Targeted isolation and cultivation of uncultivated bacteria by reverse genomics

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.

Figure 1.

Overview of targeted microbial isolation through reverse genomics. a. Diagram showing the steps in reverse genomics-enabled microbiology: (1) Identification of membrane protein-encoding genes in SAGs and MAGs, (2) selection of predicted exposed epitopes, (3) antibody production, (4) purification and fluorescent labeling, (5) staining of target cells from microbiome samples and (6) isolation for (7) genomic sequencing or (8) cultivation. b. X-ray structure of penicillin-binding protein from E. coli, with the modeled TM7 glycosyltransferase (GT) domain colored blue and the selected epitope region highlighted in yellow-brown. c. Structural model of TM7a capsular polysaccharide biosynthesis protein (cpsC), with the peptide epitope region in purple.

Figure 2.

Isolation of TM7 cells by flow cytometry cell sorting. a-b. Flow cytometry plot (forward scatter versus green fluorescence) of native (a) and TM7 antibody-labelled oral sample (b). The sort gate area indicates selection characteristics for cell sorting. Highly fluorescent particles (RFU>103) are auto-fluorescent precipitates/minerals. c. Microbial diversity in pools of 10 sorted cells, following MDA and 16S rRNA gene amplicon sequencing. Also shown for comparison is the diversity of the original subgingival sample (far right column). d. Maximum likelihood tree of human oral TM7 SSU rRNA sequences, indicating phylotypes retrieved by antibody labeling and cell sorting (blue dots) and TM7 groups (G1-G6). Circles at nodes indicate bootstrap values (not shown if <50, black>80). Scale bar = substitutions per site. FSC, forward scatter, relative units; RFU, relative fluorescence units.

Figure 3.

TM7-host colonies following single particle sorting. (a) Cells from a primary TM7 HOT346–348 positive colony were re-labeled with TM7 antibody and (b) Vertical axis shows relative fluorescence (emission filter 528–538 nm) following excitation at 488 nm. flow sorted based on the P1 gate on BHI-blood agar. The circled colonies contained pure TM7 HOT346-Cellulosimicrobium co-cultures. (c) Primary colony of the TM7 HOT351-Actinomyces sp. HOT897 co-culture on a BHI-blood agar.

Figure 4.

Diversity of cultivated and uncultivated TM7 bacteria. (a) Maximum-likelihood phylogenies of TM7 bacteria and of selected human oral Actinobacteria, based on small subunit rRNA genes. TM7 bacteria include human oral phylotypes (HOTs) as well as related host-associated lineages from open environments, identified by sequence accession numbers and associated with six operational groupings (G1–6). HOTs detected using antibodies, isolated as single cells or identified in cultures are indicated by colored stars. Circles at nodes indicate bootstrap support (>80% filled, 50–80% open, only shown for major groupings). Lines connecting TM7 and Actinobacteria species indicate epibiont-host systems identified previously (black) and in this study (green). (b) Epifluorescence microscopy images of novel TM7-host associations identified in this study using immunolabeling (TM7 HOT351-Actinomyces HOT897 and TM7 HOT346-Cellulosimicrobium cellulans) or FISH (TM7352-Actinomyces odontolyticus OR). Arrows point to green fluorescent TM7 cocci. Scale bar is 5 μm.

Figure 5.

Targeted isolation of oral SR1 bacteria. (a) Domains representation of the SR1 HOT 345 LysMpeptidoglycan-binding domain-containing protein. A heterologously expressed extracellular fragment(LysM-CHAP domains) was used as antigen. (b) Flow cytometry plot (forward scatter versus green fluorescence) of anti-SR1 antibody-labelled oral sample. The sort gate area indicates selection characteristics for cell sorting. (c) Phylogenetic diversity of human oral SR1 bacteria (GN02 bacteria is the outgroup) based on SSU rRNA gene. SR1 HOT875 (green star) was identified in enrichments of sorted cells, HOT345 genome (red) was the source for the antigen sequence.

Figure 6.

Reconstruction of central metabolic pathways for TM7 bacteria, based on completed genomes and draft SAG/MAG assemblies. Blue or green circles indicate broad presence in environmental and animal-associated TM7. Semicircles indicate occasional presence, associated with specific environments. Absent reactions are in red. The absence of genes encoding three essential pentose phosphate pathway enzymes in human oral TM7 is highlighted in gray.