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Contribution of Hydrogenase 2 to Stationary Phase H2 Production by Escherichia coli During Fermentation of Glycerol

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Abstract

Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H2 production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H2 production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H2 production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)−1 and Hyd-1 activity was 0.1 U (mg protein)−1. As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)−1, and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)−1 but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H2 production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.

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References

  1. Khanna, S., Goyal, A., & Moholkar, V. S. (2011). Microbial conversion of glycerol: Present status and future prospects. Critical Reviews in Biotechnology, 32(3), 235–262. doi:10.3109/07388551.2011.604839. Epub Sep 27.

    Article  PubMed  Google Scholar 

  2. Trchounian, K., Poladyan, A., Vassilian, A., & Trchounian, A. (2012). Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: Dependence on fermentation substrate, pH and FOF1-ATPase. Critical Reviews in Biochemistry and Molecular Biology, 47, 236–249.

    Article  PubMed  CAS  Google Scholar 

  3. Sauter, M., Bohm, R., & Bock, A. (1992). Mutational analysis of the operon (hyc) determining hydrogenase 3 formation in Escherichia coli. Molecular Microbiology, 6, 1523–1532.

    Article  PubMed  CAS  Google Scholar 

  4. Andrews, S. C., Berks, B. C., Mcclay, J., Ambler, A., Quail, M. A., Golby, P., et al. (1997). A 12-cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. Microbiology, 143, 3633–3647.

    Article  PubMed  CAS  Google Scholar 

  5. Self, W. T., Hasona, A., & Shanmugam, K. T. (2001). N-terminal truncations in the FhlA protein result in formate- and Moe-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli. Microbiology, 147, 3093–3104.

    PubMed  CAS  Google Scholar 

  6. Skibinski, D. A. G., Golby, P., Chang, Y.-S., Sargent, F., Hoffman, R., Harper, R., et al. (2002). Regulation of the hydrogenase-4 operon of Escherichia coli by the σ54-dependent transcriptional activators FhlA and HyfR. Journal of Bacteriology, 184, 6642–6653.

    Article  PubMed  CAS  Google Scholar 

  7. Bagramyan, K., Mnatsakanyan, N., Poladian, A., Vassilian, A., & Trchounian, A. (2002). The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH. FEBS Letters, 516, 172–178.

    Article  PubMed  CAS  Google Scholar 

  8. Self, W. T., Hasona, A., & Shanmugan, K. T. (2004). Expression and regulation of silent operon, hyf, coding for hydrogenase 4 isoenzyme in Escherichia coli. Journal of Bacteriology, 186, 580–587.

    Article  PubMed  CAS  Google Scholar 

  9. Maeda, T., Sanchez-Torres, V., & Wood, T. K. (2007). Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities. Applied Microbiology and Biotechnology, 76, 1036–1042.

    Article  Google Scholar 

  10. King, P. W., & Przybyla, A. E. (1999). Response of hya expression to external pH in Escherichia coli. Journal of Bacteriology, 181, 5250–5256.

    PubMed  CAS  Google Scholar 

  11. 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  CAS  Google Scholar 

  12. Menon, N. K., Robbins, J., Wendt, J. C., Shanmugan, K. T., & Przybyla, A. E. (1991). Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1. Journal of Bacteriology, 173, 4851–4861.

    PubMed  CAS  Google Scholar 

  13. Dubini, A., Pye, R. L., Jack, R. L., Palmer, T., & Sargent, F. (2002). How bacteria get energy from hydrogen: A genetic analysis of periplasmic hydrogen oxidation in Escherichia coli. International Journal of Hydrogen Energy, 27, 1413–1420.

    Article  CAS  Google Scholar 

  14. Dharmadi, Y., Murarka, A., & Gonzalez, R. (2006). Anaerobic fermentation of glycerol by Escherichia coli: A new platform for metabolic engineering. Biotechnology and Bioengineering, 94, 821–829.

    Article  PubMed  CAS  Google Scholar 

  15. Murarka, A., Dharmadi, Y., Yazdani, S. S., & Gonzalez, R. (2008). Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Applied and Environmental Microbiology, 74, 1124–1135.

    Article  PubMed  CAS  Google Scholar 

  16. Trchounian, K., & Trchounian, A. (2009). Hydrogenase 2 is most and hydrogenase 1 is less responsible for H2 production by Escherichia coli under glycerol fermentation at neutral and slightly alkaline pH. International Journal of Hydrogen Energy, 34, 8839–8845.

    Article  CAS  Google Scholar 

  17. Cintolesi, A., Comburg, J. M., Rigou, V., Zygourakis, K., & Gonzalez, R. (2011). Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli. Biotechnology and Bioengineering, 109, 187–198.

    Article  PubMed  Google Scholar 

  18. Trchounian, K., Sanchez-Torres, V., Wood, K. T., & Trchounian, A. (2011). Escherichia coli hydrogenase activity and H2 production under glycerol fermentation at a low pH. International Journal of Hydrogen Energy, 36, 4323–4331.

    Article  CAS  Google Scholar 

  19. Trchounian, K., Pinske, C., Sawers, G., & Trchounian, A. (2012). Characterization of Escherichia coli [NiFe]-hydrogenase distribution during fermentative growth at different pHs. Cell Biochemistry and Biophysics, 62, 433–440.

    Article  PubMed  CAS  Google Scholar 

  20. Redwood, M. D., Mikheenko, I. P., Sargent, F., & Macaskie, L. E. (2008). Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production. FEMS Microbiology Letters, 278, 48–55.

    Article  PubMed  CAS  Google Scholar 

  21. 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. Archives of Microbiology, 158, 444–451.

    Article  PubMed  CAS  Google Scholar 

  22. Mnatsakanyan, N., Bagramyan, K., & Trchounian, A. (2004). Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate. Cell Biochemistry and Biophysics, 41, 357–366.

    Article  PubMed  CAS  Google Scholar 

  23. Gabrielyan, L., & Trchounian, A. (2009). Relationship between molecular hydrogen production, proton transport and the FOF1-ATPase activity in Rhodobacter sphaeroides strains from mineral springs. International Journal of Hydrogen Energy, 34, 2567–2572.

    Article  CAS  Google Scholar 

  24. Gabrielyan, L., & Trchounian, A. (2012). Concentration dependent glycine effect on the photosynthetic growth and bio-hydrogen production by Rhodobacter sphaeroides from mineral springs. Biomass and Bioenergy, 36, 333–338.

    Article  CAS  Google Scholar 

  25. Eltsova, Z. A., Vasilieva, L. G., & Tsygankov, A. A. (2010). Hydrogen production by recombinant strains of Rhodobacter sphaeroides using a modified photosynthetic apparatus. Applied Biochemistry and Microbiology, 46, 487–491.

    Article  CAS  Google Scholar 

  26. Noguchi, K., Riggins, D. P., Eldahan, K. C., Kitko, R. D., & Slonczewski, J. L. (2010). Hydrogenase-3 contributes to anaerobic acid resistance of Escherichia coli. PLoS ONE, 5, 1–7.

    Google Scholar 

  27. Piskarev, I. M., Ushkanov, V. A., Aristova, N. A., Likhachev, P. P., & Myslivets, T. S. (2010). Establishment of the redox potential of water saturated with hydrogen. Biophysics, 55, 13–17.

    Article  Google Scholar 

  28. Trchounian, K., Pinske, C., Sawers, R. G., & Trchounian, A. (2011). Dependence on the F0F1-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases 1 and 2 during glucose and glycerol fermentation at high and low pH in Escherichia coli. Journal of Bioenergetics and Biomembranes, 43, 645–650.

    Article  PubMed  CAS  Google Scholar 

  29. Ballantine, S. P., & Boxer, D. H. (1986). Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli. European Journal of Biochemistry, 156, 277–284.

    Article  PubMed  CAS  Google Scholar 

  30. Lukey, M. J., Parkin, A., Roessler, M. M., Murphy, B. J., Harmer, J., Palmer, T., et al. (2010). How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. Journal of Biological Chemistry, 285, 3928–3938.

    Article  PubMed  CAS  Google Scholar 

  31. Soboh, B., Pinske, C., Kuhns, M., Waclawek, M., Ihling, C., Trchounian, K., et al. (2011). The respiratory molybdo-selenoprotein formate dehydrogenases of Escherichia coli have hydrogen: Benzyl viologen oxidoreductase activity. BMC Microbiology, 11, 173.

    Article  PubMed  CAS  Google Scholar 

  32. Zbell, A. L., & Maier, R. J. (2009). Role of the Hya hydrogenase in recycling of anaerobically produced H2 in Salmonella enterica serovar typhimurium. Applied and Environmental Microbiology, 75, 1456–1459.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The study was supported by the FEBS Summer Research Fellowship to KT (2010), by the Deutsche Forschungsgemeinschaft (Grant SA 494/3-1 to RGS), and by the Ministry of Education and Science of Armenia (Research Grant #11-F202 to AT).

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Authors and Affiliations

  1. Department of Biophysics, Yerevan State University, 1 A. Manoukian Str., 0025, Yerevan, Armenia

    Karen Trchounian & Armen Trchounian

  2. Institute of Microbiology, Martin-Luther University Halle-Wittenberg, 06120, Halle (Saale), Germany

    Karen Trchounian, Basem Soboh & R. Gary Sawers

  3. Department of Microbiology & Plants and Microbes Biotechnology, Yerevan State University, 1 A. Manoukian Str., 0025, Yerevan, Armenia

    Armen Trchounian

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  1. Karen Trchounian
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  2. Basem Soboh
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  4. Armen Trchounian
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Correspondence to Armen Trchounian.

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Trchounian, K., Soboh, B., Sawers, R.G. et al. Contribution of Hydrogenase 2 to Stationary Phase H2 Production by Escherichia coli During Fermentation of Glycerol. Cell Biochem Biophys 66, 103–108 (2013). https://doi.org/10.1007/s12013-012-9458-7

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