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Metagenomics pp 237-248 | Cite as

Methods for the Isolation of Genes Encoding Novel PHA Metabolism Enzymes from Complex Microbial Communities

  • Jiujun Cheng
  • Ricardo Nordeste
  • Maria A. Trainer
  • Trevor C. CharlesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1539)

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 bio-plastics. The specific phenotypes associated with mutations of the PHA synthesis pathway genes in Sinorhizobium meliloti and Pseudomonas putida, allows 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 the 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 pathway Sinorhizobium meliloti Pseudomonas putida Microbial community gene libraries Phenotypic complementation 

References

  1. 1.
    Henne A, Daniel R, Schmitz RA, 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 65:3901–3907PubMedPubMedCentralGoogle Scholar
  2. 2.
    Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472PubMedPubMedCentralGoogle Scholar
  3. 3.
    Zevenhuizen LPTM (1981) Cellular glycogen, β-1,2-glucan, poly-3-hydroxybutyric acid and extracellular polysaccharides in fast-growing species of Rhizobium. Antonie Van Leeuwenhoek 47:481–497CrossRefPubMedGoogle Scholar
  4. 4.
    Madison LL, Huisman GW (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63:21–53PubMedPubMedCentralGoogle Scholar
  5. 5.
    Shishatskaya EI, Voinova ON, Goreva AV, Mogilnaya OA, Volova TG (2008) Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation. J Mater Sci Mater Med 19:2493–2502CrossRefPubMedGoogle Scholar
  6. 6.
    Shishatskaya EI, Volova TG, Puzyr AP, Mogilnaya OA, Efremov SN (2004) Tissue response to the implantation of biodegradable polyhydroxyalkanoate sutures. J Mater Sci Mater Med 15:719–728CrossRefPubMedGoogle Scholar
  7. 7.
    Holmes PA (1985) Applications of PHB -- a microbially produced biodegradable thermosplastic. Phys Technol 16:32–36CrossRefGoogle Scholar
  8. 8.
    Pötter M, Steinbüchel A (2005) Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules 6:552–560CrossRefPubMedGoogle Scholar
  9. 9.
    Kadouri D, Jurkevitch E, Okon Y (2003) Involvement of the reserve material poly-β-hydroxybutyrate in Azospirillum brasilense stress endurance and root colonization. Appl Environ Microbiol 69:3244–3250CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Senior PJ, Beech GA, Ritchie GAF, Dawes EA (1972) The role of oxygen limitation in the formation of poly-3-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinckii. Biochem J 128:1193–1201CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Stam H, van Verseveld HW, de Vries W, Stouhamer AH (1986) Utilization of poly-β-hydroxybutyrate in free-living cultures of Rhizobium ORS571. FEMS Microbiol Lett 35:215–220Google Scholar
  12. 12.
    Stockdale H, Ribbons DW, Dawes EA (1968) Occurence of poly-3-hydroxybutyrate in the Azotobacteriaceae. J Bacteriol 95:1798–1803PubMedPubMedCentralGoogle Scholar
  13. 13.
    Senior PJ, Dawes EA (1971) Poly-3-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijinkereii. Biochem J 125:55–66CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Page WJ, Knosp O (1989) Hyperproduction of poly-3-Hydroxybutyrate during exponential growth of Azotobacter vinelandii UWD. Appl Environ Microbiol 55:1334–1339PubMedPubMedCentralGoogle Scholar
  15. 15.
    Schallmey M, Ly A, Wang C, Meglei G, Voget S, Streit WR et al (2011) Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening. FEMS Microbiol Lett 321:150–156CrossRefPubMedGoogle Scholar
  16. 16.
    Aneja P, Charles TC (1999) Poly-3-hydroxybutyrate degradation in Rhizobium (Sinorhizobium) meliloti: isolation and characterization of a gene encoding 3-hydroxybutyrate dehydrogenase. J Bacteriol 181:849–857PubMedPubMedCentralGoogle Scholar
  17. 17.
