Archives of Microbiology

, Volume 188, Issue 6, pp 609–617 | Cite as

Transcription and regulation of the hydrogenase(s) accessory genes, hypFCDEAB, in the cyanobacterium Lyngbya majuscula CCAP 1446/4

  • Daniela Ferreira
  • Elsa Leitão
  • Johannes Sjöholm
  • Paulo Oliveira
  • Peter Lindblad
  • Pedro Moradas-Ferreira
  • Paula TamagniniEmail author
Original Paper


Lyngbya majuscula CCAP 1446/4 is a filamentous cyanobacterium possessing both an uptake and a bi-directional hydrogenase. The presence of a single copy of the hyp operon in the cyanobacterial genomes suggests that these accessory genes might be responsible for the maturation of both hydrogenases. We investigated the concomitant transcription of hypFCDEAB with the hydrogenases structural genes—hup and hox. RT-PCRs performed with L. majuscula cells grown under different physiological conditions showed a substantial decrease in the relative amount of hupL transcript under non-N2-fixing conditions. In contrast, no significant differences were observed for the transcript levels of hypFCDEAB in all conditions tested, while minor fluctuations could be discerned for hoxH. Previously, it was demonstrated that the transcriptional regulators NtcA and LexA interact with the promoter regions of hup and hox, respectively, and that putative binding sites for both proteins are present in the hyp promoter of L. majuscula. Therefore, a putative involvement of NtcA and LexA in the regulation of the hyp transcription was investigated. Electrophoretic mobility shift assays resulted in NtcA or LexA-bound retarded fragments, suggesting the involvement of these proteins in the transcriptional regulation of hypFCDEAB.


Lyngbya majuscula hyp Hydrogenase accessory genes Transcriptional regulator factors NtcA LexA 



This work was financially supported by FCT (POCTI/BIO/44592/2002; SFRH/BD/4912/2001, SFRH/BD/16954/2004), ESF (III Quadro Comunitário de Apoio), the Swedish Research Council, the Swedish Energy Agency, the Nordic Energy Research Program (project BioHydrogen), and the EU/NEST Projects SOLAR-H (contract no. 516510) and BioModularH2 (contract no. 043340). We acknowledge Professor Enrique Flores for providing the NtcA clone and INEB (especially Eliana Vale) for providing the conditions for the anaerobic experiments.


