Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1992 Jun;174(12):3855–3866. doi: 10.1128/jb.174.12.3855-3866.1992

Isolation and characterization of the nifUSVW-rpoN gene cluster from Rhodobacter sphaeroides.

W G Meijer 1, F R Tabita 1
PMCID: PMC206092  PMID: 1317839

Abstract

The rpoN gene from Rhodobacter sphaeroides was isolated from a genomic library via complementation of a Rhodobacter capsulatus rpoN mutant. The rpoN gene was located on a 7.5-kb HindIII-EcoRI fragment. A Tn5 insertion analysis of this DNA fragment showed that a minimal DNA fragment of 5.3 kb was required for complementation. Nucleotide sequencing of the complementing region revealed the presence of nifUSVW genes upstream from rpoN. The rpoN gene was mutagenized via insertion of a gene encoding kanamycin resistance. The resulting rpoN mutant was not impaired in diazotrophic growth and was in all respects indistinguishable from the wild-type strain. Southern hybridizations using the cloned rpoN gene as a probe indicated the presence of a second rpoN gene. Deletion of the nifUS genes resulted in strongly reduced diazotrophic growth. Two conserved regions were identified in a NifV LeuA amino acid sequence alignment. Similar regions were found in pyruvate carboxylase and oxaloacetate decarboxylase. It is proposed that these conserved regions represent keto acid-binding sites.

