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Targeted isolation and cultivation of uncultivated bacteria by reverse genomics

Nature Biotechnology volume 37, pages 1314–1321 (2019)Cite this article

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Abstract

Most microorganisms from all taxonomic levels are uncultured. Single-cell genomes and metagenomes continue to increase the known diversity of Bacteria and Archaea; however, while ’omics can be used to infer physiological or ecological roles for species in a community, most of these hypothetical roles remain unvalidated. Here, we report an approach to capture specific microorganisms from complex communities into pure cultures using genome-informed antibody engineering. We apply our reverse genomics approach to isolate and sequence single cells and to cultivate three different species-level lineages of human oral Saccharibacteria (TM7). Using our pure cultures, we show that all three Saccharibacteria species are epibionts of diverse Actinobacteria. We also isolate and cultivate human oral SR1 bacteria, which are members of a lineage of previously uncultured bacteria. Reverse-genomics-enabled cultivation of microorganisms can be applied to any species from any environment and has the potential to unlock the isolation, cultivation and characterization of species from as-yet-uncultured branches of the microbial tree of life.

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Fig. 1: Overview of targeted microbial isolation through reverse genomics.
Fig. 2: Isolation of TM7 cells by flow cytometry cell sorting.
Fig. 3: TM7–host colonies following single-particle sorting.
Fig. 4: Diversity of cultivated and uncultivated TM7 bacteria.
Fig. 5: Targeted isolation of oral SR1 bacteria.
Fig. 6: Reconstruction of central metabolic pathways for TM7 bacteria, based on completed genomes and draft SAG/MAG assemblies.

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Data availability

Annotated TM7 SAGs are deposited in GenBank under the BioProject PRJNA472398. Raw and aligned SSU rRNA sequences are provided as Supplementary Datasets 1–3. MiSeq amplicon data are deposited in SRA linked to BioProject PRJNA472398.

References

  1. DeLong, E. F. & Pace, N. R. Environmental diversity of bacteria and archaea. Syst. Biol. 50, 470–478 (2001).

    Article  PubMed  CAS  Google Scholar 

  2. Hug, L. A. et al. A new view of the tree of life. Nat. Microbiol. 1, 16048 (2016).

    Article  PubMed  CAS  Google Scholar 

  3. Zaremba-Niedzwiedzka, K. et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541, 353–358 (2017).

    Article  PubMed  CAS  Google Scholar 

  4. Parks, D. H. et al. Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life. Nat. Microbiol. 2, 1533–1542 (2017).

    Article  PubMed  CAS  Google Scholar 

  5. Rinke, C. et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437 (2013).

    Article  PubMed  CAS  Google Scholar 

  6. Gutleben, J. et al. The multi-omics promise in context: from sequence to microbial isolate. Crit. Rev. Microbiol. 44, 212–229 (2017).

    Article  PubMed  Google Scholar 

  7. Epstein, S. S. The phenomenon of microbial uncultivability. Curr. Opin. Microbiol. 16, 636–642 (2013).

    Article  PubMed  CAS  Google Scholar 

  8. Overmann, J., Abt, B. & Sikorski, J. Present and future of culturing bacteria. Annu. Rev. Microbiol. 71, 711–730 (2017).

    Article  PubMed  CAS  Google Scholar 

  9. D’Onofrio, A. et al. Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chem. Biol. 17, 254–264 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Strandwitz, P. et al. GABA-modulating bacteria of the human gut microbiota. Nat. Microbiol. 4, 396–403 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Vartoukian, S. R. et al. In vitro cultivation of ‘unculturable’ oral bacteria, facilitated by community culture and media supplementation with siderophores. PLoS ONE 11, e0146926 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Cross, K. L. et al. Insights into the evolution of host association through the isolation and characterization of a novel human periodontal pathobiont, Desulfobulbus oralis. mBio 9, e02061–17 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Huber, H. et al. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417, 63–67 (2002).