  1. Antal TK, Oliveira P, Lindblad P (2006) The bidirectional hydrogenase in the cyanobacterium Synechocystis sp. strain PCC 6803. Int J Hydrogen Energy 31:1439–1444CrossRefGoogle Scholar
  2. Axelsson R, Lindblad P (2002) Transcriptional regulation of Nostoc hydrogenases: effects of oxygen, hydrogen, and nickel. Appl Environ Microbiol 68:444–447PubMedCrossRefGoogle Scholar
  3. Blokesch M, Böck A (2002) Maturation of [NiFe]-hydrogenases in Escherichia coli: the HypC cycle. J Mol Biol 324:287–296PubMedCrossRefGoogle Scholar
  4. Casalot L, Rousset M (2001) Maturation of the [NiFe] hydrogenases. Trends Microbiol 9:228–237PubMedCrossRefGoogle Scholar
  5. Chattopadhyaya R, Pal A (2004) Improved model of a LexA repressor dimer bound to recA operator. J Biomol Str Dyn 21:681–689Google Scholar
  6. Domain F, Houot L, Chauvat F, Cassier-Chauvat C (2004) Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation. Mol Microbiol 53:65–80PubMedCrossRefGoogle Scholar
  7. Gubili J, Borthakur D (1998) Organization of the hupDEAB genes within the hydrogenase gene cluster of Anabaena sp. strain PCC 7120. J Appl Phycol 10:163–167CrossRefGoogle Scholar
  8. Gutekunst K, Phunpruch S, Schwarz C, Schuchardt S, Schulz-Friedrich R, Appel J (2005) LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator. Mol Microbiol 58:810–823PubMedCrossRefGoogle Scholar
  9. Hansel A, Axelsson R, Lindberg P, Troshina OY, Wünschiers R, Lindblad P (2001) Cloning and characterisation of a hyp gene cluster in the filamentous cyanobacterium Nostoc sp. strain PCC 73102. FEMS Microbiol Lett 201:59–64PubMedCrossRefGoogle Scholar
  10. Herrero A, Muro-Pastor AM, Flores E (2001) Nitrogen control in cyanobacteria. J Bacteriol 183:411–425PubMedCrossRefGoogle Scholar
  11. Hoffmann D, Gutekunst K, Klissenbauer M, Schluz-Friedrich R, Appel J (2006) Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC 6803. Additional honologues of hypA and hypB are not active in hydrogenase maturation. FEBS J 273:4516–4527PubMedCrossRefGoogle Scholar
  12. Houchins JP (1984) The physiology and biochemistry of hydrogen metabolism in cyanobacteria. Biochim Biophys Acta 768:227–255Google Scholar
  13. Houchins JP, Burris RH (1981) Ocurrence and localization of two distinct hydrogenases in the heterocystous cyanobacterium Anabaena sp. strain 7120. J Bacteriol 146:209–214PubMedGoogle Scholar
  14. Jacobi A, Rossmann R, Böck A (1992) The hyp operon gene products are required for the maturation of catalytically active hydrogenase isoenzymes in Escherichia coli. Arch Microbiol 158:444–451PubMedCrossRefGoogle Scholar
  15. Leitão E, Oxelfelt F, Oliveira P, Moradas-Ferreira P, Tamagnini P (2005) Analysis of the hupSL operon of the non-heterocystous cyanobacterium Lyngbya majuscula CCAP 1446/4: regulation of transcription and expression under light-dark regimen. Appl Environ Microbiol 71:4567–4576PubMedCrossRefGoogle Scholar
  16. Leitão E, Pereira S, Bondoso J, Ferreira D, Pinto F, Moradas-Ferreira P, Tamagnini P (2006) Genes involved in the maturation of hydrogenase(s) in the nonheterocystous cyanobacterium Lyngbya majuscula CCAP 1446/4. Int J Hydrogen Energy 31:1469–1477CrossRefGoogle Scholar
  17. Lindberg P (2003) Cyanobacterial hydrogen metabolism—uptake hydrogenase and hydrogen production by nitrogenase in filamentous cyanobacteria. Ph.D. thesis, Uppsala University, Uppsala, Sweden, ISBN 91-554-5708-8Google Scholar
  18. Luo Y, Pfuetzner RA, Mosimann S, Paetzel M, Frey EA, Cherney M, Kim B, Little JW, Strynadka NC (2001) Crystal structure of LexA: a conformational switch for regulation of self-cleavage. Cell 106:585–594PubMedCrossRefGoogle Scholar
  19. Kim B, Little JW (1992) Dimerization of a specific DNA-binding protein on the DNA. Science 255:203–206PubMedCrossRefGoogle Scholar
  20. Magalon A, Böck A (2000a) Analysis of the HypC-HycE complex, a key intermediate in the assembly of the metal center of the Escherichia coli hydrogenase 3. J Biol Chem 275:21114–21120PubMedCrossRefGoogle Scholar
  21. Magalon A, Böck A (2000b) Dissection of the maturation reactions of the [NiFe] hydrogenase 3 from Escherichia coli taking place after nickel incorporation. FEBS Lett 473:254–258PubMedCrossRefGoogle Scholar
  22. Montesinos ML, Muro-Pastor AM, Herrero A, Flores E (1998) Ammonium/methylammonium permeases of a cyanobacterium. J Biol Chem 273:31463–31470PubMedCrossRefGoogle Scholar
  23. Mulrooney SB, Hausinger RP (2003) Nickel uptake and utilization by microorganisms. FEMS Microbiol Rev 27:239–261PubMedCrossRefGoogle Scholar
  24. Muro-Pastor AM, Valladares A, Flores E, Herrero A (1999) The hetC gene is a direct target of the NtcA transcriptional regulator in cyanobacterial heterocyst development. J Bacteriol 181:6664–6669PubMedGoogle Scholar
  25. Oliveira P, Lindblad P (2005) LexA, a transcription regulator binding in the promoter region of the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. FEMS Microbiol Lett 251:59–66PubMedCrossRefGoogle Scholar
