mol Microbiol. 1988 Nov;2(6):777–783. doi: 10.1111/j.1365-2958.1988.tb00089.x. [DOI] [PubMed] [Google Scholar]
  33. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  34. Masepohl B., Klipp W., Pühler A. Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus. Mol Gen Genet. 1988 Apr;212(1):27–37. doi: 10.1007/BF00322441. [DOI] [PubMed] [Google Scholar]
  35. McLean P. A., Dixon R. A. Requirement of nifV gene for production of wild-type nitrogenase enzyme in Klebsiella pneumoniae. Nature. 1981 Aug 13;292(5824):655–656. doi: 10.1038/292655a0. [DOI] [PubMed] [Google Scholar]
  36. Merrick M. J., Gibbins J. R., Postgate J. R. A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Escherichia coli. J Gen Microbiol. 1987 Aug;133(8):2053–2057. doi: 10.1099/00221287-133-8-2053. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Merrick M., Filser M., Kennedy C., Dixon R. Polarity of mutations induced by insertion of transposons Tn5, Tn7 and Tn10 into the nif gene cluster of Klebsiella pneumoniae. Mol Gen Genet. 1978 Sep 20;165(1):103–111. doi: 10.1007/BF00270382. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Mulligan M. E., Haselkorn R. Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon. J Biol Chem. 1989 Nov 15;264(32):19200–19207. [PubMed] [Google Scholar]
  41. 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]
  42. Pagani S., Eldridge M., Eady R. R. Nitrogenase of Klebsiella pneumoniae. Rhodanese-catalysed restoration of activity of the inactive 2Fe species of the Fe protein. Biochem J. 1987 Jun 1;244(2):485–488. doi: 10.1042/bj2440485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Paul W., Merrick M. The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis. Eur J Biochem. 1989 Jan 2;178(3):675–682. doi: 10.1111/j.1432-1033.1989.tb14497.x. [DOI] [PubMed] [Google Scholar]
  45. Ploegman J. H., Drent G., Kalk K. H., Hol W. G., Heinrikson R. L., Keim P., Weng L., Russell J. The covalent and tertiary structure of bovine liver rhodanese. Nature. 1978 May 11;273(5658):124–129. doi: 10.1038/273124a0. [DOI] [PubMed] [Google Scholar]
  46. Ricca E., Calvo J. M. The nucleotide sequence of leuA from Salmonella typhimurium. Nucleic Acids Res. 1990 Mar 11;18(5):1290–1290. doi: 10.1093/nar/18.5.1290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Robson R. L., Chesshyre J. A., Wheeler C., Jones R., Woodley P. R., Postgate J. R. Genome size and complexity in Azotobacter chroococcum. J Gen Microbiol. 1984 Jul;130(7):1603–1612. doi: 10.1099/00221287-130-7-1603. [DOI] [PubMed] [Google Scholar]
  48. Robson R. L., Postgate J. R. Oxygen and hydrogen in biological nitrogen fixation. Annu Rev Microbiol. 1980;34:183–207. doi: 10.1146/annurev.mi.34.100180.001151. [DOI] [PubMed] [Google Scholar]
  49. Robson R. L., Woodley P. R., Pau R. N., Eady R. R. Structural genes for the vanadium nitrogenase from Azotobacter chroococcum. EMBO J. 1989 Apr;8(4):1217–1224. doi: 10.1002/j.1460-2075.1989.tb03495.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Sibold L. The polar effect on nifM of mutations in the nifU,-S,-V genes of Klebsiella pneumoniae depends on their plasmid or chromosomal location. Mol Gen Genet. 1982;186(4):569–571. doi: 10.1007/BF00337966. [DOI] [PubMed] [Google Scholar]
  54. Sirko A., Hryniewicz M., Hulanicka D., Böck A. Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster. J Bacteriol. 1990 Jun;172(6):3351–3357. doi: 10.1128/jb.172.6.3351-3357.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Smith T. F., Waterman M. S. Identification of common molecular subsequences. J Mol Biol. 1981 Mar 25;147(1):195–197. doi: 10.1016/0022-2836(81)90087-5. [DOI] [PubMed] [Google Scholar]
  56. Staden R. Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 1982 Aug 11;10(15):4731–4751. doi: 10.1093/nar/10.15.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Strassman M., Ceci L. N. Enzymatic formation of homocitric acid, an intermediate in lysine biosynthesis. Biochem Biophys Res Commun. 1964;14:262–267. doi: 10.1016/0006-291x(64)90446-2. [DOI] [PubMed] [Google Scholar]
  58. Thuring R. W., Sanders J. P., Borst P. A freeze-squeeze method for recovering long DNA from agarose gels. Anal Biochem. 1975 May 26;66(1):213–220. doi: 10.1016/0003-2697(75)90739-3. [DOI] [PubMed] [Google Scholar]
  59. Tucci A. F., Ceci L. N. Homocitrate synthase from yeast. Arch Biochem Biophys. 1972 Dec;153(2):742–750. doi: 10.1016/0003-9861(72)90393-1. [DOI] [PubMed] [Google Scholar]
  60. Ugalde R. A., Imperial J., Shah V. K., Brill W. J. Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source. J Bacteriol. 1985 Dec;164(3):1081–1087. doi: 10.1128/jb.164.3.1081-1087.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. 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]
  62. White R. H. Origin of the labile sulfide in the iron-sulfur proteins of Escherichia coli. Biochem Biophys Res Commun. 1983 Apr 15;112(1):66–72. doi: 10.1016/0006-291x(83)91798-9. [DOI] [PubMed] [Google Scholar]
  63. 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]

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

RESOURCES