Construction and Screening of Marine Metagenomic Large Insert Libraries

  • Nancy Weiland-Bräuer
  • Daniela Langfeldt
  • Ruth A. Schmitz
Part of the Methods in Molecular Biology book series (MIMB, volume 1539)


The marine environment covers more than 70 % of the world’s surface. Marine microbial communities are highly diverse and have evolved during extended evolutionary processes of physiological adaptations under the influence of a variety of ecological conditions and selection pressures. They harbor an enormous diversity of microbes with still unknown and probably new physiological characteristics. In the past, marine microbes, mostly bacteria of microbial consortia attached to marine tissues of multicellular organisms, have proven to be a rich source of highly potent bioactive compounds, which represent a considerable number of drug candidates. However, to date, the biodiversity of marine microbes and the versatility of their bioactive compounds and metabolites have not been fully explored. This chapter describes sampling in the marine environment, construction of metagenomic large insert libraries from marine habitats, and exemplarily one function based screen of metagenomic clones for identification of quorum quenching activities.

Key words

Isolation of metagenomic DNA 16S rDNA phylogenetic analysis Construction of fosmid libraries Function-based screen Quorum quenching 


  1. 1.
    Kodzius R, Gojobori T (2015) Marine metagenomics as a source for bioprospecting. Mar Genomics 24(Pt 1):21–30CrossRefPubMedGoogle Scholar
  2. 2.
    DeLong EF, Karl DM (2005) Genomic perspectives in microbial oceanography. Nature 437:336–342CrossRefPubMedGoogle Scholar
  3. 3.
    Karl DM (2007) Microbial oceanography: paradigms, processes and promise. Nat Rev Microbiol 5:759–769CrossRefPubMedGoogle Scholar
  4. 4.
    Reen FJ, Gutiérrez-Barranquero JA, Dobson ADW, Adams C, O’Gara F (2015) Emerging concepts promising new horizons for marine biodiscovery and synthetic biology. Mar Drugs 13:2924–2954CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kennedy J, Marchesi JR, Dobson AD (2007) Metagenomic approaches to exploit the biotechnological potential of the microbial consortia of marine sponges. Appl Microbiol Biotechnol 75:11–20CrossRefPubMedGoogle Scholar
  6. 6.
    Zhang X, Wei W, Tan R (2015) Symbionts, a promising source of bioactive natural products. Sci China Chem 58:1097CrossRefGoogle Scholar
  7. 7.
    Bowman JP (2007) Bioactive compound synthetic capacity and ecological significance of marine bacterial genus Pseudoalteromonas. Mar Drugs 5:220–241CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Jaiganesh R, Sampath Kumar NS (2012) Marine bacterial sources of bioactive compounds. Adv Food Nutr Res 65:389–408CrossRefPubMedGoogle Scholar
  9. 9.
    Singh AJ, Field JJ, Atkinson PH, Northcote PT, Miller JH (2015) From marine organism to potential drug: using innovative techniques to identify and characterize novel compounds - a bottom-up approach. In: Bioactive natural products, chemistry and biology. Wiley-Blackwell, London, pp 443–472CrossRefGoogle Scholar
  10. 10.
    Machado H, Sonnenschein EC, Melchiorsen J, Gram L (2015) Genome mining reveals unlocked bioactive potential of marine Gram-negative bacteria. BMC Genomics 16:158CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Molina G, Pelissari FM, Pessoa MG, Pastore GM (2015) Bioactive compounds obtained through biotechnology. In: Biotechnology of bioactive compounds: sources and applications. Wiley-Blackwell, London, p 433Google Scholar
  12. 12.
    Bhakuni DS, Rawat DS (2005) Bioactive metabolites of marine algae, fungi and bacteria. In: Bioactive marine natural products. Springer, Netherlands, pp 1–25Google Scholar
  13. 13.
    Sidebottom AM, Carlson EE (2015) A reinvigorated era of bacterial secondary metabolite discovery. Curr Opin Chem Biol 24:104–111CrossRefPubMedGoogle Scholar
  14. 14.
    Blunt JW, Copp BR, Keyzers RA, Munro MHG, Prinsep MR (2014) Marine natural products. Nat Prod Rep 31:160–258CrossRefPubMedGoogle Scholar
  15. 15.
    Newman DJ, Hill RT (2006) New drugs from marine microbes: the tide is turning. J Ind Microbiol Biotechnol 33:539–544CrossRefPubMedGoogle Scholar
  16. 16.
    Roussis V, King RL, Fenical W (1993) Secondary metabolite chemistry of the Australian brown alga Encyothalia cliftonii: evidence for herbivore chemical defence. Phytochemistry 34:107–111CrossRefGoogle Scholar
  17. 17.
    Kobayashi J, Ishibashi M (1993) Bioactive metabolites of symbiotic marine microorganisms. Chem Rev 93:1753–1769CrossRefGoogle Scholar
  18. 18.
    Blunt JW, Copp BR, Keyzers RA, Munro MH, Prinsep MR (2015) Marine natural products. Nat Prod Rep 32:116–211CrossRefPubMedGoogle Scholar
  19. 19.
    Amann RI, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedPubMedCentralGoogle Scholar
  20. 20.
    Streit WR, Schmitz RA (2004) Metagenomics--the key to the uncultured microbes. Curr Opin Microbiol 7:492–498CrossRefPubMedGoogle Scholar
  21. 21.
    Lorenz P, Eck J (2005) Metagenomics and industrial applications. Nat Rev Microbiol 3:510–516CrossRefPubMedGoogle Scholar
  22. 22.
    Pham VD, Palden T, DeLong EF (2007) Large-scale screens of metagenomic libraries. J Vis Exp 201.Google Scholar
  23. 23.
    DeLong EF (2009) The microbial ocean from genomes to biomes. Nature 459:200–206CrossRefPubMedGoogle Scholar
  24. 24.
    Fu J, Leiros H-KS, de Pascale D, Johnson KA, Blencke H-M, Landfald B (2013) Functional and structural studies of a novel cold-adapted esterase from an Arctic intertidal metagenomic library. Appl Microbiol Biotechnol 97:3965–3978CrossRefPubMedGoogle Scholar
  25. 25.
    Xing M-N, Zhang X-Z, Huang H (2012) Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis. Biotechnol Adv 30:920–929CrossRefPubMedGoogle Scholar
  26. 26.
    Wang Q, Qian C, Zhang X-Z, Liu N, Yan X, Zhou Z (2012) Characterization of a novel thermostable ß-glucosidase from a metagenomic library of termite gut. Enzyme Microb Technol 51:319–324CrossRefPubMedGoogle Scholar
  27. 27.
    Nimchua T, Uengwetwanit T, Eurwilaichitr L (2012) Metagenomic analysis of novel lignocellulose-degrading enzymes from higher termite guts inhabiting microbes. J Microbiol Biotechnol 22:462–469CrossRefPubMedGoogle Scholar
  28. 28.
    Steele HL, Jaeger KE, Daniel R, Streit WR (2009) Advances in recovery of novel biocatalysts from metagenomes. J Mol Microbiol Biotechnol 16:25–37CrossRefPubMedGoogle Scholar
  29. 29.
    Beja O, Suzuki MT, Koonin EV, Aravind L, Hadd A, Nguyen LP et al (2000) Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. Environ Microbiol 2:516–529CrossRefPubMedGoogle Scholar
  30. 30.
    Beja O, Spudich E, Spudich J, Leclerc M, DeLong E (2001) Proteorhodopsin phototrophy in the ocean. Nature 411:786–789CrossRefPubMedGoogle Scholar
  31. 31.
    de la Torre JR, Christianson LM, Beja O, Suzuki MT, Karl DM, Heidelberg J, DeLong EF (2003) Proteorhodopsin genes are distributed among divergent marine bacterial taxa. Proc Natl Acad Sci U S A 100:12830–12835CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    O’Malley MA (2007) Exploratory experimentation and scientific practice: metagenomics and the proteorhodopsin case. Hist Philos Life Sci 29:337–360PubMedGoogle Scholar
  33. 33.
    Woyke T, Teeling H, Ivanova NN, Huntemann M, Richter M, Gloeckner FO et al (2006) Symbiosis insights through metagenomic analysis of a microbial consortium. Nature 443:950–955CrossRefPubMedGoogle Scholar
  34. 34.
    Wild J, Hradecna Z, Szybalski W (2002) Conditionally amplifiable BACs: switching from single-copy to high-copy vectors and genomic clones. Genome Res 12:1434–1444CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Shizuya H, Kouros-Mehr H (2001) The development and applications of the bacterial artificial chromosome cloning system. Keio J Med 50:26–30CrossRefPubMedGoogle Scholar
  36. 36.
    Liebl W, Angelov A, Juergensen J, Chow J, Loeschcke A, Drepper T et al (2014) Alternative hosts for functional (meta) genome analysis. Appl Microbiol Biotechnol 98:8099–8109CrossRefPubMedGoogle Scholar
  37. 37.
    Weiland-Bräuer N, Pinnow N, Schmitz RA (2015) Novel reporter for identification of interference with acyl homoserine lactone and autoinducer-2 quorum sensing. Appl Environ Microbiol 81:1477–1489CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Gabor EM, de Vries EJ, Janssen DB (2003) Efficient recovery of environmental DNA for expression cloning by indirect extraction methods. FEMS Microbiol Ecol 44:153–163CrossRefPubMedGoogle Scholar
  39. 39.
    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
  40. 40.
    Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR et al (2006) Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc Natl Acad Sci U S A 103:12115–12120CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    De Corte D, Yokokawa T, Varela MM, Agogue H, Herndl GJ (2009) Spatial distribution of Bacteria and Archaea and amoA gene copy numbers throughout the water column of the Eastern Mediterranean Sea. ISME J 3:147–158CrossRefPubMedGoogle Scholar
  42. 42.
    Treusch AH, Kletzin A, Raddatz G, Ochsenreiter T, Quaiser A, Meurer G et al (2004) Characterization of large-insert DNA libraries from soil for environmental genomic studies of Archaea. Environ Microbiol 6:970–980CrossRefPubMedGoogle Scholar
  43. 43.
    Metzker ML (2010) Sequencing technologies - the next generation. Nat Rev Genet 11:31–46CrossRefPubMedGoogle Scholar
  44. 44.
    Langfeldt D, Neulinger SC, Heuer W, Staufenbiel I, Kunzel S, Baines JF et al (2014) Composition of microbial oral biofilms during maturation in young healthy adults. PLoS One 9:e87449CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Weiland-Bräuer N, Neulinger SC, Pinnow N, Künzel S, Baines JF, Schmitz RA (2015) Composition of bacterial communities associated with Aurelia aurita changes with compartment, life stage, and population. Appl Environ Microbiol 81:6038–6052CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Langlois RJ, LaRoche J, Raab PA (2005) Diazotrophic diversity and distribution in the tropical and subtropical Atlantic Ocean. Appl Environ Microbiol 71:7910–7919CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Langlois RJ, Hummer D, LaRoche J (2008) Abundances and distributions of the dominant nifH phylotypes in the Northern Atlantic Ocean. Appl Environ Microbiol 74:1922–1931CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Wild J, Hradecna Z, Posfai G, Szybalski W (1996) A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct DNA sequencing. Gene 179:181–188CrossRefPubMedGoogle Scholar
  49. 49.
    Aakvik T, Degnes KF, Dahlsrud R, Schmidt F, Dam R, Yu L et al (2009) A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species. FEMS Microbiol Lett 296:149–158CrossRefPubMedGoogle Scholar
  50. 50.
    Sektas M, Szybalski W (1998) Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes. Mol Biotechnol 9:17–24CrossRefPubMedGoogle Scholar
  51. 51.
    Westenberg M, Bamps S, Soedling H, Hope IA, Dolphin CT (2010) Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host. BMC Biotechnol 10:27CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Kim BS, Kim SY, Park J, Park W, Hwang KY, Yoon YJ et al (2007) Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library. J Appl Microbiol 102:1392–1400CrossRefPubMedGoogle Scholar
  53. 53.
    Langlois R, Großkopf T, Mills M, Takeda S, LaRoche J (2015) Widespread distribution and expression of gamma A (UMB), an uncultured, diazotrophic, y-proteobacterial nifH phylotype. PLoS One 10:e0128912CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Venter JC, Remington K, Heidelberg JF, Halpern AL, Rusch D, Eisen JA et al (2004) Environmental genome shotgun sequencing of the Sargasso Sea. Science 304:66–74CrossRefPubMedGoogle Scholar
  55. 55.
    Gonzalez A, Knight R (2012) Advancing analytical algorithms and pipelines for billions of microbial sequences. Curr Opin Biotechnol 23:64–71CrossRefPubMedGoogle Scholar
  56. 56.
    Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N et al (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621–1624CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Lasken RS (2013) Single-cell sequencing in its prime. Nat Biotechnol 31:211–212CrossRefPubMedGoogle Scholar
  58. 58.
    Hicks MA, Prather KL (2014) Bioprospecting in the genomic age. Adv Appl Microbiol 87(87):111–146CrossRefPubMedGoogle Scholar
  59. 59.
    Gillespie DE, Brady SF, Bettermann AD, Cianciotto NP, Liles MR, Rondon MR et al (2002) Isolation of antibiotics turbomycin a and B from a metagenomic library of soil microbial DNA. Appl Environ Microbiol 68:4301–4306CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Brady SF, Chao CJ, Clardy J (2002) New natural product families from an environmental DNA (eDNA) gene cluster. J Am Chem Soc 124:9968–9969CrossRefPubMedGoogle Scholar
  61. 61.
    Banik JJ, Brady SF (2010) Recent application of metagenomic approaches toward the discovery of antimicrobials and other bioactive small molecules. Curr Opin Microbiol 13:603–609CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    MacNeil IA, Tiong CL, Minor C, August PR, Grossman TH, Loiacono KA et al (2001) Expression and isolation of antimicrobial small molecules from soil DNA libraries. J Mol Microbiol Biotechnol 3:301–308PubMedGoogle Scholar
  63. 63.
    Madalozzo AD, Martini VP, Kuniyoshi KK, de Souza EM, Pedrosa FO, Glogauer A et al (2015) Immobilization of LipC12, a new lipase obtained by metagenomics, and its application in the synthesis of biodiesel esters. J Mol Catal B: Enzym 116:45–51CrossRefGoogle Scholar
  64. 64.
    Selvin J, Kennedy J, Lejon DPH, Kiran GS, Dobson ADW (2012) Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans. Microb Cell Fact 11:72CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Henne A, Schmitz RA, Bomeke M, Gottschalk G, Daniel R (2000) Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl Environ Microbiol 66:3113–3116CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Cretoiu MS, Kielak AM, Al-Soud WA, Sörensen SJ, van Elsas JD (2012) Mining of unexplored habitats for novel chitinases - chiA as a helper gene proxy in metagenomics. Appl Microbiol Biotechnol 94:1347–1358CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Cottrell MT, Moore JA, Kirchman DL (1999) Chitinases from uncultured marine microorganisms. Appl Environ Microbiol 65:2553–2557PubMedPubMedCentralGoogle Scholar
  68. 68.
    Majernik A, Gottschalk G, Daniel R (2001) Screening of environmental DNA libraries for the presence of genes conferring Na(+)(Li(+))/H(+) antiporter activity on Escherichia coli: characterization of the recovered genes and the corresponding gene products. J Bacteriol 183:6645–6653CrossRefPubMedPubMedCentralGoogle Scholar
  69. 69.
    Dickschat JS (2010) Quorum sensing and bacterial biofilms. Nat Prod Rep 27:343–369CrossRefPubMedGoogle Scholar
  70. 70.
    Shrout JD, Tolker-Nielsen T, Givskov M, Parsek MR (2011) The contribution of cell-cell signaling and motility to bacterial biofilm formation. MRS Bull 36:367–373CrossRefPubMedPubMedCentralGoogle Scholar
  71. 71.
    Landini P, Antoniani D, Burgess JG, Nijland R (2010) Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal. Appl Microbiol Biotechnol 86:813CrossRefPubMedGoogle Scholar
  72. 72.
    Liu L, Tan X, Jia A (2012) Relationship between bacterial quorum sensing and biofilm formation--a review. Wei Sheng Wu Xue Bao 52:271–278PubMedGoogle Scholar
  73. 73.
    Moreira CG, Sperandio V (2010) The epinephrine/norepinephrine/autoinducer-3 interkingdom signaling system in Escherichia coli O157:H7. In: Lyte M, Cryan JF (eds) Microbial endocrinology. Springer, New York, NY, pp 213–227CrossRefGoogle Scholar
  74. 74.
    Zohar B-A, Kolodkin-Gal I (2015) Quorum sensing in Escherichia coli: interkingdom, inter-and intraspecies dialogues, and a suicide-inducing peptide. In: Quorum sensing vs quorum quenching: a battle with no end in sight. Springer, New York, NY, pp 85–99Google Scholar
  75. 75.
    Higgins DA, Pomianek ME, Kraml CM, Taylor RK, Semmelhack MF, Bassler BL (2007) The major Vibrio cholerae autoinducer and its role in virulence factor production. Nature 450:883–886CrossRefPubMedGoogle Scholar
  76. 76.
    Donlan RM, Costerton JW (2002) Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 15:167–193CrossRefPubMedPubMedCentralGoogle Scholar
  77. 77.
    Elias S, Banin E (2012) Multi-species biofilms: living with friendly neighbors. FEMS Microbiol Rev 36:990CrossRefPubMedGoogle Scholar
  78. 78.
    Dong YH, Zhang LH (2005) Quorum sensing and quorum-quenching enzymes. J Microbiol 43(Spec No):101–109PubMedGoogle Scholar
  79. 79.
    Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O (2010) Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents 35:322–332CrossRefPubMedGoogle Scholar
  80. 80.
    Romero M, Acuna L, Otero A (2012) Patents on quorum quenching: interfering with bacterial communication as a strategy to fight infections. Recent Pat Biotechnol 6:2–12CrossRefPubMedGoogle Scholar
  81. 81.
    Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH (2001) Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813–817CrossRefPubMedGoogle Scholar
  82. 82.
    Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen TB, Bagge N et al (2003) Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J 22:3803–3815CrossRefPubMedPubMedCentralGoogle Scholar
  83. 83.
    Zhang LH (2003) Quorum quenching and proactive host defense. Trends Plant Sci 8:238–244CrossRefPubMedGoogle Scholar
  84. 84.
    Zhang LH, Dong YH (2004) Quorum sensing and signal interference: diverse implications. Mol Microbiol 53:1563–1571CrossRefPubMedGoogle Scholar
  85. 85.
    Kalia VC, Purohit HJ (2011) Quenching the quorum sensing system: potential antibacterial drug targets. Crit Rev Microbiol 37:121–140CrossRefPubMedGoogle Scholar
  86. 86.
    Inoue H, Nojima H, Okayama H (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Nancy Weiland-Bräuer
    • 1
  • Daniela Langfeldt
    • 1
  • Ruth A. Schmitz
    • 1
  1. 1.Institut für Allgemeine MikrobiologieChristian-Albrechts-Universität zu KielKielGermany

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