    Article  PubMed  CAS  Google Scholar 

  14. St. John, E. et al. A new symbiotic nanoarchaeote (Candidatus Nanoclepta minutus) and its host (Zestosphaera tikiterensis gen. nov., sp. nov.) from a New Zealand hot spring. Syst. Appl. Microbiol. 42, 94–106 (2018).

    Article  Google Scholar 

  15. Wurch, L. et al. Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment. Nat. Commun. 7, 1–10 (2016).

    Article  Google Scholar 

  16. Pulschen, A. A. et al. Isolation of uncultured bacteria from antarctica using long incubation periods and low nutritional media. Front. Microbiol. 8, 1346 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Oliver, J. D. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol. Rev. 34, 415–425 (2010).

    Article  PubMed  CAS  Google Scholar 

  18. Terekhov, S. S. et al. Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity. Proc. Natl Acad. Sci. USA 114, 2550–2555 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Zengler, K. et al. High-throughput cultivation of microorganisms using microcapsules. Methods Enzymol. 397, 124–130 (2005).

    Article  PubMed  CAS  Google Scholar 

  20. Browne, H. P. et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533, 543–546 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Lagier, J. C. et al. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat. Microbiol. 1, 16203 (2016).

    Article  PubMed  CAS  Google Scholar 

  22. Oberhardt, M. A. et al. Harnessing the landscape of microbial culture media to predict new organism-media pairings. Nat. Commun. 6, 8493 (2015).

    Article  PubMed  CAS  Google Scholar 

  23. Berdy, B., Spoering, A. L., Ling, L. L. & Epstein, S. S. In situ cultivation of previously uncultivable microorganisms using the ichip. Nat. Protoc. 12, 2232–2242 (2017).

    Article  PubMed  Google Scholar 

  24. Sizova, M. V. et al. New approaches for isolation of previously uncultivated oral bacteria. Appl. Environ. Microbiol. 78, 194–203 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Rheims, H., Rainey, F. A. & Stackebrandt, E. A molecular approach to search for diversity among bacteria in the environment. J. Ind. Microbiol. 17, 159–169 (1996).

    CAS  Google Scholar 

  26. Hugenholtz, P., Goebel, B. M. & Pace, N. R. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, 4765–4774 (1998).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Albertsen, M. et al. Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nat. Biotechnol. 31, 533–538 (2013).

    Article  PubMed  CAS  Google Scholar 

  28. Dewhirst, F. E. et al. The human oral microbiome. J. Bacteriol. 192, 5002–5017 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Brinig, M. M., Lepp, P. W., Ouverney, C. C., Armitage, G. C. & Relman, D. A. Prevalence of bacteria of division TM7 in human subgingival plaque and their association with disease. Appl. Environ. Microbiol. 69, 1687–1694 (2003).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Kuehbacher, T. et al. Intestinal TM7 bacterial phylogenies in active inflammatory bowel disease. J. Med. Microbiol. 57, 1569–1576 (2008).

    Article  PubMed  CAS  Google Scholar 

  31. Dewhirst, F. E. et al. The feline oral microbiome: a provisional 16S rRNA gene based taxonomy with full-length reference sequences. Vet. Microbiol. 175, 294–303 (2015).

    Article  PubMed  CAS  Google Scholar 

  32. Dewhirst, F. E. et al. The canine oral microbiome. PLoS ONE 7, e36067 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Dudek, N. K. et al. Novel microbial diversity and functional potential in the marine mammal oral microbiome. Curr. Biol. 27, 3752–3762.e6 (2017).

    Article  PubMed  CAS  Google Scholar 

  34. Marcy, Y. et al. Dissecting biological ‘dark matter’ with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc. Natl Acad. Sci. USA 104, 11889–11894 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. He, X. et al. Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle. Proc. Natl Acad. Sci. USA 112, 244–249 (2015).

    Article  PubMed  CAS  Google Scholar 

  36. Haghighat, S., Siadat, S. D., Sorkhabadi, S. M. R., Sepahi, A. A. & Mahdavi, M. A novel recombinant vaccine candidate comprising PBP2a and autolysin against Methicillin Resistant Staphylococcus aureus confers protection in the experimental mice. Mol. Immunol. 91, 1–7 (2017).

