Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Oct;175(19):6276–6286. doi: 10.1128/jb.175.19.6276-6286.1993

Characterization of genes for an alternative nitrogenase in the cyanobacterium Anabaena variabilis.

T Thiel 1
PMCID: PMC206724  PMID: 8407800

Abstract

Anabaena variabilis ATCC 29413 is a heterotrophic, nitrogen-fixing cyanobacterium that has been reported to fix nitrogen and reduce acetylene to ethane in the absence of molybdenum. DNA from this strain hybridized well at low stringency to the nitrogenase 2 (vnfDGK) genes of Azotobacter vinelandii. The hybridizing region was cloned from a lambda EMBL3 genomic library of A. variabilis, mapped, and sequenced. The deduced amino acid sequences of the vnfD and vnfK genes of A. variabilis showed only about 56% similarity to the nifDK genes of Anabaena sp. strain PCC 7120 but were 76 to 86% similar to the anfDK or vnfDK genes of A. vinelandii. The organization of the vnf gene cluster in A. variabilis was similar to that of A. vinelandii. However, in A. variabilis, the vnfG gene was fused to vnfD; hence, this gene is designated vnfDG. A vnfH gene was not contiguous with the vnfDG gene and has not yet been identified. A mutant strain, in which a neomycin resistance cassette was inserted into the vnf cluster, grew well in a medium lacking a source of fixed nitrogen in the presence of molybdenum but grew poorly when vanadium replaced molybdenum. In contrast, the parent strain grew equally well in media containing either molybdenum or vanadium. The vnf genes were transcribed in the absence of molybdenum, with or without vanadium. The vnf gene cluster did not hybridize to chromosomal DNA from Anabaena sp. strain PCC 7120 or from the heterotrophic strains, Nostoc sp. strain Mac and Nostoc sp. strain ATCC 29150. A hybridizing ClaI fragment very similar in size to the A. variabilis ClaI fragment was present in DNA isolated from several independent, cultured isolates of Anabaena sp. from the Azolla symbiosis.

Full text

PDF
6276

Images in this article

6277Image
on p.6277
6282Image
on p.6282
6283Image
on p.6283
6283Image
on p.6283

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. Arnon D. I., McSwain B. D., Tsujimoto H. Y., Wada K. Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin. Biochim Biophys Acta. 1974 Aug 23;357(2):231–245. doi: 10.1016/0005-2728(74)90063-2. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  6. 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]
  7. Böhme H., Haselkorn R. Molecular cloning and nucleotide sequence analysis of the gene coding for heterocyst ferredoxin from the cyanobacterium Anabaena sp. strain PCC 7120. Mol Gen Genet. 1988 Oct;214(2):278–285. doi: 10.1007/BF00337722. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. Elhai J., Wolk C. P. Conjugal transfer of DNA to cyanobacteria. Methods Enzymol. 1988;167:747–754. doi: 10.1016/0076-6879(88)67086-8. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Gebhardt J. S., Nierzwicki-Bauer S. A. Identification of a common cyanobacterial symbiont associated with Azolla spp. through molecular and morphological characterization of free-living and symbiotic cyanobacteria. Appl Environ Microbiol. 1991 Aug;57(8):2141–2146. doi: 10.1128/aem.57.8.2141-2146.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Golden J. W., Mulligan M. E., Haselkorn R. Different recombination site specificity of two developmentally regulated genome rearrangements. Nature. 1987 Jun 11;327(6122):526–529. doi: 10.1038/327526a0. [DOI] [PubMed] [Google Scholar]
  19. Golden J. W., Robinson S. J., Haselkorn R. Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena. Nature. 1985 Apr 4;314(6010):419–423. doi: 10.1038/314419a0. [DOI] [PubMed] [Google Scholar]
  20. Golden J. W., Wiest D. R. Genome rearrangement and nitrogen fixation in Anabaena blocked by inactivation of xisA gene. Science. 1988 Dec 9;242(4884):1421–1423. doi: 10.1126/science.3144039. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. Jacobitz S., Bishop P. E. Regulation of nitrogenase-2 in Azotobacter vinelandii by ammonium, molybdenum, and vanadium. J Bacteriol. 1992 Jun;174(12):3884–3888. doi: 10.1128/jb.174.12.3884-3888.1992. [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., 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]
  26. Joerger R. D., Jacobson M. R., Premakumar R., Wolfinger E. D., Bishop P. E. Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii. J Bacteriol. 1989 Feb;171(2):1075–1086. doi: 10.1128/jb.171.2.1075-1086.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Lammers P. J., Haselkorn R. Sequence of the nifD gene coding for the alpha subunit of dinitrogenase from the cyanobacterium Anabaena. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4723–4727. doi: 10.1073/pnas.80.15.4723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Luque F., Pau R. N. Transcriptional regulation by metals of structural genes for Azotobacter vinelandii nitrogenases. Mol Gen Genet. 1991 Jul;227(3):481–487. doi: 10.1007/BF00273941. [DOI] [PubMed] [Google Scholar]
  30. Mazur B. J., Chui C. F. Sequence of the gene coding for the beta-subunit of dinitrogenase from the blue-green alga Anabaena. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6782–6786. doi: 10.1073/pnas.79.22.6782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meeks J. C., Joseph C. M., Haselkorn R. Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros. Arch Microbiol. 1988 May;150(1):61–71. doi: 10.1007/BF00409719. [DOI] [PubMed] [Google Scholar]
  32. Mulligan M. E., Buikema W. J., Haselkorn R. Bacterial-type ferredoxin genes in the nitrogen fixation regions of the cyanobacterium Anabaena sp. strain PCC 7120 and Rhizobium meliloti. J Bacteriol. 1988 Sep;170(9):4406–4410. doi: 10.1128/jb.170.9.4406-4410.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Murry M. A., Horne A. J., Benemann J. R. Physiological Studies of Oxygen Protection Mechanisms in the Heterocysts of Anabaena cylindrica. Appl Environ Microbiol. 1984 Mar;47(3):449–454. doi: 10.1128/aem.47.3.449-454.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pau R. N., Mitchenall L. A., Robson R. L. Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium. J Bacteriol. 1989 Jan;171(1):124–129. doi: 10.1128/jb.171.1.124-129.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Peterson R. B., Dolan E., Calvert H. E., Ke B. Energy transfer from phycobiliproteins to photosystem I in vegetative cells and heterocysts of Anabaena variabilis. Biochim Biophys Acta. 1981 Feb 12;634(2):237–248. doi: 10.1016/0005-2728(81)90142-0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Rice D., Mazur B. J., Haselkorn R. Isolation and physical mapping of nitrogen fixation genes from the cyanobacterium Anabaena 7120. J Biol Chem. 1982 Nov 10;257(21):13157–13163. [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. 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]
  43. Vega-Palas M. A., Madueño F., Herrero A., Flores E. Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol. 1990 Feb;172(2):643–647. doi: 10.1128/jb.172.2.643-647.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wei T. F., Ramasubramanian T. S., Pu F., Golden J. W. Anabaena sp. strain PCC 7120 bifA gene encoding a sequence-specific DNA-binding protein cloned by in vivo transcriptional interference selection. J Bacteriol. 1993 Jul;175(13):4025–4035. doi: 10.1128/jb.175.13.4025-4035.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wolk C. P., Wojciuch E. Simple methods for plating single vegetative cells of, and for replica-plating, filamentous blue-green algae. Arch Mikrobiol. 1973 May 24;91(2):91–95. doi: 10.1007/BF00424753. [DOI] [PubMed] [Google Scholar]

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

RESOURCES