1887

Abstract

HynSL from ‘deep ecotype’ (AltDE) is an oxygen-tolerant and thermostable [NiFe] hydrogenase. Its two structural genes (), encoding small and large hydrogenase subunits, are surrounded by eight genes (, and ) predicted to encode accessory proteins involved in maturation of the hydrogenase. A 13 kb fragment containing the ten structural and accessory genes along with three additional adjacent genes (, and ) was cloned into an IPTG-inducible expression vector and transferred into an mutant strain lacking its native hydrogenases. Upon induction, HynSL from AltDE was expressed in and was active, as determined by an hydrogen evolution assay. Subsequent genetic analysis revealed that , , and are not essential for assembling an active hydrogenase. However, and can enhance the activity of the heterologously expressed hydrogenase. We used this genetic system to compare maturation mechanisms between AltDE HynSL and its homologue. When the structural genes for the hydrogenase, , were expressed along with known accessory genes ( and ), no active hydrogenase was produced. Further, co-expression of AltDE accessory genes and with the entire set of the genes did not produce an active hydrogenase. However, co-expression of all AltDE accessory genes with the structural genes generated an active hydrogenase. This result demonstrates that the accessory genes from AltDE can complement their counterparts from and that the two hydrogenases share similar maturation mechanisms.

Funding
This study was supported by the:
  • Synthetic Genomics, Inc.
  • US Department of Energy, the Hydrogen, Fuel Cells, and Infrastructure Technology Program (Award DE-FG36-05GO15027)
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2011-05-01
2024-12-05
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References

  1. Barrett E. L., Kwan H. S., Macy J. ( 1984). Anaerobiosis, formate, nitrate, and pyrA are involved in the regulation of formate hydrogenlyase in Salmonella typhimurium. J Bacteriol 158:972–977[PubMed]
    [Google Scholar]
  2. Báscones E., Imperial J., Ruiz-Argüeso T., Palacios J. M. ( 2000). Generation of new hydrogen-recycling Rhizobiaceae strains by introduction of a novel Hup minitransposon. Appl Environ Microbiol 66:4292–4299 [View Article][PubMed]
    [Google Scholar]
  3. Benner S. A., Sismour A. M. ( 2005). Synthetic biology. Nat Rev Genet 6:533–543 [View Article][PubMed]
    [Google Scholar]
  4. Bernhard M., Schwartz E., Rietdorf J., Friedrich B. ( 1996). The Alcaligenes eutrophus membrane-bound hydrogenase gene locus encodes functions involved in maturation and electron transport coupling. J Bacteriol 178:4522–4529[PubMed]
    [Google Scholar]
  5. Böck A., King P. W., Blokesch M., Posewitz M. C. ( 2006). Maturation of hydrogenases. Adv Microb Physiol 51:1–71 [View Article][PubMed]
    [Google Scholar]
  6. Bogorov L. V. ( 1974). [The properties of Thiocapsa roseopersicina, strain BBS, isolated from an estuary of the White Sea]. Mikrobiologiia 43:326–332 (in Russian) [PubMed]
    [Google Scholar]
  7. Casadaban M. J. ( 1976). Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol 104:541–555 [View Article][PubMed]
    [Google Scholar]
  8. Casalot L., Rousset M. ( 2001). Maturation of the [NiFe] hydrogenases. Trends Microbiol 9:228–237 [View Article][PubMed]
    [Google Scholar]
  9. Elhai J., Wolk C. P. ( 1988). A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68:119–138 [View Article][PubMed]
    [Google Scholar]
  10. Fodor B., Rákhely G., Kovács A. T., Kovács K. L. ( 2001). Transposon mutagenesis in purple sulfur photosynthetic bacteria: identification of hypF, encoding a protein capable of processing [NiFe] hydrogenases in alpha, beta, and gamma subdivisions of the proteobacteria. Appl Environ Microbiol 67:2476–2483 [View Article][PubMed]