    Article  PubMed  CAS  Google Scholar 

  37. Lovering, A. L., de Castro, L. H., Lim, D. & Strynadka, N. C. Structural insight into the transglycosylation step of bacterial cell-wall biosynthesis. Science 315, 1402–1405 (2007).

    Article  PubMed  CAS  Google Scholar 

  38. Simons, K. T. et al. Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins. Proteins 34, 82–95 (1999).

    Article  PubMed  CAS  Google Scholar 

  39. Palmer, R. J., Jr. et al. Interbacterial adhesion networks within early oral biofilms of single human hosts. Appl. Environ. Microbiol. 83 (2017).

  40. Mark Welch, J. L., Rossetti, B. J., Rieken, C. W., Dewhirst, F. E. & Borisy, G. G. Biogeography of a human oral microbiome at the micron scale. Proc. Natl Acad. Sci. USA 113, E791–E800 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  41. Jakubovics, N. S., Yassin, S. A. & Rickard, A. H. Community interactions of oral streptococci. Adv. Appl. Microbiol. 87, 43–110 (2014).

    Article  PubMed  CAS  Google Scholar 

  42. Heym, B. et al. Molecular detection of Cellulosimicrobium cellulans as the etiological agent of a chronic tongue ulcer in a human immunodeficiency virus-positive patient. J. Clin. Microbiol. 43, 4269–4271 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Bor, B. et al. Phenotypic and physiological characterization of the epibiotic interaction between TM7x and its basibiont actinomyces. Microb. Ecol. 71, 243–255 (2016).

    Article  PubMed  CAS  Google Scholar 

  44. Campbell, J. H. et al. UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota. Proc. Natl Acad. Sci. USA 110, 5540–5545 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Podar, M. et al. Targeted access to the genomes of low-abundance organisms in complex microbial communities. Appl. Environ. Microbiol. 73, 3205–3214 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Campbell, A. G. et al. Diversity and genomic insights into the uncultured Chloroflexi from the human microbiota. Environ. Microbiol. 16, 2635–2643 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Castelle, C. J. et al. Biosynthetic capacity, metabolic variety and unusual biology in the CPR and DPANN radiations. Nat. Rev. Microbiol. 16, 629–645 (2018).

    Article  PubMed  CAS  Google Scholar 

  48. Ferrari, B. C., Binnerup, S. J. & Gillings, M. Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria. Appl. Environ. Microbiol. 71, 8714–8720 (2005).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. Soro, V. et al. Axenic culture of a candidate division TM7 bacterium from the human oral cavity and biofilm interactions with other oral bacteria. Appl. Environ. Microbiol. 80, 6480–6489 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Zengler, K. et al. Cultivating the uncultured. Proc. Natl Acad. Sci. USA 99, 15681–15686 (2002).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  51. Markowitz, V. M. et al. The integrated microbial genomes (IMG) system. Nucleic Acids Res. 34, D344–D348 (2006).

    Article  PubMed  CAS  Google Scholar 

  52. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J. Mol. Biol. 305, 567–580 (2001).

    Article  PubMed  CAS  Google Scholar 

  53. Sauvage, E., Kerff, F., Terrak, M., Ayala, J. A. & Charlier, P. The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis. FEMS Microbiol. Rev. 32, 234–258 (2008).

    Article  PubMed  CAS  Google Scholar 

  54. Haghighat, S., Siadat, S. D., Sorkhabadi, S. M., Sepahi, A. A. & Mahdavi, M. Cloning, expression and purification of penicillin binding Protein2a (PBP2a) from Methicillin resistant Staphylococcus aureus: a study on immunoreactivity in Balb/C mouse. Avicenna J. Med. Biotechnol. 5, 204–211 (2013).

    PubMed  PubMed Central  CAS  Google Scholar 

  55. Zarantonelli, M. L. et al. Immunogenicity of meningococcal PBP2 during natural infection and protective activity of anti-PBP2 antibodies against meningococcal bacteraemia in mice. J. Antimicrob. Chemother. 57, 924–930 (2006).

