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Metagenomics pp 235-246 | Cite as

Methods for the Isolation of Genes Encoding Novel PHB Cycle Enzymes from Complex Microbial Communities

  • Ricardo F. Nordeste
  • Maria A. Trainer
  • Trevor C. CharlesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 668)

Abstract

Development of different PHAs as alternatives to petrochemically derived plastics can be facilitated by mining metagenomic libraries for diverse PHA cycle genes that might be useful for synthesis of bioplastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti allows for the use of powerful selection and screening tools to identify complementing novel PHA synthesis genes. Identification of novel genes through their function rather than sequence facilitates finding functional proteins that may otherwise have been excluded through sequence-only screening methodology. We present here methods that we have developed for the isolation of clones expressing novel PHA metabolism genes from metagenomic libraries.

Key words

PHA/PHB cycle Sinorhizobium meliloti Microbial community gene libraries Phenotypic complementation 

References

  1. 1.
    Henne, A., Daniel, R., Schmitz, R.A., and Gottschalk, G. (1999) Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Appl Environ Microbiol 65, 3901–3907.PubMedGoogle Scholar
  2. 2.
    Anderson, A.J. and Dawes, E.A. (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54, 450–472.PubMedGoogle Scholar
  3. 3.
    Zevenhuizen, L.P.T.M. (1981) Cellular glycogen, β-1,2-glucan, poly-3-hydroxybutyic acid and extracellular polysaccharides in fast-growing species of Rhizobium. Antonie Van Leeuwenhoek 47, 481–497.PubMedCrossRefGoogle Scholar
  4. 4.
    Madison, L.L. and Huisman, G.W. (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63, 21–53.PubMedGoogle Scholar
  5. 5.
    Shishatskaya, E.I., Voinova, O.N., Goreva, A.V., Mogilnaya, O.A., and Volova, T.G. (2008) Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation. J Mater Sci Mater Med 19, 2493–2502.PubMedCrossRefGoogle Scholar
  6. 6.
    Shishatskaya, E.I., Volova, T.G., Puzyr, A.P., Mogilnaya, O.A., and Efremov, S.N. (2004) Tissue response to the implantation of biodegradable polyhydroxyalkanoate sutures. J Mater Sci Mater Med 15, 719–728.PubMedCrossRefGoogle Scholar
  7. 7.
    Holmes, P.A. (1985) Applications of PHB – a microbially produced biodegradable thermosplastic. Phys Technol 16, 32–36.CrossRefGoogle Scholar
  8. 8.
    Pötter, M. and Steinbüchel, A. (2005) Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules 6, 552–560.PubMedCrossRefGoogle Scholar
  9. 9.
    Kadouri, D., Jurkevitch, E., and Okon, Y. (2003) Involvement of the reserve material poly-β-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization. Appl Environ Microbiol 69, 3244–3250.PubMedCrossRefGoogle Scholar
  10. 10.
    Senior, P.J., Beech, G.A., Ritchie, G.A.F., and Dawes, E.A. (1972) The role of oxygen limitation in the formation of poly-3-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii. Biochem J 128, 1193–1201.PubMedGoogle Scholar
  11. 11.
    Stam, H., van Verseveld, H.W., de Vries, W., and Stouhamer, A.H. (1986) Utilization of poly-β-hydroxybutyrate in free-living cultures of Rhizobium ORS571. FEMS Microbiol Lett 35, 215–220.Google Scholar
  12. 12.
    Stockdale, H., Ribbons, D.W., and Dawes, E.A. (1968) Occurrence of poly-3-hydroxybutyrate in the Azotobacteriaceae. J Bacteriol 95, 1798–1803.PubMedGoogle Scholar
  13. 13.
    Senior, P.J. and Dawes, E.A. (1971) Poly-3-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijinkereii. Biochem J 125, 55–66.PubMedGoogle Scholar
  14. 14.
    Page, W.J. and Knosp, O. (1989) Hyperproduction of poly-3-hydroxybutyrate during exponential growth of Azotobacter vinelandii UWD. Appl Environ Microbiol 55, 1334–1339.PubMedGoogle Scholar
  15. 15.
    Aneja, P. and Charles, T.C. (1999) Poly-3-hydroxybutyrate degradation in Rhizobium (Sinorhizobium) meliloti: isolation and characterization of a gene encoding 3-hydroxybutyrate dehydrogenase. J Bacteriol 181, 849–857.PubMedGoogle Scholar
  16. 16.
    Aneja, P., Dziak, R., Cai, G.Q., and Charles, T.C. (2002) Identification of an acetoacetyl coenzyme-A synthetase-dependent pathway for utilization of l-(+)-3-hydroxybutyrate in Sinorhizobium meliloti. J Bacteriol 184, 1571–1577.PubMedCrossRefGoogle Scholar
  17. 17.
