Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Aug;178(15):4493–4499. doi: 10.1128/jb.178.15.4493-4499.1996

Isolation and characterization of the VnfEN genes of the cyanobacterium Anabaena variabilis.

T Thiel 1
PMCID: PMC178215  PMID: 8755876

Abstract

The filamentous cyanobacterium Anabaena variabilis fixes nitrogen in the presence of vanadium (V) and in the absence of molybdenum (Mo), using a V-dependent nitrogenase (V-nitrogenase) encoded by the vnfDGK genes. Downstream from these genes are two genes that are similar to the vnfEN genes of Azotobacter vinelandii. Like the vnfDGK genes, the vnfEN genes were transcribed in the absence of Mo, whether or not V was present. A mutant with an insertion in the vnfN gene lacked V-nitrogenase activity; thus, the vnfEN genes were essential for the V-nitrogenase system in A. variabilis. Growth and acetylene reduction assays with wild-type and mutant strains suggested that the V-nitrogenase reduced dinitrogen better than acetylene. The similarity of the vnfEN genes of A. variabilis and A. vinelandii was not strong. The vnfEN genes of A. variabilis showed greater similarity to the vnfDK genes just upstream than to the A. vinelandii vnfEN genes. Sequence comparisons provide support for the idea that if the vnf genes were transferred laterally among bacterial strains, the vnf cluster was not transferred intact. It appears likely that the structural genes were transferred before a duplication event led to the evolution of the vnfEN genes independently in the two strains. The divergence of the vnfEN genes from the vnfDK genes suggests that this duplication, and hence the transfer of vnf genes, was an ancient event.

Full Text

The Full Text of this article is available as a PDF (381.9 KB).