    Article  PubMed  CAS  Google Scholar 

  56. Byrne, J. P., Morona, J. K., Paton, J. C. & Morona, R. Identification of Streptococcus pneumoniae Cps2C residues that affect capsular polysaccharide polymerization, cell wall ligation, and Cps2D phosphorylation. J. Bacteriol. 193, 2341–2346 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Toniolo, C. et al. Streptococcus agalactiae capsule polymer length and attachment is determined by the proteins CpsABCD. J. Biol. Chem. 290, 9521–9532 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  58. Morona, R., Purins, L., Tocilj, A., Matte, A. & Cygler, M. Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins. Trends Biochem. Sci. 34, 78–84 (2009).

    Article  PubMed  CAS  Google Scholar 

  59. Saha, S. & Raghava, G. P. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins 65, 40–48 (2006).

    Article  PubMed  CAS  Google Scholar 

  60. Larsen, J. E., Lund, O. & Nielsen, M. Improved method for predicting linear B-cell epitopes. Immunome Res. 2, 2 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Heimerl, T. et al. A complex endomembrane system in the archaeon Ignicoccus hospitalis tapped by Nanoarchaeum equitans. Front. Microbiol. 8, 1072 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Miller, L. D. et al. Establishment and metabolic analysis of a model microbial community for understanding trophic and electron accepting interactions of subsurface anaerobic environments. BMC Microbiol. 10, 149 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Campbell, A. G. et al. Multiple single-cell genomes provide insight into functions of uncultured Deltaproteobacteria in the human oral cavity. PloS ONE 8, e59361 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Podar, M. et al. Insights into archaeal evolution and symbiosis from the genomes of a nanoarchaeon and its inferred crenarchaeal host from Obsidian Pool, Yellowstone National Park. Biol. Direct 8, 9 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  65. Rinke, C. et al. Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics. Nat. Protoc. 9, 1038–1048 (2014).

    Article  PubMed  CAS  Google Scholar 

  66. Wong, L. & Sissons, C. A comparison of human dental plaque microcosm biofilms grown in an undefined medium and a chemically defined artificial saliva. Arch. Oral Biol. 46, 477–486 (2001).

    Article  PubMed  CAS  Google Scholar 

  67. Pernthaler, A. & Pernthaler, J. Fluorescence in situ hybridization for the identification of environmental microbes. Methods Mol. Biol. 353, 153–164 (2007).

    PubMed  Google Scholar 

  68. Lundberg, D. S., Yourstone, S., Mieczkowski, P., Jones, C. D. & Dangl, J. L. Practical innovations for high-throughput amplicon sequencing. Nat. Methods 10, 999–1002 (2013).

    Article  PubMed  CAS  Google Scholar 

  69. Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).

    Article  Google Scholar 

  70. Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).

    Article  PubMed  CAS  Google Scholar 

  71. Caporaso, J., Kuczynski, J., Stombaugh, J. & Bittinger, K. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  72. Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Brown, C. T. et al. Unusual biology across a group comprising more than 15% of domain Bacteria. Nature 523, 208–211 (2015).

    Article  PubMed  CAS  Google Scholar 

  74. Hug, L. A. et al. Critical biogeochemical functions in the subsurface are associated with bacteria from new phyla and little studied lineages. Environ. Microbiol. 18, 159–173 (2015).

    Article  PubMed  Google Scholar 

  75. Parks, D. H., Imelfort, M., Skennerton, C. T., Hugenholtz, P. & Tyson, G. W. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 25, 1043–1055 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  76. Kearse, M. et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28, 1647–1649 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  77. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014).

    Article  PubMed  CAS  Google Scholar 

  78. Anantharaman, K. et al. Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system. Nat. Commun. 7, 13219 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230 (2014).

    Article  PubMed  CAS  Google Scholar 

  80. Huerta-Cepas, J. et al. eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences. Nucleic Acids Res. 44, D286–D293 (2016).

