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Expansion of Cultured Bacterial Diversity by Large-Scale Dilution-to-Extinction Culturing from a Single Seawater Sample

  • Microbiology of Aquatic Systems
  • Published:
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Abstract

High-throughput cultivation (HTC) based on a dilution-to-extinction method has been applied broadly to the cultivation of marine bacterial groups, which has often led to the repeated isolation of abundant lineages such as SAR11 and oligotrophic marine gammaproteobacteria (OMG). In this study, to expand the phylogenetic diversity of HTC isolates, we performed a large-scale HTC with a single surface seawater sample collected from the East Sea, the Western Pacific Ocean. Phylogenetic analyses of the 16S rRNA genes from 847 putative pure cultures demonstrated that some isolates were affiliated with not-yet-cultured clades, including the OPB35 and Puniceicoccaceae marine group of Verrucomicrobia and PS1 of Alphaproteobacteria. In addition, numerous strains were obtained from abundant clades, such as SAR11, marine Roseobacter clade, OMG (e.g., SAR92 and OM60), OM43, and SAR116, thereby increasing the size of available culture resources for representative marine bacterial groups. Comparison between the composition of HTC isolates and the bacterial community structure of the seawater sample used for HTC showed that diverse marine bacterial groups exhibited various growth capabilities under our HTC conditions. The growth response of many bacterial groups, however, was clearly different from that observed with conventional plating methods, as exemplified by numerous isolates of the SAR11 clade and Verrucomicrobia. This study showed that a large number of novel bacterial strains could be obtained by an extensive HTC from even a small number of samples.

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References

  1. Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S, Wu D, Eisen JA, Hoffman JM, Remington K, Beeson K, Tran B, Smith H, Baden-Tillson H, Stewart C, Thorpe J, Freeman J, Andrews-Pfannkoch C, Venter JE, Li K, Kravitz S, Heidelberg JF, Utterback T, Rogers Y-H, Falcón LI, Souza V, Bonilla-Rosso G, Eguiarte LE, Karl DM, Sathyendranath S, Platt T, Bermingham E, Gallardo V, Tamayo-Castillo G, Ferrari MR, Strausberg RL, Nealson K, Friedman R, Frazier M, Venter JC (2007) The sorcerer II global ocean sampling expedition: northwest Atlantic through eastern tropical pacific. PLoS Biol 5:e77

    Article  PubMed Central  PubMed  Google Scholar 

  2. Swan BK, Tupper B, Sczyrba A, Lauro FM, Martinez-Garcia M, González JM, Luo H, Wright JJ, Landry ZC, Hanson NW, Thompson BP, Poulton NJ, Schwientek P, Acinas SG, Giovannoni SJ, Moran MA, Hallam SJ, Cavicchioli R, Woyke T, Stepanauskas R (2013) Prevalent genome streamlining and latitudinal divergence of planktonic bacteria in the surface ocean. Proc Natl Acad Sci U S A 110:11463–11468. doi:10.1073/pnas.1304246110

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  3. Karl DM, Beversdorf L, Bjorkman KM, Church MJ, Martinez A, Delong EF (2008) Aerobic production of methane in the sea. Nat Geosci 1:473–478

    Article  CAS  Google Scholar 

  4. Metcalf WW, Griffin BM, Cicchillo RM, Gao J, Janga SC, Cooke HA, Circello BT, Evans BS, Martens-Habbena W, Stahl DA, van der Donk WA (2012) Synthesis of methylphosphonic acid by marine microbes: a source for methane in the aerobic ocean. Science 337:1104–1107. doi:10.1126/science.1219875

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  5. Carini P, White AE, Campbell EO, Giovannoni SJ (2014) Methane production by phosphate-starved SAR11 chemoheterotrophic marine bacteria. Nat Commun 5:4346. doi:10.1038/ncomms5346

