Targeted isolation and cultivation of uncultivated bacteria by reverse genomics


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.

Data availability

Annotated TM7 SAGs are deposited in GenBank under the BioProject PRJNA472398. Raw and aligned SSU rRNA sequences are provided as Supplementary Datasets 13. MiSeq amplicon data are deposited in SRA linked to BioProject PRJNA472398.


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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). S.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.

Author information




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. S.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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The authors declare no competing interests.

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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).

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