    [Google Scholar]
  11. Fontecilla-Camps J. C., Volbeda A., Cavazza C., Nicolet Y. ( 2007). Structure/function relationships of [NiFe]- and [FeFe]-hydrogenases. Chem Rev 107:4273–4303 [View Article][PubMed]
    [Google Scholar]
  12. Forzi L., Sawers R. G. ( 2007). Maturation of [NiFe]-hydrogenases in Escherichia coli. Biometals 20:565–578 [View Article][PubMed]
    [Google Scholar]
  13. Friedrich B., Friedrich C. G., Meyer M., Schlegel H. G. ( 1984). Expression of hydrogenase in Alcaligenes spp. is altered by interspecific plasmid exchange. J Bacteriol 158:331–333[PubMed]
    [Google Scholar]
  14. Gibson D. G., Young L., Chuang R. Y., Venter J. C., Hutchison C. A. III, Smith H. O. ( 2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345 [View Article][PubMed]
    [Google Scholar]
  15. Ivars-Martinez E., Martin-Cuadrado A.-B., D’Auria G., Mira A., Ferriera S., Johnson J., Friedman R., Rodriguez-Valera F. ( 2008). Comparative genomics of two ecotypes of the marine planktonic copiotroph Alteromonas macleodii suggests alternative lifestyles associated with different kinds of particulate organic matter. ISME J 2:1194–1212 [View Article][PubMed]
    [Google Scholar]
  16. Kovács K. L., Tigyi G., Thanh L. T., Lakatos S., Kiss Z., Bagyinka C. ( 1991). Structural rearrangements in active and inactive forms of hydrogenase from Thiocapsa roseopersicina. J Biol Chem 266:947–951[PubMed]
    [Google Scholar]
  17. Kovács K. L., Fodor B., Kovács A. T., Csanádi G., Maróti G., Balogh J., Arvani S., Rákhely G. ( 2002). Hydrogenases, accessory genes and the regulation of [NiFe] hydrogenase biosynthesis in Thiocapsa roseopersicina. Int J Hydrogen Energy 27:1463–1469 [View Article]
    [Google Scholar]
  18. Lenz O., Gleiche A., Strack A., Friedrich B. ( 2005). Requirements for heterologous production of a complex metalloenzyme: the membrane-bound [NiFe] hydrogenase. J Bacteriol 187:6590–6595 [View Article][PubMed]
    [Google Scholar]
  19. López-López A., Bartual S. G., Stal L., Onyshchenko O., Rodríguez-Valera F. ( 2005). Genetic analysis of housekeeping genes reveals a deep-sea ecotype of Alteromonas macleodii in the Mediterranean Sea. Environ Microbiol 7:649–659 [View Article][PubMed]
    [Google Scholar]
  20. Ludwig M., Schubert T., Zebger I., Wisitruangsakul N., Saggu M., Strack A., Lenz O., Hildebrandt P., Friedrich B. ( 2009). Concerted action of two novel auxiliary proteins in assembly of the active site in a membrane-bound [NiFe] hydrogenase. J Biol Chem 284:2159–2168 [View Article][PubMed]
    [Google Scholar]
  21. Manyani H., Rey L., Palacios J. M., Imperial J., Ruiz-Argüeso T. ( 2005). Gene products of the hupGHIJ operon are involved in maturation of the iron-sulfur subunit of the [NiFe] hydrogenase from Rhizobium leguminosarum bv. viciae. J Bacteriol 187:7018–7026 [View Article][PubMed]
    [Google Scholar]
  22. Maróti G., Fodor B. D., Rákhely G., Kovács A. T., Arvani S., Kovács K. L. ( 2003). Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina. Eur J Biochem 270:2218–2227 [View Article][PubMed]
    [Google Scholar]
  23. Maróti G., Tong Y., Yooseph S., Baden-Tillson H., Smith H. O., Kovács K. L., Frazier M., Venter J. C., Xu Q. ( 2009). Discovery of [NiFe] hydrogenase genes in metagenomic DNA: cloning and heterologous expression in Thiocapsa roseopersicina. Appl Environ Microbiol 75:5821–5830 [View Article][PubMed]
    [Google Scholar]
  24. Maróti G., Rákhely G., Maróti J., Dorogházi E., Klement E., Medzihradszky K. F., Kovács K. L. ( 2010). Specificity and selectivity of HypC chaperonins and endopeptidases in the molecular assembly machinery of [NiFe] hydrogenases of Thiocapsa roseopersicina. Int J Hydrogen Energy 35:3358–3370 [View Article]