  26. Oliveira P, Leitão E, Tamagnini P, Moradas-Ferreira P, Oxelfelt F (2004) Characterization and transcriptional analysis of hupSLW in Gloeothece sp. ATCC 27152: an uptake hydrogenase from a unicellular cyanobacterium. Microbiology 150:3647–3655PubMedCrossRefGoogle Scholar
  27. Olson JW, Mehta NS, Maier RJ (2001) Requirement of nickel metabolism proteins HypA and HypB for full activity of both hydrogenase and urease in Helicobacter pylori. Mol Microbiol 39:176–182PubMedCrossRefGoogle Scholar
  28. Omoregie EO, Crumbliss LL, Bebout BM, Zehr JP (2004) Determination of nitrogen-fixing phylotypes in Lyngbya sp. and Microcoleus chthonoplastes cyanobacterial mats from Guerrero Negro, Baja California, Mexico. Appl Environ Microbiol 70:2119–2128PubMedCrossRefGoogle Scholar
  29. Osborne NJ, Webb PM, Shaw GR (2001) The toxins of Lyngbya majuscula and their human and ecological health effects. Environ Int 27:381–392PubMedCrossRefGoogle Scholar
  30. Oxelfelt F, Tamagnini P, Salema R, Lindblad P (1995) Hydrogen uptake in Nostoc strain PCC 73102: effects of nickel, hydrogen, carbon and nitrogen. Plant Physiol Biochem 33:617–623Google Scholar
  31. Paschos A, Bauer A, Zimmermann A, Zehelein E, Böck A (2002) HypF, a carbamoyl phosphate-converting enzyme involved in [NiFe] hydrogenase maturation. J Biol Chem 277:49945–49951PubMedCrossRefGoogle Scholar
  32. Patterson-Fortin LM, Colvin KR, Owttrim GW (2006) A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR. Nuclic Acids Res 34:3446–3454CrossRefGoogle Scholar
  33. Reissmann S, Hochleitner E, Wang H, Paschos A, Lottspeich F, Glass RS, Böck A (2003) Taming of a poison: biosynthesis of the NiFe-hydrogenase cyanide ligands. Science 299:1067–1070PubMedCrossRefGoogle Scholar
  34. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  35. Schnarr M, Granger-Schnarr M, Hurstel S, Pouyet J (1988) The carboxy-terminal domain of the LexA repressor oligomerises essentially as the entire protein. FEBS Lett 234:56–60PubMedCrossRefGoogle Scholar
  36. Schütz K, Happe T, Troshina O, Lindblad P, Leitão E, Oliveira P, Tamagnini P (2004) Cyanobacterial H2 production—a comparative analysis. Planta 218:350–359PubMedCrossRefGoogle Scholar
  37. Serebriakova L, Zorin NA, Lindblad P (1994) Reversible hydrogenase in Anabaena variabilis ATCC 29413: presence and localization in non-N2-fixing cells. Arch Microbiol 161:140–144Google Scholar
  38. Serebryakova LT, Sheremetieva M, Tsygankov AA (1998) Reversible hydrogenase activity of Gloeocapsa alpicola in continuous culture. FEMS Microbiol Lett 166:89–94CrossRefGoogle Scholar
  39. Serebryakova LT, Sheremetieva M, Lindblad P (2000) H2-uptake and evolution in the unicellular cyanobacterium Chroococcidiopsis thermalis CALU 758. Plant Physiol Biochem 38:525–530CrossRefGoogle Scholar
  40. Sheremetieva ME, Troshina OY, Serebryakova LT, Lindblad P (2002) Identification of hox genes and analysis of their transcription in the unicellular cyanobacterium Gloeocapsa alpicola CALU 743 growing under nitrate-limiting conditions. FEMS Microbiol Lett 214:229–233PubMedCrossRefGoogle Scholar
  41. Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35:171–205PubMedGoogle Scholar
  42. Tamagnini P, Troshina O, Oxelfelt F, Salema R, Lindblad P (1997) Hydrogenases in Nostoc sp. strain PCC 73102, a strain lacking a bidirectional enzyme. Appl Environ Microbiol 63:1801–1807PubMedGoogle Scholar
  43. Tamagnini P, Axelsson R, Lindberg P, Oxelfelt F, Wünschiers R, Lindblad P (2002) Hydrogenases and hydrogen metabolism of cyanobacteria. Microbiol Mol Biol Rev 66:1–20PubMedCrossRefGoogle Scholar
  44. Tamagnini P, Leitão E, Oxelfelt F (2005) Uptake hydrogenases in cyanobacteria—novel input from non-heterocystous strains. Biochem Soc Trans 33:67–69PubMedCrossRefGoogle Scholar
  45. Theodoratou E, Huber R, Böck A (2005) [NiFe]-hydrogenase maturation endopeptidase: structure and function. Biochem Soc Trans 33:108–111PubMedCrossRefGoogle Scholar
  46. Vignais PM, Colbeau A (2004) Molecular biology of microbial hydrogenases. Curr Issues Mol Biol 6:159–188PubMedGoogle Scholar
  47. Watkinson AJ, O’Neil JM, Dennison WC (2005) Ecophysiology of the marine cyanobacterium, Lyngbya majuscula (Oscillatoriaceae) in Moreton Bay, Australia. Harmful Algae 4:697–715CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Daniela Ferreira
    • 1
    • 2
  • Elsa Leitão
    • 1
  • Johannes Sjöholm
    • 4
  • Paulo Oliveira
    • 4
  • Peter Lindblad
    • 4
  • Pedro Moradas-Ferreira
    • 1
    • 3
  • Paula Tamagnini
    • 1
    • 2
    Email author
  1. 1.IBMC—Instituto de Biologia Molecular e CelularUniversidade do PortoPortoPortugal
  2. 2.Departamento de Botânica, Faculdade de CiênciasUniversidade do PortoPortoPortugal
  3. 3.Instituto de Ciências Biomédicas Abel Salazar (ICBAS)Universidade do PortoPortoPortugal
  4. 4.Department of Photochemistry and Molecular Science, The Ångström LaboratoriesUppsala UniversityUppsalaSweden

Personalised recommendations