Selected References

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

  1. Allen M. B., Arnon D. I. Studies on Nitrogen-Fixing Blue-Green Algae. I. Growth and Nitrogen Fixation by Anabaena Cylindrica Lemm. Plant Physiol. 1955 Jul;30(4):366–372. doi: 10.1104/pp.30.4.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen R. M., Chatterjee R., Ludden P. W., Shah V. K. Incorporation of iron and sulfur from NifB cofactor into the iron-molybdenum cofactor of dinitrogenase. J Biol Chem. 1995 Nov 10;270(45):26890–26896. doi: 10.1074/jbc.270.45.26890. [DOI] [PubMed] [Google Scholar]
  3. Black T. A., Cai Y., Wolk C. P. Spatial expression and autoregulation of hetR, a gene involved in the control of heterocyst development in Anabaena. Mol Microbiol. 1993 Jul;9(1):77–84. doi: 10.1111/j.1365-2958.1993.tb01670.x. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Brusca J. S., Hale M. A., Carrasco C. D., Golden J. W. Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts. J Bacteriol. 1989 Aug;171(8):4138–4145. doi: 10.1128/jb.171.8.4138-4145.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Currier T. C., Wolk C. P. Characteristics of Anabaena variabilis influencing plaque formation by cyanophage N-1. J Bacteriol. 1979 Jul;139(1):88–92. doi: 10.1128/jb.139.1.88-92.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Dilworth M. J., Eady R. R., Eldridge M. E. The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane. Biochem J. 1988 Feb 1;249(3):745–751. doi: 10.1042/bj2490745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Drummond M., Walmsley J., Kennedy C. Expression from the nifB promoter of Azotobacter vinelandii can be activated by NifA, VnfA, or AnfA transcriptional activators. J Bacteriol. 1996 Feb;178(3):788–792. doi: 10.1128/jb.178.3.788-792.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elhai J., Wolk C. P. A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene. 1988 Aug 15;68(1):119–138. doi: 10.1016/0378-1119(88)90605-1. [DOI] [PubMed] [Google Scholar]
  11. Elhai J., Wolk C. P. Developmental regulation and spatial pattern of expression of the structural genes for nitrogenase in the cyanobacterium Anabaena. EMBO J. 1990 Oct;9(10):3379–3388. doi: 10.1002/j.1460-2075.1990.tb07539.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  13. Fleming H., Haselkorn R. Differentiation in Nostoc muscorum: nitrogenase is synthesized in heterocysts. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2727–2731. doi: 10.1073/pnas.70.10.2727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frise E., Green A., Drummond M. Chimeric transcriptional activators generated in vivo from VnfA and AnfA of Azotobacter vinelandii: N-terminal domain of AnfA is responsible for dependence on nitrogenase Fe protein. J Bacteriol. 1994 Nov;176(21):6545–6549. doi: 10.1128/jb.176.21.6545-6549.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Golden S. S., Brusslan J., Haselkorn R. Genetic engineering of the cyanobacterial chromosome. Methods Enzymol. 1987;153:215–231. doi: 10.1016/0076-6879(87)53055-5. [DOI] [PubMed] [Google Scholar]
  16. Herrero A., Wolk C. P. Genetic mapping of the chromosome of the cyanobacterium, Anabaena variabilis. Proximity of the structural genes for nitrogenase and ribulose-bisphosphate carboxylase. J Biol Chem. 1986 Jun 15;261(17):7748–7754. [PubMed] [Google Scholar]
  17. Joerger R. D., Jacobson M. R., Bishop P. E. Two nifA-like genes required for expression of alternative nitrogenases by Azotobacter vinelandii. J Bacteriol. 1989 Jun;171(6):3258–3267. doi: 10.1128/jb.171.6.3258-3267.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Joerger R. D., Loveless T. M., Pau R. N., Mitchenall L. A., Simon B. H., Bishop P. E. Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii. J Bacteriol. 1990 Jun;172(6):3400–3408. doi: 10.1128/jb.172.6.3400-3408.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lyons E. M., Thiel T. Characterization of nifB, nifS, and nifU genes in the cyanobacterium Anabaena variabilis: NifB is required for the vanadium-dependent nitrogenase. J Bacteriol. 1995 Mar;177(6):1570–1575. doi: 10.1128/jb.177.6.1570-1575.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mylona P. V., Premakumar R., Pau R. N., Bishop P. E. Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii. J Bacteriol. 1996 Jan;178(1):204–208. doi: 10.1128/jb.178.1.204-208.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Peterson R. B., Wolk C. P. High recovery of nitrogenase activity and of Fe-labeled nitrogenase in heterocysts isolated from Anabaena variabilis. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6271–6275. doi: 10.1073/pnas.75.12.6271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Roll J. T., Shah V. K., Dean D. R., Roberts G. P. Characteristics of NIFNE in Azotobacter vinelandii strains. Implications for the synthesis of the iron-molybdenum cofactor of dinitrogenase. J Biol Chem. 1995 Mar 3;270(9):4432–4437. doi: 10.1074/jbc.270.9.4432. [DOI] [PubMed] [Google Scholar]
  25. Schneider K., Müller A., Johannes K. U., Diemann E., Kottmann J. Selective removal of molybdenum traces from growth media of N2-fixing bacteria. Anal Biochem. 1991 Mar 2;193(2):292–298. doi: 10.1016/0003-2697(91)90024-n. [DOI] [PubMed] [Google Scholar]
  26. Schrautemeier B., Neveling U., Schmitz S. Distinct and differently regulated Mo-dependent nitrogen-fixing systems evolved for heterocysts and vegetative cells of Anabaena variabilis ATCC 29413: characterization of the fdxH1/2 gene regions as part of the nif1/2 gene clusters. Mol Microbiol. 1995 Oct;18(2):357–369. doi: 10.1111/j.1365-2958.1995.mmi_18020357.x. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Thiel T., Bramble J., Rogers S. Optimum conditions for growth of cyanobacteria on solid media. FEMS Microbiol Lett. 1989 Oct 1;52(1-2):27–31. doi: 10.1016/0378-1097(89)90164-x. [DOI] [PubMed] [Google Scholar]
  29. Thiel T. Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis. J Bacteriol. 1993 Oct;175(19):6276–6286. doi: 10.1128/jb.175.19.6276-6286.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thiel T., Lyons E. M., Erker J. C., Ernst A. A second nitrogenase in vegetative cells of a heterocyst-forming cyanobacterium. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9358–9362. doi: 10.1073/pnas.92.20.9358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ugalde R. A., Imperial J., Shah V. K., Brill W. J. Biosynthesis of iron-molybdenum cofactor in the absence of nitrogenase. J Bacteriol. 1984 Sep;159(3):888–893. doi: 10.1128/jb.159.3.888-893.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Walmsley J., Toukdarian A., Kennedy C. The role of regulatory genes nifA, vnfA, anfA, nfrX, ntrC, and rpoN in expression of genes encoding the three nitrogenases of Azotobacter vinelandii. Arch Microbiol. 1994;162(6):422–429. doi: 10.1007/BF00282107. [DOI] [PubMed] [Google Scholar]
  33. Waugh S. I., Paulsen D. M., Mylona P. V., Maynard R. H., Premakumar R., Bishop P. E. The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii. J Bacteriol. 1995 Mar;177(6):1505–1510. doi: 10.1128/jb.177.6.1505-1510.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wolfinger E. D., Bishop P. E. Nucleotide sequence and mutational analysis of the vnfENX region of Azotobacter vinelandii. J Bacteriol. 1991 Dec;173(23):7565–7572. doi: 10.1128/jb.173.23.7565-7572.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES