skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Targeted isolation and cultivation of uncultivated bacteria by reverse genomics

Abstract

The majority of 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 a technique 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.

Authors:
 [1];  [2];  [3];  [2];  [1];  [4];  [5]; ORCiD logo [3]; ORCiD logo [3];  [4];  [3]; ORCiD logo [1]; ORCiD logo [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Northwest Missouri State Univ., Maryville, MO (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  4. The Ohio State Univ., Columbus, OH (United States)
  5. Knoxville Periodontics, Knoxville, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE; National Institutes of Health (NIH); National Science Foundation (NSF)
OSTI Identifier:
1570133
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Nature Biotechnology
Additional Journal Information:
Journal Volume: 37; Journal Issue: 11; Journal ID: ISSN 1087-0156
Publisher:
Springer Nature
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Cross, Karissa L., Campbell, James H., Balachandran, Manasi, Campbell, Alisha G., Cooper, Sarah J., Griffen, Ann, Heaton, Matthew, Joshi, Snehal, Klingeman, Dawn, Leys, Eugene, Yang, Zamin, Parks, Jerry M., and Podar, Mircea. Targeted isolation and cultivation of uncultivated bacteria by reverse genomics. United States: N. p., 2019. Web. doi:10.1038/s41587-019-0260-6.
Cross, Karissa L., Campbell, James H., Balachandran, Manasi, Campbell, Alisha G., Cooper, Sarah J., Griffen, Ann, Heaton, Matthew, Joshi, Snehal, Klingeman, Dawn, Leys, Eugene, Yang, Zamin, Parks, Jerry M., & Podar, Mircea. Targeted isolation and cultivation of uncultivated bacteria by reverse genomics. United States. doi:10.1038/s41587-019-0260-6.
Cross, Karissa L., Campbell, James H., Balachandran, Manasi, Campbell, Alisha G., Cooper, Sarah J., Griffen, Ann, Heaton, Matthew, Joshi, Snehal, Klingeman, Dawn, Leys, Eugene, Yang, Zamin, Parks, Jerry M., and Podar, Mircea. Mon . "Targeted isolation and cultivation of uncultivated bacteria by reverse genomics". United States. doi:10.1038/s41587-019-0260-6. https://www.osti.gov/servlets/purl/1570133.
@article{osti_1570133,
title = {Targeted isolation and cultivation of uncultivated bacteria by reverse genomics},
author = {Cross, Karissa L. and Campbell, James H. and Balachandran, Manasi and Campbell, Alisha G. and Cooper, Sarah J. and Griffen, Ann and Heaton, Matthew and Joshi, Snehal and Klingeman, Dawn and Leys, Eugene and Yang, Zamin and Parks, Jerry M. and Podar, Mircea},
abstractNote = {The majority of 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 a technique 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.},
doi = {10.1038/s41587-019-0260-6},
journal = {Nature Biotechnology},
number = 11,
volume = 37,
place = {United States},
year = {2019},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 19 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

The phenomenon of microbial uncultivability
journal, October 2013


The Canine Oral Microbiome
journal, April 2012


Anvi’o: an advanced analysis and visualization platform for ‘omics data
journal, January 2015

  • Eren, A. Murat; Esen, Özcan C.; Quince, Christopher
  • PeerJ, Vol. 3
  • DOI: 10.7717/peerj.1319

Asgard archaea illuminate the origin of eukaryotic cellular complexity
journal, January 2017

  • Zaremba-Niedzwiedzka, Katarzyna; Caceres, Eva F.; Saw, Jimmy H.
  • Nature, Vol. 541, Issue 7637
  • DOI: 10.1038/nature21031

Microcolony Cultivation on a Soil Substrate Membrane System Selects for Previously Uncultured Soil Bacteria
journal, December 2005


Cutadapt removes adapter sequences from high-throughput sequencing reads
journal, May 2011


Prediction of continuous B-cell epitopes in an antigen using recurrent neural network
journal, August 2006