    [Google Scholar]
  25. Menon N. K., Robbins J., Wendt J. C., Shanmugam K. T., Przybyla A. E. ( 1991). Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1. J Bacteriol 173:4851–4861[PubMed]
    [Google Scholar]
  26. Palágyi-Mészáros L. S., Maróti J., Latinovics D., Balogh T., Klement E., Medzihradszky K. F., Rákhely G., Kovács K. L. ( 2009). Electron-transfer subunits of the NiFe hydrogenases in Thiocapsa roseopersicina BBS. FEBS J 276:164–174 [View Article][PubMed]
    [Google Scholar]
  27. Peters J. E., Thate T. E., Craig N. L. ( 2003). Definition of the Escherichia coli MC4100 genome by use of a DNA array. J Bacteriol 185:2017–2021 [View Article][PubMed]
    [Google Scholar]
  28. Redwood M. D., Mikheenko I. P., Sargent F., Macaskie L. E. ( 2008). Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production. FEMS Microbiol Lett 278:48–55 [View Article][PubMed]
    [Google Scholar]
  29. Richard D. J., Sawers G., Sargent F., McWalter L., Boxer D. H. ( 1999). Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli. Microbiology 145:2903–2912[PubMed]
    [Google Scholar]
  30. Rousset M., Magro V., Forget N., Guigliarelli B., Belaich J. P., Hatchikian E. C. ( 1998). Heterologous expression of the Desulfovibrio gigas [NiFe] hydrogenase in Desulfovibrio fructosovorans MR400. J Bacteriol 180:4982–4986[PubMed]
    [Google Scholar]
  31. Sambrook J., Russell D. W. ( 2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  32. Shirshikova G. N., Khusnutdinova A. N., Postnikova O. A., Patrusheva E. V., Butanaev A. M., Tsygankov A. A. ( 2009). Expression of Ni-Fe hydrogenase structural genes derived from Thiocapsa roseopersicina in Escherichia coli. Dokl Biochem Biophys 425:124–126 [View Article][PubMed]
    [Google Scholar]
  33. Studier F. W. ( 2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234 [View Article][PubMed]
    [Google Scholar]
  34. Sun J., Hopkins R. C., Jenney F. E., McTernan P. M., Adams M. W. ( 2010). Heterologous expression and maturation of an NADP-dependent [NiFe]-hydrogenase: a key enzyme in biofuel production. PLoS ONE 5:e10526 [View Article][PubMed]
    [Google Scholar]
  35. Vargas W. A., Weyman P. D., Tong Y., Smith H. O., Xu Q. ( 2011). A [NiFe]-hydrogenase from Alteromonas macleodii with unusual stability in the presence of oxygen and high temperature. Appl Environ Microbiol 77:1990–1998 [View Article][PubMed]
    [Google Scholar]
  36. Vasala A., Panula J., Bollók M., Illmann L., Hälsig C., Neubauer P. ( 2006). A new wireless system for decentralised measurement of physiological parameters from shake flasks. Microb Cell Fact 5:8 [View Article][PubMed]
    [Google Scholar]
  37. Vignais P. M., Billoud B. ( 2007). Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 107:4206–4272 [View Article][PubMed]
    [Google Scholar]
  38. Xu Y., Mori T., Johnson C. H. ( 2003). Cyanobacterial circadian clockwork: roles of KaiA, KaiB and the kaiBC promoter in regulating KaiC. EMBO J 22:2117–2126 [View Article][PubMed]
    [Google Scholar]
  39. Yagi K., Min H., Urushihara M., Manabe Y., Umeda F., Miura Y. ( 1986). Isolation of hydrogen-oxidation gene from Alcaligenes hydrogenophilus and its expression in Pseudomonas oxalaticus. Biochem Biophys Res Commun 137:114–119 [View Article][PubMed]
    [Google Scholar]
  40. Zhang J. W., Butland G., Greenblatt J. F., Emili A., Zamble D. B. ( 2005). A role for SlyD in the Escherichia coli hydrogenase biosynthetic pathway. J Biol Chem 280:4360–4366 [View Article][PubMed]
    [Google Scholar]
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