Full text

PDF
3855

Images in this article

3864Image
on p.3864

Selected References

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

  1. Arigoni F., Kaminski P. A., Hennecke H., Elmerich C. Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW. Mol Gen Genet. 1991 Mar;225(3):514–520. doi: 10.1007/BF00261695. [DOI] [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. Avtges P., Kranz R. G., Haselkorn R. Isolation and organization of genes for nitrogen fixation in Rhodopseudomonas capsulata. Mol Gen Genet. 1985;201(3):363–369. doi: 10.1007/BF00331324. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Berger D. K., Woods D. R., Rawlings D. E. Complementation of Escherichia coli sigma 54 (NtrA)-dependent formate hydrogenlyase activity by a cloned Thiobacillus ferrooxidans ntrA gene. J Bacteriol. 1990 Aug;172(8):4399–4406. doi: 10.1128/jb.172.8.4399-4406.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Beynon J., Cannon M., Buchanan-Wollaston V., Ally A., Setterquist R., Dean D., Cannon F. The nucleotide sequence of the nifT, nifY, nifX and nifW genes of K. pneumoniae. Nucleic Acids Res. 1988 Oct 25;16(20):9860–9860. doi: 10.1093/nar/16.20.9860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Borthakur D., Basche M., Buikema W. J., Borthakur P. B., Haselkorn R. Expression, nucleotide sequence and mutational analysis of two open reading frames in the nif gene region of Anabaena sp. strain PCC7120. Mol Gen Genet. 1990 Apr;221(2):227–234. doi: 10.1007/BF00261725. [DOI] [PubMed] [Google Scholar]
  11. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  12. Evans D. J., Jones R., Woodley P. R., Wilborn J. R., Robson R. L. Nucleotide sequence and genetic analysis of the Azotobacter chroococcum nifUSVWZM gene cluster, including a new gene (nifP) which encodes a serine acetyltransferase. J Bacteriol. 1991 Sep;173(17):5457–5469. doi: 10.1128/jb.173.17.5457-5469.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Figurski D., Meyer R., Miller D. S., Helinski D. R. Generation in vitro of deletions in the broad host range plasmid RK2 using phage Mu insertions and a restriction endonuclease. Gene. 1976;1(1):107–119. doi: 10.1016/0378-1119(76)90010-x. [DOI] [PubMed] [Google Scholar]
  14. Gibson J. L., Chen J. H., Tower P. A., Tabita F. R. The form II fructose 1,6-bisphosphatase and phosphoribulokinase genes form part of a large operon in Rhodobacter sphaeroides: primary structure and insertional mutagenesis analysis. Biochemistry. 1990 Sep 4;29(35):8085–8093. doi: 10.1021/bi00487a014. [DOI] [PubMed] [Google Scholar]
  15. Gibson J. L., Falcone D. L., Tabita F. R. Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides. J Biol Chem. 1991 Aug 5;266(22):14646–14653. [PubMed] [Google Scholar]
  16. Gibson J. L., Tabita F. R. Localization and mapping of CO2 fixation genes within two gene clusters in Rhodobacter sphaeroides. J Bacteriol. 1988 May;170(5):2153–2158. doi: 10.1128/jb.170.5.2153-2158.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gibson J. L., Tabita F. R. Organization of phosphoribulokinase and ribulose bisphosphate carboxylase/oxygenase genes in Rhodopseudomonas (Rhodobacter) sphaeroides. J Bacteriol. 1987 Aug;169(8):3685–3690. doi: 10.1128/jb.169.8.3685-3690.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gribskov M., McLachlan A. D., Eisenberg D. Profile analysis: detection of distantly related proteins. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4355–4358. doi: 10.1073/pnas.84.13.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Helmann J. D., Chamberlin M. J. Structure and function of bacterial sigma factors. Annu Rev Biochem. 1988;57:839–872. doi: 10.1146/annurev.bi.57.070188.004203. [DOI] [PubMed] [Google Scholar]
  20. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  21. Higgins C. F., Hiles I. D., Salmond G. P., Gill D. R., Downie J. A., Evans I. J., Holland I. B., Gray L., Buckel S. D., Bell A. W. A family of related ATP-binding subunits coupled to many distinct biological processes in bacteria. Nature. 1986 Oct 2;323(6087):448–450. doi: 10.1038/323448a0. [DOI] [PubMed] [Google Scholar]
  22. Hirschman J., Wong P. K., Sei K., Keener J., Kustu S. Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7525–7529. doi: 10.1073/pnas.82.22.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Hoover T. R., Shah V. K., Roberts G. P., Ludden P. W. nifV-dependent, low-molecular-weight factor required for in vitro synthesis of iron-molybdenum cofactor of nitrogenase. J Bacteriol. 1986 Sep;167(3):999–1003. doi: 10.1128/jb.167.3.999-1003.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Inouye S., Yamada M., Nakazawa A., Nakazawa T. Cloning and sequence analysis of the ntrA (rpoN) gene of Pseudomonas putida. Gene. 1989 Dec 21;85(1):145–152. doi: 10.1016/0378-1119(89)90474-5. [DOI] [PubMed] [Google Scholar]
  27. Ishimoto K. S., Lory S. Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase. Proc Natl Acad Sci U S A. 1989 Mar;86(6):1954–1957. doi: 10.1073/pnas.86.6.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Jones R., Haselkorn R. The DNA sequence of the Rhodobacter capsulatus ntrA, ntrB and ntrC gene analogues required for nitrogen fixation. Mol Gen Genet. 1989 Feb;215(3):507–516. doi: 10.1007/BF00427050. [DOI] [PubMed] [Google Scholar]