  • Saha, Sudipto; Raghava, G. P. S.
  • Proteins: Structure, Function, and Bioinformatics, Vol. 65, Issue 1
  • DOI: 10.1002/prot.21078

eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences
journal, November 2015

  • Huerta-Cepas, Jaime; Szklarczyk, Damian; Forslund, Kristoffer
  • Nucleic Acids Research, Vol. 44, Issue D1
  • DOI: 10.1093/nar/gkv1248

Crystal structure of a peptidoglycan glycosyltransferase suggests a model for processive glycan chain synthesis
journal, March 2007

  • Yuan, Y.; Barrett, D.; Zhang, Y.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 13
  • DOI: 10.1073/pnas.0701160104

Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation
journal, May 2016

  • Browne, Hilary P.; Forster, Samuel C.; Anonye, Blessing O.
  • Nature, Vol. 533, Issue 7604
  • DOI: 10.1038/nature17645

Targeted Access to the Genomes of Low-Abundance Organisms in Complex Microbial Communities
journal, March 2007

  • Podar, M.; Abulencia, C. B.; Walcher, M.
  • Applied and Environmental Microbiology, Vol. 73, Issue 10
  • DOI: 10.1128/AEM.02985-06

Genomics-informed isolation and characterization of a symbiotic Nanoarchaeota system from a terrestrial geothermal environment
journal, July 2016

  • Wurch, Louie; Giannone, Richard J.; Belisle, Bernard S.
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms12115

Axenic Culture of a Candidate Division TM7 Bacterium from the Human Oral Cavity and Biofilm Interactions with Other Oral Bacteria
journal, August 2014

  • Soro, Valeria; Dutton, Lindsay C.; Sprague, Susan V.
  • Applied and Environmental Microbiology, Vol. 80, Issue 20
  • DOI: 10.1128/AEM.01827-14

Crystal structure of Staphylococcus aureus transglycosylase in complex with a lipid II analog and elucidation of peptidoglycan synthesis mechanism
journal, April 2012

  • Huang, C. -Y.; Shih, H. -W.; Lin, L. -Y.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 17
  • DOI: 10.1073/pnas.1203900109

The multi-omics promise in context: from sequence to microbial isolate
journal, May 2017

  • Gutleben, Johanna; Chaib De Mares, Maryam; van Elsas, Jan Dirk
  • Critical Reviews in Microbiology, Vol. 44, Issue 2
  • DOI: 10.1080/1040841X.2017.1332003

Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins
journal, February 2009


Practical innovations for high-throughput amplicon sequencing
journal, September 2013

  • Lundberg, Derek S.; Yourstone, Scott; Mieczkowski, Piotr
  • Nature Methods, Vol. 10, Issue 10
  • DOI: 10.1038/nmeth.2634

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis
journal, March 2008


Present and Future of Culturing Bacteria
journal, September 2017


Assessing the utility of coevolution-based residue-residue contact predictions in a sequence- and structure-rich era
journal, September 2013

  • Kamisetty, H.; Ovchinnikov, S.; Baker, D.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 39
  • DOI: 10.1073/pnas.1314045110

Hidden Markov model speed heuristic and iterative HMM search procedure
journal, August 2010


Improved recognition of native-like protein structures using a combination of sequence-dependent and sequence-independent features of proteins
journal, January 1999


A new symbiotic nanoarchaeote (Candidatus Nanoclepta minutus) and its host (Zestosphaera tikiterensis gen. nov., sp. nov.) from a New Zealand hot spring
journal, January 2019


Recent findings on the viable but nonculturable state in pathogenic bacteria
journal, July 2010


Protein structure determination using metagenome sequence data
journal, January 2017


Environmental Diversity of Bacteria and Archaea
journal, August 2001


The integrated microbial genomes (IMG) system
journal, January 2006


Immunogenicity of meningococcal PBP2 during natural infection and protective activity of anti-PBP2 antibodies against meningococcal bacteraemia in mice
journal, March 2006

  • Zarantonelli, Maria Leticia; Antignac, Aude; Lancellotti, Marcelo
  • Journal of Antimicrobial Chemotherapy, Vol. 57, Issue 5
  • DOI: 10.1093/jac/dkl066

CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes
journal, May 2015

  • Parks, Donovan H.; Imelfort, Michael; Skennerton, Connor T.
  • Genome Research, Vol. 25, Issue 7
  • DOI: 10.1101/gr.186072.114

Biosynthetic capacity, metabolic variety and unusual biology in the CPR and DPANN radiations
journal, September 2018

  • Castelle, Cindy J.; Brown, Christopher T.; Anantharaman, Karthik
  • Nature Reviews Microbiology, Vol. 16, Issue 10
  • DOI: 10.1038/s41579-018-0076-2

Crystal structure of the membrane-bound bifunctional transglycosylase PBP1b from Escherichia coli
journal, May 2009

  • Sung, M. -T.; Lai, Y. -T.; Huang, C. -Y.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 22
  • DOI: 10.1073/pnas.0904030106

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity
journal, February 2017

  • Terekhov, Stanislav S.; Smirnov, Ivan V.; Stepanova, Anastasiya V.
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 10
  • DOI: 10.1073/pnas.1621226114

A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont
journal, May 2002

  • Huber, Harald; Hohn, Michael J.; Rachel, Reinhard
  • Nature, Vol. 417, Issue 6884
  • DOI: 10.1038/417063a

The Human Oral Microbiome
journal, July 2010

  • Dewhirst, F. E.; Chen, T.; Izard, J.
  • Journal of Bacteriology, Vol. 192, Issue 19
  • DOI: 10.1128/JB.00542-10

Generalized Fragment Picking in Rosetta: Design, Protocols and Applications
journal, August 2011


Interbacterial Adhesion Networks within Early Oral Biofilms of Single Human Hosts
journal, March 2017

  • Palmer, Robert J.; Shah, Nehal; Valm, Alex
  • Applied and Environmental Microbiology, Vol. 83, Issue 11
  • DOI: 10.1128/AEM.00407-17

GABA-modulating bacteria of the human gut microbiota
journal, December 2018


UGA is an additional glycine codon in uncultured SR1 bacteria from the human microbiota
journal, March 2013

  • Campbell, J. H.; O'Donoghue, P.; Campbell, A. G.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 14, p. 5540-5545
  • DOI: 10.1073/pnas.1303090110

Search and clustering orders of magnitude faster than BLAST
journal, August 2010


In Vitro Cultivation of ‘Unculturable’ Oral Bacteria, Facilitated by Community Culture and Media Supplementation with Siderophores
journal, January 2016


In situ cultivation of previously uncultivable microorganisms using the ichip
journal, September 2017

  • Berdy, Brittany; Spoering, Amy L.; Ling, Losee L.
  • Nature Protocols, Vol. 12, Issue 10
  • DOI: 10.1038/nprot.2017.074

InterProScan 5: genome-scale protein function classification
journal, January 2014


Prokka: rapid prokaryotic genome annotation
journal, March 2014


Intestinal TM7 bacterial phylogenies in active inflammatory bowel disease
journal, December 2008

  • Kuehbacher, T.; Rehman, A.; Lepage, P.
  • Journal of Medical Microbiology, Vol. 57, Issue 12
  • DOI: 10.1099/jmm.0.47719-0

Multiple Single-Cell Genomes Provide Insight into Functions of Uncultured Deltaproteobacteria in the Human Oral Cavity
journal, March 2013


Establishment and metabolic analysis of a model microbial community for understanding trophic and electron accepting interactions of subsurface anaerobic environments
journal, January 2010

  • Miller, Lance D.; Mosher, Jennifer J.; Venkateswaran, Amudhan
  • BMC Microbiology, Vol. 10, Issue 1
  • DOI: 10.1186/1471-2180-10-149

Siderophores from Neighboring Organisms Promote the Growth of Uncultured Bacteria
journal, March 2010


Biogeography of a human oral microbiome at the micron scale
journal, January 2016

  • Mark Welch, Jessica L.; Rossetti, Blair J.; Rieken, Christopher W.
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 6
  • DOI: 10.1073/pnas.1522149113

New Approaches for Isolation of Previously Uncultivated Oral Bacteria
journal, November 2011

  • Sizova, M. V.; Hohmann, T.; Hazen, A.
  • Applied and Environmental Microbiology, Vol. 78, Issue 1
  • DOI: 10.1128/AEM.06813-11

The feline oral microbiome: A provisional 16S rRNA gene based taxonomy with full-length reference sequences
journal, February 2015


SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing
journal, May 2012

  • Bankevich, Anton; Nurk, Sergey; Antipov, Dmitry
  • Journal of Computational Biology, Vol. 19, Issue 5
  • DOI: 10.1089/cmb.2012.0021

Distinctive Attributes of β-Lactam Target Proteins in Acinetobacter baumannii Relevant to Development of New Antibiotics
journal, December 2011

  • Han, Seungil; Caspers, Nicole; Zaniewski, Richard P.
  • Journal of the American Chemical Society, Vol. 133, Issue 50
  • DOI: 10.1021/ja208835z

Isolation of Uncultured Bacteria from Antarctica Using Long Incubation Periods and Low Nutritional Media
journal, July 2017

  • Pulschen, Andre A.; Bendia, Amanda G.; Fricker, Ashwana D.
  • Frontiers in Microbiology, Vol. 8
  • DOI: 10.3389/fmicb.2017.01346

Linking pangenomes and metagenomes: the Prochlorococcus metapangenome
journal, January 2018


Unusual biology across a group comprising more than 15% of domain Bacteria
journal, June 2015

  • Brown, Christopher T.; Hug, Laura A.; Thomas, Brian C.
  • Nature, Vol. 523, Issue 7559
  • DOI: 10.1038/nature14486

Genome sequences of rare, uncultured bacteria obtained by differential coverage binning of multiple metagenomes
journal, May 2013

  • Albertsen, Mads; Hugenholtz, Philip; Skarshewski, Adam
  • Nature Biotechnology, Vol. 31, Issue 6
  • DOI: 10.1038/nbt.2579

Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle
journal, December 2014

  • He, Xuesong; McLean, Jeffrey S.; Edlund, Anna
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 1
  • DOI: 10.1073/pnas.1419038112

Learning generative models for protein fold families
journal, January 2011

  • Balakrishnan, Sivaraman; Kamisetty, Hetunandan; Carbonell, Jaime G.
  • Proteins: Structure, Function, and Bioinformatics, Vol. 79, Issue 4
  • DOI: 10.1002/prot.22934

Prevalence of Bacteria of Division TM7 in Human Subgingival Plaque and Their Association with Disease
journal, March 2003


Nonlinear partial differential equations and applications: Cultivating the uncultured
journal, November 2002

  • Zengler, K.; Toledo, G.; Rappe, M.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 24
  • DOI: 10.1073/pnas.252630999

A new view of the tree of life
journal, April 2016


A Complex Endomembrane System in the Archaeon Ignicoccus hospitalis Tapped by Nanoarchaeum equitans
journal, June 2017


Predicting transmembrane protein topology with a hidden markov model: application to complete genomes11Edited by F. Cohen
journal, January 2001

  • Krogh, Anders; Larsson, Björn; von Heijne, Gunnar
  • Journal of Molecular Biology, Vol. 305, Issue 3
  • DOI: 10.1006/jmbi.2000.4315

Culture of previously uncultured members of the human gut microbiota by culturomics
journal, November 2016

  • Lagier, Jean-Christophe; Khelaifia, Saber; Alou, Maryam Tidjani
  • Nature Microbiology, Vol. 1, Issue 12
  • DOI: 10.1038/nmicrobiol.2016.203

Harnessing the landscape of microbial culture media to predict new organism–media pairings
journal, October 2015

  • Oberhardt, Matthew A.; Zarecki, Raphy; Gronow, Sabine
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms9493

QIIME allows analysis of high-throughput community sequencing data
journal, April 2010

  • Caporaso, J. Gregory; Kuczynski, Justin; Stombaugh, Jesse
  • Nature Methods, Vol. 7, Issue 5
  • DOI: 10.1038/nmeth.f.303

Novel Microbial Diversity and Functional Potential in the Marine Mammal Oral Microbiome
journal, December 2017


A novel recombinant vaccine candidate comprising PBP2a and autolysin against Methicillin Resistant Staphylococcus aureus confers protection in the experimental mice
journal, November 2017


HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment
journal, December 2011

  • Remmert, Michael; Biegert, Andreas; Hauser, Andreas
  • Nature Methods, Vol. 9, Issue 2
  • DOI: 10.1038/nmeth.1818

Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life
journal, September 2017

  • Parks, Donovan H.; Rinke, Christian; Chuvochina, Maria
  • Nature Microbiology, Vol. 2, Issue 11
  • DOI: 10.1038/s41564-017-0012-7

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics
journal, April 2014


Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system
journal, October 2016

  • Anantharaman, Karthik; Brown, Christopher T.; Hug, Laura A.
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms13219

Insights into the phylogeny and coding potential of microbial dark matter
journal, July 2013

  • Rinke, Christian; Schwientek, Patrick; Sczyrba, Alexander
  • Nature, Vol. 499, Issue 7459
  • DOI: 10.1038/nature12352

Pfam: the protein families database
journal, November 2013

  • Finn, Robert D.; Bateman, Alex; Clements, Jody
  • Nucleic Acids Research, Vol. 42, Issue D1
  • DOI: 10.1093/nar/gkt1223

Phenotypic and Physiological Characterization of the Epibiotic Interaction Between TM7x and Its Basibiont Actinomyces
journal, November 2015


Diversity and genomic insights into the uncultured C hloroflexi from the human microbiota : Uncultured human-associated
journal, April 2014

  • Campbell, Alisha G.; Schwientek, Patrick; Vishnivetskaya, Tatiana
  • Environmental Microbiology, Vol. 16, Issue 9
  • DOI: 10.1111/1462-2920.12461

Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth
journal, July 2007

  • Marcy, Y.; Ouverney, C.; Bik, E. M.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 29, p. 11889-11894
  • DOI: 10.1073/pnas.0704662104

Streptococcus agalactiae Capsule Polymer Length and Attachment Is Determined by the Proteins CpsABCD
journal, February 2015

  • Toniolo, Chiara; Balducci, Evita; Romano, Maria Rosaria
  • Journal of Biological Chemistry, Vol. 290, Issue 15
  • DOI: 10.1074/jbc.M114.631499

Structural Insight into the Transglycosylation Step of Bacterial Cell-Wall Biosynthesis
journal, March 2007


Environmental Diversity of Bacteria and Archaea
journal, August 2001


    Works referencing / citing this record:

    Next-generation physiology approaches to study microbiome function at single cell level
    journal, February 2020

    • Hatzenpichler, Roland; Krukenberg, Viola; Spietz, Rachel L.
    • Nature Reviews Microbiology, Vol. 18, Issue 4
    • DOI: 10.1038/s41579-020-0323-1

    Culturing the uncultured
    journal, October 2019


    Genome‐inferred spatio‐temporal resolution of an uncultivated Roizmanbacterium reveals its ecological preferences in groundwater
    journal, December 2019

    • Geesink, Patricia; Wegner, Carl‐Eric; Probst, Alexander J.
    • Environmental Microbiology, Vol. 22, Issue 2
    • DOI: 10.1111/1462-2920.14865

    Methane oxidation and methylotroph population dynamics in groundwater mesocosms
    journal, April 2020

    • Kuloyo, Olukayode; Ruff, S. Emil; Cahill, Aaron
    • Environmental Microbiology, Vol. 22, Issue 4
    • DOI: 10.1111/1462-2920.14929

    Insights Obtained by Culturing Saccharibacteria With Their Bacterial Hosts
    journal, February 2020