    Article  PubMed  CAS  Google Scholar 

  6. Drüppel K, Hensler M, Trautwein K, Koßmehl S, Wöhlbrand L, Schmidt-Hohagen K, Ulbrich M, Bergen N, Meier-Kolthoff JP, Göker M, Klenk H-P, Schomburg D, Rabus R (2014) Pathways and substrate-specific regulation of amino acid degradation in Phaeobacter inhibens DSM 17395 (archetype of the marine Roseobacter clade). Environ Microbiol 16:218–238. doi:10.1111/1462-2920.12276

    Article  PubMed  Google Scholar 

  7. Wiegmann K, Hensler M, Wöhlbrand L, Ulbrich M, Schomburg D, Rabus R (2014) Carbohydrate catabolism in Phaeobacter inhibens DSM 17395, a member of the marine Roseobacter clade. Appl Environ Microbiol 80:4725–4737. doi:10.1128/aem.00719-14doi:10.1128/aem.00719-14

    Article  PubMed Central  PubMed  Google Scholar 

  8. Zhao Y, Temperton B, Thrash JC, Schwalbach MS, Vergin KL, Landry ZC, Ellisman M, Deerinck T, Sullivan MB, Giovannoni SJ (2013) Abundant SAR11 viruses in the ocean. Nature 494:357–360. doi:10.1038/nature11921

    Article  PubMed  CAS  Google Scholar 

  9. Kang I, Oh H-M, Kang D, Cho J-C (2013) Genome of a SAR116 bacteriophage shows the prevalence of this phage type in the oceans. Proc Natl Acad Sci U S A 110:12343–12348. doi:10.1073/pnas.1219930110

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  10. Yooseph S, Nealson KH, Rusch DB, McCrow JP, Dupont CL, Kim M, Johnson J, Montgomery R, Ferriera S, Beeson K, Williamson SJ, Tovchigrechko A, Allen AE, Zeigler LA, Sutton G, Eisenstadt E, Rogers Y-H, Friedman R, Frazier M, Venter JC (2010) Genomic and functional adaptation in surface ocean planktonic prokaryotes. Nature 468:60–66

    Article  PubMed  CAS  Google Scholar 

  11. Gifford SM, Sharma S, Booth M, Moran MA (2013) Expression patterns reveal niche diversification in a marine microbial assemblage. ISME J 7:281–298. doi:10.1038/ismej.2012.96

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  12. Rappé MS (2013) Stabilizing the foundation of the house that omics builds: the evolving value of cultured isolates to marine microbiology. Curr Opin Microbiol 16:618–624. doi:10.1016/j.mib.2013.09.009

    Article  PubMed  Google Scholar 

  13. Staley JT, Konopka A (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol 39:321–346. doi:10.1146/annurev.mi.39.100185.001541

    Article  PubMed  CAS  Google Scholar 

  14. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    PubMed Central  PubMed  CAS  Google Scholar 

  15. Zengler K, Toledo G, Rappé M, Elkins J, Mathur EJ, Short JM, Keller M (2002) Cultivating the uncultured. Proc Natl Acad Sci U S A 99:15681–15686. doi:10.1073/pnas.252630999

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  16. Kaeberlein T, Lewis K, Epstein SS (2002) Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296:1127–1129. doi:10.1126/science.1070633

    Article  PubMed  CAS  Google Scholar 

  17. Aoi Y, Kinoshita T, Hata T, Ohta H, Obokata H, Tsuneda S (2009) Hollow-fiber membrane chamber as a device for in situ environmental cultivation. Appl Environ Microbiol 75:3826–3833. doi:10.1128/aem.02542-08

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  18. Steinert G, Whitfield S, Taylor M, Thoms C, Schupp P (2014) Application of diffusion growth chambers for the cultivation of marine sponge-associated bacteria. Mar Biotechnol 16:594–603. doi:10.1007/s10126-014-9575-y