    Charles, T.C., Cai, G.Q., and Aneja, P. (1997) Megaplasmid and chromosomal loci for the PHB degradation pathway in Rhizobium (Sinorhizobium) meliloti. Genetics 146, 1211–1220.PubMedGoogle Scholar
  18. 18.
    Willis, L.B. and Walker, G.C. (1998) The phbC (poly-β-hydroxybutyrate synthase) gene of Rhizobium (Sinorhizobium) meliloti and characterization of phbC mutants. Can J Microbiol 44, 554–564.PubMedGoogle Scholar
  19. 19.
    Aneja, P., Dai, M., Lacorre, D.A., Pillon, B., and Charles, T.C. (2004) Heterologous complementation of the exopolysaccharide synthesis and carbon utilization phenotypes of Sinorhizobium meliloti Rm1021 polyhydroxyalkanoate synthesis mutants. FEMS Microbiol Lett 239, 277–283.PubMedCrossRefGoogle Scholar
  20. 20.
    Ostle, A.G. and Holt, J.G. (1982) Nile blue as a fluorescent stain for poly-β-hydroxybutyrate. Appl Environ Microbiol 44, 238–241.PubMedGoogle Scholar
  21. 21.
    Kranz, R.G., Gabbert, K.K., and Madigan, M.T. (1997) Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification. Appl Environ Microbiol 63, 3010–3013.PubMedGoogle Scholar
  22. 22.
    Povolo, S., Tombolini, R., Morea, A., Anderson, A.J., Casella, S., and Nuti, M.P. (1994) Isolation and characterization of mutants of Rhizobium meliloti unable to synthesize poly-3-hydroxybutyrate (PHB). Can J Microbiol 40, 823–829.CrossRefGoogle Scholar
  23. 23.
    Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York.Google Scholar
  24. 24.
    Beringer, J.E. (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84, 188–198.PubMedGoogle Scholar
  25. 25.
    Miller, J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
  26. 26.
    Dowling, D.N., Samrey, U., Stanley, J., and Broughton, W.J. (1987) Cloning of Rhizobium leguminosarum genes for competitive nodulation blocking on peas. J Bacteriol 169, 1345–1348.PubMedGoogle Scholar
  27. 27.
    Jones, J.D. and Gutterson, N. (1987) An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a. Gene 61, 299–306.PubMedCrossRefGoogle Scholar
  28. 28.
    Wang, C., Meek, D.J., Panchal, P., Boruvka, N., Archibald, F.S., Driscoll, B.T., et al. (2006) Isolation of poly-3-hydroxbutyrate metabolism genes from complex microbial communities by phenotypic complementation of bacterial mutants. Appl Environ Microbiol 72, 384–391.PubMedCrossRefGoogle Scholar
  29. 29.
    Law, J. and Slepecky, R. (1961) Assay of poly-3-hydroxybutyric acid. J Bacteriol 82, 33–36.PubMedGoogle Scholar
  30. 30.
    Kovach, M.E., Elzer, P.H., Hill, D.S., Robertson, G.T., Farris, M.A., Roop, R.M., et al. (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176.PubMedCrossRefGoogle Scholar
  31. 31.
    Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, Z., Miller, W., and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.PubMedCrossRefGoogle Scholar
  32. 32.
    Meade, H.M., Long, S.R., Ruvkun, G.B., Brown, S.E., and Ausubel, F.M. (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149, 114–122.PubMedGoogle Scholar
  33. 33.
    Cai, G., Driscoll, B.T., and Charles, T.C. (2000) Requirement for the enzymes acetoacetyl coenzyme-A synthetase and poly-3-hydroxybutyrate (PHB) synthase for growth of Sinorhizobium meliloti on PHB cycle intermediates. J Bacteriol 182, 2113–2118.PubMedCrossRefGoogle Scholar
  34. 34.
    Aneja, P., Zachertowska, A., and Charles, T.C. (2005) Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti PHB synthesis and degradation pathway mutants. Can J Microbiol 51, 599–604.PubMedCrossRefGoogle Scholar
  35. 35.
    Wang, C.X., Sheng, X.Y., Equi, R.C., Trainer, M.A., Charles, T.C., and Sobral, B.W.S. (2007) Influence of the poly-3-hydroxybutyrate (PHB) granule-associated proteins (PhaP1 and PhaP2) on PHB accumulation and symbiotic nitrogen fixation in Sinorhizobium meliloti Rm1021. J Bacteriol 189, 9050–9056.PubMedCrossRefGoogle Scholar
  36. 36.
    Leigh, J.A., Signer, E.R., and Walker, G.C. (1985) Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci U S A 82, 6231–6235.PubMedCrossRefGoogle Scholar
  37. 37.
    Miller-Williams, M., Loewen, P.C., and Oresnik, I.J. (2006) Isolation of salt-sensitive mutants of Sinorhizobium meliloti strain Rm1021. Microbiology 152, 2049–2059.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2010

Authors and Affiliations

  • Ricardo F. Nordeste
    • 1
  • Maria A. Trainer
    • 1
  • Trevor C. Charles
    • 1
    Email author
  1. 1.Department of BiologyUniversity of WaterlooWaterlooCanada

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