    Aneja P, Dziak R, Cai GQ, Charles TC (2002) Identification of an acetoacetyl coenzyme-A synthetase-dependent pathway for utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti. J Bacteriol 184:1571–1577CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Charles TC, Cai GQ, Aneja P (1997) Megaplasmid and chromosomal loci for the PHB degradation pathway in Rhizobium (Sinorhizobium) meliloti. Genetics 146:1211–1220PubMedPubMedCentralGoogle Scholar
  19. 19.
    Willis LB, Walker GC (1998) The phbC (poly-β-hydroxybutyrate synthase) gene of Rhizobium (Sinorhizobium) meliloti and characterization of phbC mutants. Can J Microbiol 44:554–564CrossRefPubMedGoogle Scholar
  20. 20.
    Aneja P, Dai M, Lacorre DA, Pillon B, Charles TC (2004) Heterologous complementation of the exopolysaccharide synthesis and carbon utilization phenotypes of Sinorhizobium meliloti Rm1021 polyhydroxyalkanoate synthesis mutants. FEMS Microbiol Lett 239:277–283CrossRefPubMedGoogle Scholar
  21. 21.
    Ostle AG, Holt JG (1982) Nile blue as a fluorescent stain for poly-β-hydroxybutyrate. Appl Environ Microbiol 44:238–241PubMedPubMedCentralGoogle Scholar
  22. 22.
    Kranz RG, Gabbert KK, Madigan MT (1997) Positive selection systems for discovery of novel polyester biosynthesis genes based on fatty acid detoxification. Appl Environ Microbiol 63:3010–3013PubMedPubMedCentralGoogle Scholar
  23. 23.
    Povolo S, Tombolini R, Morea A, Anderson AJ, Casella S, Nuti MP (1994) Isolation and characterization of mutants of Rhizobium meliloti unable to synthesize poly-3-hydroxybutyrate (PHB). Can J Microbiol 40:823–829CrossRefGoogle Scholar
  24. 24.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NYGoogle Scholar
  25. 25.
    Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198PubMedGoogle Scholar
  26. 26.
    Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  27. 27.
    Dowling DN, Samrey U, Stanley J, Broughton WJ (1987) Cloning of Rhizobium leguminosarum genes for competitive nodulation blocking on peas. J Bacteriol 169:1345–1348CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Cheng J, Pinnell L, Engel K, Neufeld JD, Charles TC (2014) Versatile broad-host-range cosmids for construction of high quality metagenomic libraries. J Microbiol Methods 99:27–34CrossRefPubMedGoogle Scholar
  29. 29.
    Jones JD, 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 61:299–306CrossRefPubMedGoogle Scholar
  30. 30.
    Wang C, Meek DJ, Panchal P, Boruvka N, Archibald FS, Driscoll BT, Charles TC (2006) Isolation of poly-3-hydroxbutyrate metabolism genes from complex microbial communities by phenotypic complementation of bacterial mutants. Appl Environ Microbiol 72:384–391CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Law J, Slepecky R (1961) Assay of poly-3-hydroxybutyric acid. J Bacteriol 82:33–36PubMedPubMedCentralGoogle Scholar
  32. 32.
    Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176CrossRefPubMedGoogle Scholar
  33. 33.
    Altschul SF, Madden TL, Schäffer AA, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122PubMedPubMedCentralGoogle Scholar
  35. 35.
    Cai G, Driscoll BT, Charles TC (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 182:2113–2118CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Aneja P, Zachertowska A, Charles TC (2005) Comparison of the symbiotic and competition phenotypes of Sinorhizobium meliloti PHB synthesis and degradation pathway mutants. Can J Microbiol 51:599–604CrossRefPubMedGoogle Scholar
  37. 37.
    Wang CX, Sheng XY, Equi RC, Trainer MA, Charles TC, Sobral BWS (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 189:9050–9056CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Leigh JA, Signer ER, Walker GC (1985) Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci U S A 82:6231–6235CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Miller-Williams M, Loewen PC, Oresnik IJ (2006) Isolation of salt-sensitive mutants of Sinorhizobium meliloti strain Rm1021. Microbiology 152:2049–2059CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

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

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