  31. Kay R., McPherson J. Hybrid pUC vectors for addition of new restriction enzyme sites to the ends of DNA fragments. Nucleic Acids Res. 1987 Mar 25;15(6):2778–2778. doi: 10.1093/nar/15.6.2778. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Klipp W., Masepohl B., Pühler A. Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region. J Bacteriol. 1988 Feb;170(2):693–699. doi: 10.1128/jb.170.2.693-699.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kullik I., Fritsche S., Knobel H., Sanjuan J., Hennecke H., Fischer H. M. Bradyrhizobium japonicum has two differentially regulated, functional homologs of the sigma 54 gene (rpoN). J Bacteriol. 1991 Feb;173(3):1125–1138. doi: 10.1128/jb.173.3.1125-1138.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kustu S., Santero E., Keener J., Popham D., Weiss D. Expression of sigma 54 (ntrA)-dependent genes is probably united by a common mechanism. Microbiol Rev. 1989 Sep;53(3):367–376. doi: 10.1128/mr.53.3.367-376.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Köhler T., Harayama S., Ramos J. L., Timmis K. N. Involvement of Pseudomonas putida RpoN sigma factor in regulation of various metabolic functions. J Bacteriol. 1989 Aug;171(8):4326–4333. doi: 10.1128/jb.171.8.4326-4333.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lim F., Morris C. P., Occhiodoro F., Wallace J. C. Sequence and domain structure of yeast pyruvate carboxylase. J Biol Chem. 1988 Aug 15;263(23):11493–11497. [PubMed] [Google Scholar]
  37. 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]
  38. Mathews F. S. The structure, function and evolution of cytochromes. Prog Biophys Mol Biol. 1985;45(1):1–56. doi: 10.1016/0079-6107(85)90004-5. [DOI] [PubMed] [Google Scholar]
  39. Merrick M. J., Gibbins J. R. The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. Nucleic Acids Res. 1985 Nov 11;13(21):7607–7620. doi: 10.1093/nar/13.21.7607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Merrick M. J., Stewart W. D. Studies on the regulation and function of the Klebsiella pneumoniae ntrA gene. Gene. 1985;35(3):297–303. doi: 10.1016/0378-1119(85)90008-3. [DOI] [PubMed] [Google Scholar]
  41. Merrick M., Gibbins J., Toukdarian A. The nucleotide sequence of the sigma factor gene ntrA (rpoN) of Azotobacter vinelandii: analysis of conserved sequences in NtrA proteins. Mol Gen Genet. 1987 Dec;210(2):323–330. doi: 10.1007/BF00325701. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. ORMEROD J. G., ORMEROD K. S., GEST H. Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch Biochem Biophys. 1961 Sep;94:449–463. doi: 10.1016/0003-9861(61)90073-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. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  46. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Ronson C. W., Nixon B. T., Albright L. M., Ausubel F. M. Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions. J Bacteriol. 1987 Jun;169(6):2424–2431. doi: 10.1128/jb.169.6.2424-2431.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Römermann D., Warrelmann J., Bender R. A., Friedrich B. An rpoN-like gene of Alcaligenes eutrophus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxidation. J Bacteriol. 1989 Feb;171(2):1093–1099. doi: 10.1128/jb.171.2.1093-1099.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sasse-Dwight S., Gralla J. D. Role of eukaryotic-type functional domains found in the prokaryotic enhancer receptor factor sigma 54. Cell. 1990 Sep 7;62(5):945–954. doi: 10.1016/0092-8674(90)90269-k. [DOI] [PubMed] [Google Scholar]
  51. Schwarz E., Oesterhelt D., Reinke H., Beyreuther K., Dimroth P. The sodium ion translocating oxalacetate decarboxylase of Klebsiella pneumoniae. Sequence of the biotin-containing alpha-subunit and relationship to other biotin-containing enzymes. J Biol Chem. 1988 Jul 15;263(20):9640–9645. [PubMed] [Google Scholar]
  52. Suwanto A., Kaplan S. Physical and genetic mapping of the Rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. J Bacteriol. 1989 Nov;171(11):5850–5859. doi: 10.1128/jb.171.11.5850-5859.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tabita F. R., Gibson J. L., Falcone D. L., Lee B. G., Chen J. H. Recent studies on the molecular biology and biochemistry of CO2 fixation in phototrophic bacteria. FEMS Microbiol Rev. 1990 Dec;7(3-4):437–443. doi: 10.1111/j.1574-6968.1990.tb04950.x. [DOI] [PubMed] [Google Scholar]
  54. Tabita F. R. Molecular and cellular regulation of autotrophic carbon dioxide fixation in microorganisms. Microbiol Rev. 1988 Jun;52(2):155–189. doi: 10.1128/mr.52.2.155-189.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  56. Wall J. D., Love J., Quinn S. P. Spontaneous Nif- mutants of Rhodopseudomonas capsulata. J Bacteriol. 1984 Aug;159(2):652–657. doi: 10.1128/jb.159.2.652-657.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weaver K. E., Tabita F. R. Complementation of a Rhodopseudomonas sphaeroides ribulose bisphosphate carboxylase-oxygenase regulatory mutant from a genomic library. J Bacteriol. 1985 Oct;164(1):147–154. doi: 10.1128/jb.164.1.147-154.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Weaver K. E., Tabita F. R. Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase. J Bacteriol. 1983 Nov;156(2):507–515. doi: 10.1128/jb.156.2.507-515.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. van Slooten J. C., Cervantes E., Broughton W. J., Wong C. H., Stanley J. Sequence and analysis of the rpoN sigma factor gene of rhizobium sp. strain NGR234, a primary coregulator of symbiosis. J Bacteriol. 1990 Oct;172(10):5563–5574. doi: 10.1128/jb.172.10.5563-5574.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES