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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DeLong, E. F. & Pace, N. R. Environmental diversity of bacteria and archaea. Syst. Biol. 50, 470–478 (2001).
Hug, L. A. et al. A new view of the tree of life. Nat. Microbiol. 1, 16048 (2016).
Zaremba-Niedzwiedzka, K. et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541, 353–358 (2017).
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).
Rinke, C. et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437 (2013).
Gutleben, J. et al. The multi-omics promise in context: from sequence to microbial isolate. Crit. Rev. Microbiol. 44, 212–229 (2017).
Epstein, S. S. The phenomenon of microbial uncultivability. Curr. Opin. Microbiol. 16, 636–642 (2013).
Overmann, J., Abt, B. & Sikorski, J. Present and future of culturing bacteria. Annu. Rev. Microbiol. 71, 711–730 (2017).
D’Onofrio, A. et al. Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chem. Biol. 17, 254–264 (2010).
Strandwitz, P. et al. GABA-modulating bacteria of the human gut microbiota. Nat. Microbiol. 4, 396–403 (2018).
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).
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).
Huber, H. et al. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417, 63–67 (2002).
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).
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).
Pulschen, A. A. et al. Isolation of uncultured bacteria from antarctica using long incubation periods and low nutritional media. Front. Microbiol. 8, 1346 (2017).
Oliver, J. D. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbiol. Rev. 34, 415–425 (2010).
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).
Zengler, K. et al. High-throughput cultivation of microorganisms using microcapsules. Methods Enzymol. 397, 124–130 (2005).
Browne, H. P. et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533, 543–546 (2016).
Lagier, J. C. et al. Culture of previously uncultured members of the human gut microbiota by culturomics. Nat. Microbiol. 1, 16203 (2016).
Oberhardt, M. A. et al. Harnessing the landscape of microbial culture media to predict new organism-media pairings. Nat. Commun. 6, 8493 (2015).
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).
Sizova, M. V. et al. New approaches for isolation of previously uncultivated oral bacteria. Appl. Environ. Microbiol. 78, 194–203 (2012).
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).
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).
Albertsen, M. et al. Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes. Nat. Biotechnol. 31, 533–538 (2013).
Dewhirst, F. E. et al. The human oral microbiome. J. Bacteriol. 192, 5002–5017 (2010).
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).
Kuehbacher, T. et al. Intestinal TM7 bacterial phylogenies in active inflammatory bowel disease. J. Med. Microbiol. 57, 1569–1576 (2008).
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).
Dewhirst, F. E. et al. The canine oral microbiome. PLoS ONE 7, e36067 (2012).
Dudek, N. K. et al. Novel microbial diversity and functional potential in the marine mammal oral microbiome. Curr. Biol. 27, 3752–3762.e6 (2017).
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).
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).
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).
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).
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).
Palmer, R. J., Jr. et al. Interbacterial adhesion networks within early oral biofilms of single human hosts. Appl. Environ. Microbiol. 83 (2017).
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).
Jakubovics, N. S., Yassin, S. A. & Rickard, A. H. Community interactions of oral streptococci. Adv. Appl. Microbiol. 87, 43–110 (2014).
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).
Bor, B. et al. Phenotypic and physiological characterization of the epibiotic interaction between TM7x and its basibiont actinomyces. Microb. Ecol. 71, 243–255 (2016).
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).
Podar, M. et al. Targeted access to the genomes of low-abundance organisms in complex microbial communities. Appl. Environ. Microbiol. 73, 3205–3214 (2007).
Campbell, A. G. et al. Diversity and genomic insights into the uncultured Chloroflexi from the human microbiota. Environ. Microbiol. 16, 2635–2643 (2014).
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).
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).
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).
Zengler, K. et al. Cultivating the uncultured. Proc. Natl Acad. Sci. USA 99, 15681–15686 (2002).
Markowitz, V. M. et al. The integrated microbial genomes (IMG) system. Nucleic Acids Res. 34, D344–D348 (2006).
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).
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).
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).
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).
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).
Toniolo, C. et al. Streptococcus agalactiae capsule polymer length and attachment is determined by the proteins CpsABCD. J. Biol. Chem. 290, 9521–9532 (2015).
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).
Saha, S. & Raghava, G. P. Prediction of continuous B-cell epitopes in an antigen using recurrent neural network. Proteins 65, 40–48 (2006).
Larsen, J. E., Lund, O. & Nielsen, M. Improved method for predicting linear B-cell epitopes. Immunome Res. 2, 2 (2006).
Heimerl, T. et al. A complex endomembrane system in the archaeon Ignicoccus hospitalis tapped by Nanoarchaeum equitans. Front. Microbiol. 8, 1072 (2017).
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).
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).
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).
Rinke, C. et al. Obtaining genomes from uncultivated environmental microorganisms using FACS-based single-cell genomics. Nat. Protoc. 9, 1038–1048 (2014).
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).
Pernthaler, A. & Pernthaler, J. Fluorescence in situ hybridization for the identification of environmental microbes. Methods Mol. Biol. 353, 153–164 (2007).
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).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).
Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).
Caporaso, J., Kuczynski, J., Stombaugh, J. & Bittinger, K. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).
Bankevich, A. et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 19, 455–477 (2012).
Brown, C. T. et al. Unusual biology across a group comprising more than 15% of domain Bacteria. Nature 523, 208–211 (2015).
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).
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).
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).
Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014).
Anantharaman, K. et al. Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system. Nat. Commun. 7, 13219 (2016).
Finn, R. D. et al. Pfam: the protein families database. Nucleic Acids Res. 42, D222–D230 (2014).
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).
Jones, P. et al. InterProScan 5: genome-scale protein function classification. Bioinformatics 30, 1236–1240 (2014).
Delmont, T. O. & Eren, A. M. Linking pangenomes and metagenomes: the Prochlorococcus metapangenome. PeerJ 6, e4320 (2018).
Eren, A. M. et al. Anvi’o: an advanced analysis and visualization platform for ‘omics data. PeerJ 3, e1319 (2015).
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).
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).
Ovchinnikov, S. et al. Protein structure determination using metagenome sequence data. Science 355, 294–298 (2017).
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).
Johnson, L. S., Eddy, S. R. & Portugaly, E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinformatics 11, 431 (2010).
Burley, S. K. et al. Protein Data Bank (PDB): the single global macromolecular structure archive. Methods Mol. Biol. 1607, 627–641 (2017).
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).
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).
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).
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).
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).
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.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
(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.
(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).
Comparison of representative oral TM7 genomes spanning four phylogenetic groups using Anvi’o.
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.
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.
(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.
Sanger sequencing chromatogram of rRNA amplicon from a culture containing SR1 HOT875.
Supplementary Figs. 1–8 and Note.
TM7 16S sequences.
Actinobacteria 16S sequences.
Sanger raw data sequences.
MiSeq sequence processing commands.
TM7 16S rRNA V4 region sequences.
Structural alignment of PBP2.
3D model structure of TM7a PBP2.
3D model structure of TM7x PBP2.
3D model structure of CpsC.
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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