    Article  PubMed  CAS  Google Scholar 

  19. Connon SA, Giovannoni SJ (2002) High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl Environ Microbiol 68:3878–3885. doi:10.1128/aem.68.8.3878-3885.2002

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  20. Tandogan N, Abadian PN, Epstein S, Aoi Y, Goluch ED (2014) Isolation of microorganisms using sub-micrometer constrictions. PLoS ONE 9:e101429. doi:10.1371/journal.pone.0101429

    Article  PubMed Central  PubMed  Google Scholar 

  21. Nichols D, Cahoon N, Trakhtenberg EM, Pham L, Mehta A, Belanger A, Kanigan T, Lewis K, Epstein SS (2010) Use of Ichip for high-throughput in situ cultivation of “uncultivable” microbial species. Appl Environ Microbiol 76:2445–2450. doi:10.1128/aem.01754-09

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  22. Jung D, Seo E-Y, Epstein SS, Joung Y, Han J, Parfenova VV, Belykh OI, Gladkikh AS, Ahn TS (2014) Application of a new cultivation technology, I-tip, for studying microbial diversity in freshwater sponges of lake Baikal, Russia. FEMS Microbiol Ecol 90:417–423. doi:10.1111/1574-6941.12399

    PubMed  CAS  Google Scholar 

  23. Rappe MS, Connon SA, Vergin KL, Giovannoni SJ (2002) Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 418:630–633

    Article  PubMed  CAS  Google Scholar 

  24. Cho J-C, Giovannoni SJ (2004) Cultivation and growth characteristics of a diverse group of oligotrophic marine Gammaproteobacteria. Appl Environ Microbiol 70:432–440. doi:10.1128/aem.70.1.432-440.2004

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  25. Cho J-C, Vergin KL, Morris RM, Giovannoni SJ (2004) Lentisphaera araneosa gen. nov., sp. nov, a transparent exopolymer producing marine bacterium, and the description of a novel bacterial phylum, Lentisphaerae. Environ Microbiol 6:611–621. doi:10.1111/j.1462-2920.2004.00614.x

    Article  PubMed  CAS  Google Scholar 

  26. Giovannoni SJ, Hayakawa DH, Tripp HJ, Stingl U, Givan SA, Cho J-C, Oh H-M, Kitner JB, Vergin KL, Rappé MS (2008) The small genome of an abundant coastal ocean methylotroph. Environ Microbiol 10:1771–1782. doi:10.1111/j.1462-2920.2008.01598.x

    Article  PubMed  CAS  Google Scholar 

  27. Grote J, Bayindirli C, Bergauer K, De Moraes PC, Chen H, D’Ambrosio L, Edwards B, Fernández-Gómez B, Hamisi M, Logares R, Nguyen D, Rii Y, Saeck E, Schutte C, Widner B, Church M, Steward G, Karl D, DeLong E, Eppley J, Schuster SC, Kyrpides N, Rappe M (2011) Complete genome sequence of strain HIMB100, a cultured representative of the SAR116 clade of marine Alphaproteobacteria. Stand Genomic Sci 5:269–278

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  28. Marshall KT, Morris RM (2012) Isolation of an aerobic sulfur oxidizer from the SUP05/Arctic96BD-19 clade. ISME J 7:452–455. doi:10.1038/ismej.2012.78

    Article  PubMed Central  PubMed  Google Scholar 

  29. Stingl U, Tripp HJ, Giovannoni SJ (2007) Improvements of high-throughput culturing yielded novel SAR11 strains and other abundant marine bacteria from the Oregon coast and the Bermuda Atlantic Time Series study site. ISME J 1:361–371

    PubMed  CAS  Google Scholar 

  30. Song J, Oh H-M, Cho J-C (2009) Improved culturability of SAR11 strains in dilution-to-extinction culturing from the East Sea, West Pacific Ocean. FEMS Microbiol Lett 295:141–147

    Article  PubMed  CAS  Google Scholar 

  31. Lane D (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175

    Google Scholar 

  32. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. In: Nucleic acids symposium series, Vol. 41, pp 95-98

  33. Giovannoni SJ, Rappé MS, Vergin KL, Adair NL (1996) 16S rRNA genes reveal stratified open ocean bacterioplankton populations related to the green non-sulfur bacteria. Proc Natl Acad Sci U S A 93:7979–7984

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  34. Turner S, Pryer KM, Miao VPW, Palmer JD (1999) Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J Eukaryot Microbiol 46:327–338. doi:10.1111/j.1550-7408.1999.tb04612.x

    Article  PubMed  CAS  Google Scholar 

  35. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. doi:10.1128/aem.01541-09

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  36. Schloss PD, Gevers D, Westcott SL (2011) Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS ONE 6:e27310. doi:10.1371/journal.pone.0027310

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  37. Ludwig W, Strunk O, Westram R, Richter L, Meier H, Buchner A, Yadhukumar, Lai T, Steppi S, Jobb G, Förster W, Brettske I, Gerber S, Ginhart AW, Gross O, Grumann S, Hermann S, Jost R, König A, Liss T, Lüßmann R, May M, Nonhoff B, Reichel B, Strehlow R, Stamatakis A, Stuckmann N, Vilbig A, Lenke M, Ludwig T, Bode A, Schleifer KH (2004) ARB: a software environment for sequence data. Nucleic Acids Res 32:1363–1371

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  38. Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL (2008) NCBI BLAST: a better web interface. Nucleic Acids Res 36:W5–W9. doi:10.1093/nar/gkn201

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  39. Kim O-S, Cho Y-J, Lee K, Yoon S-H, Kim M, Na H, Park S-C, Jeon YS, Lee J-H, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721. doi:10.1099/ijs.0.038075-0

    Article  PubMed  CAS  Google Scholar 

  40. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. doi:10.1093/molbev/msr121

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  41. Button DK, Schut F, Quang P, Martin R, Robertson BR (1993) Viability and isolation of marine bacteria by dilution culture: theory, procedures, and initial results. Appl Environ Microbiol 59:881–891

    PubMed Central  PubMed  CAS  Google Scholar 

  42. Vartoukian SR, Palmer RM, Wade WG (2010) Strategies for culture of ‘unculturable’ bacteria. FEMS Microbiol Lett 309:1–7. doi:10.1111/j.1574-6968.2010.02000.x

    PubMed  CAS  Google Scholar 

  43. Morris RM, Rappe MS, Connon SA, Vergin KL, Siebold WA, Carlson CA, Giovannoni SJ (2002) SAR11 clade dominates ocean surface bacterioplankton communities. Nature 420:806–810

    Article  PubMed  CAS  Google Scholar 

  44. Grote J, Thrash JC, Huggett MJ, Landry ZC, Carini P, Giovannoni SJ, Rappé MS (2012) Streamlining and core genome conservation among highly divergent members of the SAR11 clade. mBio 3:e00252-12. doi:10.1128/mBio.00252-12

  45. Thrash JC, Boyd A, Huggett MJ, Grote J, Carini P, Yoder RJ, Robbertse B, Spatafora JW, Rappe MS, Giovannoni SJ (2011) Phylogenomic evidence for a common ancestor of mitochondria and the SAR11 clade. Sci Rep 1:13

    Article  PubMed Central  PubMed  Google Scholar 

  46. Viklund J, Martijn J, Ettema TJG, Andersson SGE (2013) Comparative and phylogenomic evidence that the alphaproteobacterium HIMB59 is not a member of the oceanic SAR11 Clade. PLoS ONE 8:e78858. doi:10.1371/journal.pone.0078858

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  47. Luo H (2015) Evolutionary origin of a streamlined marine bacterioplankton lineage. ISME J 9:1423–1433. doi:10.1038/ismej.2014.227

    Article  PubMed  Google Scholar 

  48. Buchan A, González JM, Moran MA (2005) Overview of the marine Roseobacter lineage. Appl Environ Microbiol 71:5665–5677. doi:10.1128/aem.71.10.5665-5677.2005

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  49. Suzuki MT, Preston CM, Béjà O, de la Torre JR, Steward GF, DeLong EF (2004) Phylogenetic screening of ribosomal RNA gene-containing clones in bacterial artificial chromosome (BAC) libraries from different depths in Monterey Bay. Microb Ecol 48:473–488. doi:10.1007/s00248-004-0213-5

    Article  PubMed  CAS  Google Scholar 

  50. Obernosterer I, Catala P, Lebaron P, West NJ (2011) Distinct bacterial groups contribute to carbon cycling during a naturally iron fertilized phytoplankton bloom in the Southern Ocean. Limnol Oceanogr 56:2391–2401

    Article  CAS  Google Scholar 

  51. Teeling H, Fuchs BM, Becher D, Klockow C, Gardebrecht A, Bennke CM, Kassabgy M, Huang S, Mann AJ, Waldmann J, Weber M, Klindworth A, Otto A, Lange J, Bernhardt J, Reinsch C, Hecker M, Peplies J, Bockelmann FD, Callies U, Gerdts G, Wichels A, Wiltshire KH, Glöckner FO, Schweder T, Amann R (2012) Substrate-controlled succession of marine bacterioplankton populations induced by a phytoplankton bloom. Science 336:608–611. doi:10.1126/science.1218344

    Article  PubMed  CAS  Google Scholar 

  52. Giovannoni SJ, Vergin KL (2012) Seasonality in ocean microbial communities. Science 335:671–676. doi:10.1126/science.1198078

    Article  PubMed  CAS  Google Scholar 

  53. Oh H-M, Kwon KK, Kang I, Kang SG, Lee J-H, Kim S-J, Cho J-C (2010) Complete genome sequence of “Candidatus puniceispirillum marinum” IMCC1322, a representative of the SAR116 clade in the Alphaproteobacteria. J Bacteriol 192:3240–3241. doi:10.1128/jb.00347-10

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  54. Treusch AH, Vergin KL, Finlay LA, Donatz MG, Burton RM, Carlson CA, Giovannoni SJ (2009) Seasonality and vertical structure of microbial communities in an ocean gyre. ISME J 3:1148–1163

    Article  PubMed  Google Scholar 

  55. Jimenez-Infante F, Ngugi DK, Alam I, Rashid M, Baalawi W, Kamau AA, Bajic VB, Stingl U (2014) Genomic differentiation among two strains of the PS1 clade isolated from geographically separated marine habitats. FEMS Microbiol Ecol 89:181–197. doi:10.1111/1574-6941.12348

    Article  PubMed  CAS  Google Scholar 

  56. Morris RM, Vergin KL, Cho JC, Rappé MS, Carlson CA, Giovannoni SJ (2005) Temporal and spatial response of bacterioplankton lineages to annual convective overturn at the Bermuda Atlantic Time-Series Study site. Limnol Oceanogr 50:1687–1696

    Article  CAS  Google Scholar 

  57. Iverson V, Morris RM, Frazar CD, Berthiaume CT, Morales RL, Armbrust EV (2012) Untangling genomes from metagenomes: revealing an uncultured class of marine Euryarchaeota. Science 335:587–590. doi:10.1126/science.1212665

    Article  PubMed  CAS  Google Scholar 

  58. Yang S-J, Kang I, Cho J-C (2012) Genome sequence of strain IMCC14465, isolated from the East Sea, belonging to the PS1 clade of Alphaproteobacteria. J Bacteriol 194:6952–6953. doi:10.1128/jb.01888-12

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  59. Stingl U, Desiderio RA, Cho J-C, Vergin KL, Giovannoni SJ (2007) The SAR92 clade: an abundant coastal clade of culturable marine bacteria possessing proteorhodopsin. Appl Environ Microbiol 73:2290–2296. doi:10.1128/aem.02559-06

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  60. Yan S, Fuchs BM, Lenk S, Harder J, Wulf J, Jiao N-Z, Amann R (2009) Biogeography and phylogeny of the NOR5/OM60 clade of Gammaproteobacteria. Syst Appl Microbiol 32:124–139

    Article  PubMed  CAS  Google Scholar 

  61. Huggett MJ, Rappé MS (2012) Genome sequence of strain HIMB55, a novel marine gammaproteobacterium of the OM60/NOR5 clade. J Bacteriol 194:2393–2394. doi:10.1128/jb.00171-12

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  62. Pham VD, Konstantinidis KT, Palden T, DeLong EF (2008) Phylogenetic analyses of ribosomal DNA-containing bacterioplankton genome fragments from a 4000 m vertical profile in the North Pacific Subtropical Gyre. Environ Microbiol 10:2313–2330. doi:10.1111/j.1462-2920.2008.01657.x

    Article  PubMed  CAS  Google Scholar 

  63. Wilkins D, Yau S, Williams TJ, Allen MA, Brown MV, DeMaere MZ, Lauro FM, Cavicchioli R (2013) Key microbial drivers in Antarctic aquatic environments. FEMS Microbiol Rev 37:303–335. doi:10.1111/1574-6976.12007

    Article  PubMed  CAS  Google Scholar 

  64. Nelson CE, Carlson CA (2012) Tracking differential incorporation of dissolved organic carbon types among diverse lineages of Sargasso sea bacterioplankton. Environ Microbiol 14:1500–1516. doi:10.1111/j.1462-2920.2012.02738.x

    Article  PubMed  CAS  Google Scholar 

  65. Wemheuer B, Güllert S, Billerbeck S, Giebel H-A, Voget S, Simon M, Daniel R (2014) Impact of a phytoplankton bloom on the diversity of the active bacterial community in the southern North Sea as revealed by metatranscriptomic approaches. FEMS Microbiol Ecol 87:378–389. doi:10.1111/1574-6941.12230

    Article  PubMed  CAS  Google Scholar 

  66. Sowell SM, Abraham PE, Shah M, Verberkmoes NC, Smith DP, Barofsky DF, Giovannoni SJ (2011) Environmental proteomics of microbial plankton in a highly productive coastal upwelling system. ISME J 5:856–865

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  67. Spring S, Riedel T, Sproer C, Yan S, Harder J, Fuchs B (2013) Taxonomy and evolution of bacteriochlorophyll a-containing members of the OM60/NOR5 clade of marine gammaproteobacteria: description of Luminiphilus syltensis gen. nov., sp. nov., reclassification of Haliea rubra as Pseudohaliea rubra gen. nov., comb. nov., and emendation of Chromatocurvus halotolerans. BMC Microbiol 13:118

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  68. Cho J-C, Stapels MD, Morris RM, Vergin KL, Schwalbach MS, Givan SA, Barofsky DF, Giovannoni SJ (2007) Polyphyletic photosynthetic reaction centre genes in oligotrophic marine Gammaproteobacteria. Environ Microbiol 9:1456–1463. doi:10.1111/j.1462-2920.2007.01264.x

    Article  PubMed  CAS  Google Scholar 

  69. Jang Y, Oh H-M, Kang I, Lee K, Yang S-J, Cho J-C (2011) Genome sequence of strain IMCC3088, a proteorhodopsin-containing marine bacterium belonging to the OM60/NOR5 clade. J Bacteriol 193:3415–3416. doi:10.1128/jb.05111-11

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  70. Spring S, Scheuner C, Göker M, Klenk H-P (2015) A taxonomic framework for emerging groups of ecologically important marine gammaproteobacteria based on the reconstruction of evolutionary relationships using genome-scale data. Front Microbiol 9(6):281. doi:10.3389/fmicb.2015.00281

    Google Scholar 

  71. Kang I, Kang D, Oh H-M, Kim H, Kim H-J, Kang T-W, Kim S-Y, Cho J-C (2011) Genome sequence of strain IMCC2047, a novel marine member of the Gammaproteobacteria. J Bacteriol 193:3688–3689. doi:10.1128/jb.05226-11

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  72. Rappé MS, Kemp PF, Giovannoni SJ (1997) Phylogenetic diversity of marine coastal picoplankton 16s rRNA genes cloned from the continental shelf off Cape Hatteras, North Carolina. Limnol Oceanogr 42:811–826

    Article  Google Scholar 

  73. Huggett M, Hayakawa D, Rappe M (2012) Genome sequence of strain HIMB624, a cultured representative from the OM43 clade of marine Betaproteobacteria. Stand Genomic Sci 6:11–20

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  74. Freitas S, Hatosy S, Fuhrman JA, Huse SM, Mark Welch DB, Sogin ML, Martiny AC (2012) Global distribution and diversity of marine Verrucomicrobia. ISME J 6:1499–1505

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  75. Choo Y-J, Lee K, Song J, Cho J-C (2007) Puniceicoccus vermicola gen. nov., sp. nov., a novel marine bacterium, and description of Puniceicoccaceae fam. nov., Puniceicoccales ord. nov., Opitutaceae fam. nov., Opitutales ord. nov. and Opitutae classis nov. In the phylum ‘Verrucomicrobia’. Int J Syst Evol Microbiol 57:532–537. doi:10.1099/ijs.0.64616-0

    Article  PubMed  CAS  Google Scholar 

  76. Yoon J, Yasumoto-Hirose M, Katsuta A, Sekiguchi H, Matsuda S, Kasai H, Yokota A (2007) Coraliomargarita akajimensis gen. nov., sp. nov., a novel member of the phylum ‘Verrucomicrobia’ isolated from seawater in Japan. Int J Syst Evol Microbiol 57:959–963. doi:10.1099/ijs.0.64755-0

    Article  PubMed  CAS  Google Scholar 

  77. Kant R, van Passel MWJ, Sangwan P, Palva A, Lucas S, Copeland A, Lapidus A, Glavina del Rio T, Dalin E, Tice H, Bruce D, Goodwin L, Pitluck S, Chertkov O, Larimer FW, Land ML, Hauser L, Brettin TS, Detter JC, Han S, de Vos WM, Janssen PH, Smidt H (2011) Genome sequence of “Pedosphaera parvula” Ellin514, an aerobic verrucomicrobial isolate from pasture soil. J Bacteriol 193:2900–2901. doi:10.1128/jb.00299-11

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  78. Martinez-Garcia M, Brazel DM, Swan BK, Arnosti C, Chain PSG, Reitenga KG, Xie G, Poulton NJ, Gomez ML, Masland DED, Thompson B, Bellows WK, Ziervogel K, Lo C-C, Ahmed S, Gleasner CD, Detter CJ, Stepanauskas R (2012) Capturing single cell genomes of active polysaccharide degraders: an unexpected contribution of Verrucomicrobia. PLoS ONE 7:e35314. doi:10.1371/journal.pone.0035314

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  79. Choi A, Yang S-J, Rhee K-H, Cho J-C (2013) Lentisphaera marina sp. nov., and emended description of the genus Lentisphaera. Int J Syst Evol Microbiol 63:1540–1544. doi:10.1099/ijs.0.046433-0

    Article  PubMed  CAS  Google Scholar 

  80. Yilmaz P, Iversen MH, Hankeln W, Kottmann R, Quast C, Glöckner FO (2012) Ecological structuring of bacterial and archaeal taxa in surface ocean waters. FEMS Microbiol Ecol 81:373–385. doi:10.1111/j.1574-6941.2012.01357.x

    Article  PubMed  CAS  Google Scholar 

  81. Zinger L, Boetius A, Ramette A (2014) Bacterial taxa–area and distance–decay relationships in marine environments. Mol Ecol 23:954–964. doi:10.1111/mec.12640

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  82. Cottrell MT, Kirchman DL (2000) Natural assemblages of marine Proteobacteria and members of the Cytophaga-Flavobacter cluster consuming low- and high-molecular-weight dissolved organic matter. Appl Environ Microbiol 66:1692–1697. doi:10.1128/aem.66.4.1692-1697.2000

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  83. Fernandez-Gomez B, Richter M, Schuler M, Pinhassi J, Acinas SG, Gonzalez JM, Pedros-Alio C (2013) Ecology of marine Bacteroidetes: a comparative genomics approach. ISME J 7:1026–1037. doi:10.1038/ismej.2012.169

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  84. Morris RM, Frazar CD, Carlson CA (2012) Basin-scale patterns in the abundance of SAR11 subclades, marine Actinobacteria (OM1), members of the Roseobacter clade and OCS116 in the South Atlantic. Environ Microbiol 14:1133–1144. doi:10.1111/j.1462-2920.2011.02694.x

    Article  PubMed  CAS  Google Scholar 

  85. Halsey KH, Carter AE, Giovannoni SJ (2011) Synergistic metabolism of a broad range of C1 compounds in the marine methylotrophic bacterium HTCC2181. Environ Microbiol 14:630–640. doi:10.1111/j.1462-2920.2011.02605.x

    Article  PubMed  Google Scholar 

  86. Sosa OA, Gifford SM, Repeta DJ, DeLong EF (2015) High molecular weight dissolved organic matter enrichment selects for methylotrophs in dilution to extinction cultures. ISME J. doi:10.1038/ismej.2015.68

    PubMed  Google Scholar 

  87. Newton RJ, Griffin LE, Bowles KM, Meile C, Gifford S, Givens CE, Howard EC, King E, Oakley CA, Reisch CR, Rinta-Kanto JM, Sharma S, Sun S, Varaljay V, Vila-Costa M, Westrich JR, Moran MA (2010) Genome characteristics of a generalist marine bacterial lineage. ISME J 4:784–798

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This research was supported by a grant from the Marine Biotechnology Program (PJT200620, Genome Analysis of Marine Organisms and Development of Functional Applications) funded by the Ministry of Oceans and Fisheries, Korea and also by Mid-Career Research Program through National Research Foundation funded by the Ministry of Science, ICT and Future Planning (NRF-2013R1A2A2A01068004). The authors declare that they have no conflicts of interest.

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    Authors and Affiliations

    1. Department of Biological Sciences, Inha University, Incheon, 402-751, Republic of Korea

      Seung-Jo Yang, Ilnam Kang & Jang-Cheon Cho

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    1. Seung-Jo Yang
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    2. Ilnam Kang
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    3. Jang-Cheon Cho
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    Correspondence to Jang-Cheon Cho.

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    Seung-Jo Yang and Ilnam Kang contributed equally to this work.

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    Fig. S1

    Neighbor-joining phylogenetic tree of the HTC isolates based on 16S rRNA gene sequences. This tree is a combined and extended version of the two trees presented in Fig. 2 and includes strain ID of all HTC isolates (PDF 364 kb)

    Table S1

    Detailed taxonomic information on the HTC isolates (spreadsheet 1) and representatives of pyrosequencing reads from the pooled HTC cultures (spreadsheet 2) (XLSX 78 kb)

    Table S2

    The number of genus-level taxa found in the three sets of growth-positive HTC wells (PDF 74 kb)

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    Yang, SJ., Kang, I. & Cho, JC. Expansion of Cultured Bacterial Diversity by Large-Scale Dilution-to-Extinction Culturing from a Single Seawater Sample. Microb Ecol 71, 29–43 (2016). https://doi.org/10.1007/s00248-015-0695-3

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    • DOI: https://doi.org/10.1007/s00248-015-0695-3

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