    Article  PubMed  CAS  Google Scholar 

  81. Jones, P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236–1240 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  82. Delmont, T. O. & Eren, A. M. Linking pangenomes and metagenomes: the Prochlorococcus metapangenome. PeerJ 6, e4320 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  83. Eren, A. M. et al. Anvi’o: an advanced analysis and visualization platform for ‘omics data. PeerJ 3, e1319 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  84. Balakrishnan, S., Kamisetty, H., Carbonell, J. G., Lee, S. I. & Langmead, C. J. Learning generative models for protein fold families. Proteins 79, 1061–1078 (2011).

    Article  PubMed  CAS  Google Scholar 

  85. Kamisetty, H., Ovchinnikov, S. & Baker, D. Assessing the utility of coevolution-based residue–residue contact predictions in a sequence- and structure-rich era. Proc. Natl Acad. Sci. USA 110, 15674–15679 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  86. Ovchinnikov, S. et al. Protein structure determination using metagenome sequence data. Science 355, 294–298 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  87. Remmert, M., Biegert, A., Hauser, A. & Soding, J. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat. Methods 9, 173–175 (2011).

    Article  PubMed  Google Scholar 

  88. Johnson, L. S., Eddy, S. R. & Portugaly, E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinformatics 11, 431 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  89. Burley, S. K. et al. Protein Data Bank (PDB): the single global macromolecular structure archive. Methods Mol. Biol. 1607, 627–641 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  90. Sung, M. T. et al. Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli. Proc. Natl Acad. Sci. USA 106, 8824–8829 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  91. Han, S. et al. Distinctive attributes of beta-lactam target proteins in Acinetobacter baumannii relevant to development of new antibiotics. J. Am. Chem. Soc. 133, 20536–20545 (2011).

    Article  PubMed  CAS  Google Scholar 

  92. Yuan, Y. et al. Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis. Proc. Natl Acad. Sci. USA 104, 5348–5353 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  93. Huang, C. Y. et al. Crystal structure of Staphylococcus aureus transglycosylase in complex with a lipid II analog and elucidation of peptidoglycan synthesis mechanism. Proc. Natl Acad. Sci. USA 109, 6496–6501 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  94. Gront, D., Kulp, D. W., Vernon, R. M., Strauss, C. E. & Baker, D. Generalized fragment picking in Rosetta: design, protocols and applications. PloS ONE 6, e23294 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

We thank S. Allman, S. Kauffman, S. Lebreux, L. Sukharnikov and M. Robeson for technical assistance. Support for this work was provided by grants (nos. R56DE021567 and R01DE024463) from the National Institute of Dental and Craniofacial Research of the US National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.M.P. was supported by the Laboratory Directed Research and Development program at Oak Ridge National Laboratory, which is managed by UT-Battelle, LLC for the US Department of Energy (contract no. DE-AC05-00OR22725). C.J.C. was supported by a National Science Foundation Graduate Research Fellowship (grant no. 2017219379). This work used resources of the Compute and Data Environment for Science at Oak Ridge National Laboratory.

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Author notes

  1. James H. Campbell & Alisha G. Campbell

    Present address: Department of Natural Sciences, Northwest Missouri State University, Maryville, MO, USA

  2. These authors contributed equally: Karissa L. Cross, James H. Campbell.

Authors and Affiliations

  1. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA

    Karissa L. Cross, James H. Campbell, Manasi Balachandran, Alisha G. Campbell, Connor J. Cooper, Snehal Joshi, Dawn Klingeman, Zamin Yang, Jerry M. Parks & Mircea Podar

  2. Department of Microbiology, University of Tennessee, Knoxville, TN, USA

    Karissa L. Cross & Mircea Podar

  3. Genome Science and Technology Program, University of Tennessee, Knoxville, TN, USA

    Alisha G. Campbell, Connor J. Cooper, Jerry M. Parks & Mircea Podar

  4. College of Dentistry, The Ohio State University, Columbus, OH, USA

    Ann Griffen & Eugene Leys

  5. Knoxville Periodontics, Knoxville, TN, USA

    Matthew Heaton

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Contributions

M.P. conceived the study, designed and performed experiments and analyzed data. K.L.C. and J.H.C. designed and performed experiments and analyzed data. M.B., A.G.C., S.J., D.K. and Z.Y. designed and performed experiments. C.C. and J.P. performed protein sequence analyses and structure modeling. M.P., M.H., A.G. and E.L. recruited human subjects and collected oral samples. K.L.C. and M.P. wrote the manuscript.

Corresponding author

Correspondence to Mircea Podar.

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Integrated supplementary information

Supplementary Figure 1 Antigenic peptide selection.

(a). Alignment of TM7a PBP2 with the E.coli homologue based on 3D structure (3vma). Alpha helices are in red, beta sheets are shown as green arrows. The location of the predicted antigenic peptides (epitope 1-7) are in gray, and the selected epitope for antibody production is in blue. Three-dimensional structure (3vma) on the right shows the location of epitopes 2, 5 (selected) and 7 in gold color. (b). Predicted secondary structure and topology of TM7a CpsC. The four predicted antigenic peptides (epitope 1-4) are indicated, the selected one is in blue.

Supplementary Figure 2 SSU rRNA V4 taxonomic resolution.

(a) Phylogenetic tree (Jukes Cantor-corrected distances) of human oral TM7 reference sequences (HOTs) and MiSeq OTUs. Most OTUs can be unambiguoulsy assigned to known HOT types. (b) Pairwise sequence identity matrix for human oral TM7 HOTs (SSU rRNA V4 region).

Supplementary Figure 3

Comparison of representative oral TM7 genomes spanning four phylogenetic groups using Anvi’o.

Supplementary Figure 4 Completion level and contamination analysis of oral TM7 SAGs.

Analysis was based on CheckM and using 42 single copy genes conserved in bacteria from the ‘Candidate Phyla Radiation’. Four closed TM7 genomes (TM7x and 3 environmental TM7) were also analyzed for comparison.

Supplementary Figure 5

Abundance of COG categories between environmental and human oral TM7 bacteria.

Supplementary Figure 6 Maximum likelihood phylogeny of concatenated proteins from TM7 bacteria based on SAGs, MAGs and TM7x.

SAGs in red were sequenced as part of this study. Other host-associated TM7 are indicated by gray dots. Black dots at nodes indicate bootstrap support >80, white circles are support values of 50-80%. Scale bar is inferred number of substitutions per site.

Supplementary Figure 7 Structure modelling of TM7 PBP.

(a) Structural templates for the GT Domain of PBP2 from TM7a and TM7x identified by HHsearch. (b) Target sequences for structural modeling. Structural models of PBP2 were generated for the regions in black text. The epitope region in TM7a is boxed. (c) Final models of the glycosyltransferase domain of PBP2 from TM7a (left) and TM7x (right). Residue contacts predicted from coevolution analysis are shown as yellow lines. The models of the GT domain satisfy the coevolution restraints well and show that the overall folds of the two PBP variants are similar.

Supplementary Figure 8

Sanger sequencing chromatogram of rRNA amplicon from a culture containing SR1 HOT875.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Note.

Reporting Summary

Supplementary Dataset 1

TM7 16S sequences.

Supplementary Dataset 2

Actinobacteria 16S sequences.

Supplementary Dataset 3

Sanger raw data sequences.

Supplementary Dataset 4

MiSeq sequence processing commands.

Supplementary Dataset 5

TM7 16S rRNA V4 region sequences.

Supplementary Dataset 6

Structural alignment of PBP2.

Supplementary Dataset 7

3D model structure of TM7a PBP2.

Supplementary Dataset 8

3D model structure of TM7x PBP2.

Supplementary Dataset 9

3D model structure of CpsC.

Supplementary Table 1

Supplementary Table 1.

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Cross, K.L., Campbell, J.H., Balachandran, M. et al. Targeted isolation and cultivation of uncultivated bacteria by reverse genomics. Nat Biotechnol 37, 1314–1321 (2019). https://doi.org/10.1038/s41587-019-0260-6

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