Insights Obtained by Culturing Saccharibacteria With Their Bacterial Hosts

Abstract

Oral microbiome research has moved from asking "Who's there?" to "What are they doing?" Understanding what microbes "do" involves multiple approaches, including obtaining genomic information and examining the interspecies interactions. Recently we isolated a human oral Saccharibacteria (TM7) bacterium, HMT-952, strain TM7x, which is an ultrasmall parasite of the oral bacterium Actinomyces odontolyticus. The host-parasite interactions, such as phage-bacterium or Saccharibacteria-host bacterium, are understudied areas with large potential for insight. The Saccharibacteria phylum is a member of Candidate Phyla Radiation, a large lineage previously devoid of cultivated members. However, expanding our understanding of Saccharibacteria-host interactions requires examining multiple phylogenetically distinct Saccharibacteria-host pairs. Here we report the isolation of 3 additional Saccharibacteria species from the human oral cavity in binary coculture with their bacterial hosts. They were obtained by filtering ultrasmall Saccharibacteria cells free of other larger bacteria and inoculating them into cultures of potential host bacteria. The binary cocultures obtained could be stably passaged and studied. Complete closed genomes were obtained and allowed full genome analyses. All have small genomes (<1 Mb) characteristic of parasitic species and dramatically limited de novo synthetic pathway capabilities but include either restriction modification or CRISPR-Cas systems as part of an innate defense against foreign DNA. High levels of gene synteny exist among Saccharibacteria species. Having isolates growing in coculture with their hosts allowed time course studies of growth and parasite-host interactions by phase contrast, fluorescence in situ hybridization, and scanning electron microscopy. The cells of the 4 oral Saccharibacteria species are ultrasmall and could be seen attached to their larger Actinobacteria hosts. Parasite attachment appears to lead to host cell death and lysis. The successful cultivation of Saccharibacteria species has significantly expanded our understanding of these ultrasmall Candidate Phyla Radiation bacteria.

Keywords: culture technique; genomics; human; microbiota; mouth; parasite.

Figure 1.

Neighbor-joining 16S rRNA phylogenetic gene tree for candidate division Saccharibacteria. Blue (group G-1A) and red (group G-1B) are separately rooted monophyletic clades that both fall into group 1 (Camanocha and Dewhirst 2014). Blue and red bold strains are currently isolated strains and used in this study. The scale is 2.5 substitutions per site.

Figure 2.

Saccharibacteria genome comparisons. (A) Closed contigs for 4 oral (top) and 3 environmental (bottom) genomes were aligned after reassigning the start position for all genomes using dnaA with progressive Mauve (Darling et al. 2004). Syntenic blocks are colored to locate regions in each genome that are similar. The bar chart within the blocks shows the percentage of Blastn identity. (B) Venn diagram shows unique and shared genes of 4 oral Saccharibacteria strains. See Appendix Tables 4 and 6 for specific gene list.

Figure 3.

Restriction-modification (RM) and CRISPR-Cas system of cultivated Saccharibacteria strains. (A) Schematic diagram of TM7x (Type I/Type I), BB001 (Type III), and AC001 (Type II/Type III) RM systems. Hypothetical (H) and non-RM system proteins are shown in gray. The MTase (blue) and specificity subunit S (green) form an active multisubunit protein that methylates bipartite DNA recognition sequence. In the presence of REase subunit (orange), the complex can also act as an endonuclease. (B) Predicted recognition sequences of each RM system types shown in panel A. In each motif, base modifications that were detected during PacBio SMRT sequencing are shown in bold. (C) Gene cluster organization of PM004 CRISPR-Cas loci includes genes for the Cas9, Cas1, and Cas2 proteins with the CRISPR array.

Figure 4.

Phenotypic characterization of isolated Saccharibacteria strains. (A) Phase contrast, (B) fluorescence in situ hybridization (FISH), and (C) scanning electron microscope images were taken for each Saccharibacteria strain–host binary coculture and compared with that of the monocultures (see Appendix Fig. 3). In the FISH images, red and green represent Saccharibacteria and host bacteria, respectively. Scale bars are 10 µm for phase contrast and FISH images and 1 µm for the scanning electron microscope images. Red arrows point out the “bowling pin”–like morphology of TM7x. (D, E) Growth curve of 2 Saccharibacteria–host binary cocultures (TM7x, BB001) and host monocultures were acquired with image-based cell density measurement by oCelloScope. Generation time (gt) for each mono- and coculture was determined (see Methods and Appendix Table 9) and presented next to the line graph in matching color. Values are means of 3 independent generation times with SD as an error bar.

Figure 5.

Host range of cultivated isolates. Phylogenetic tree was created with the 16S rRNA sequences of different Actinomyces sp. (red and green), Pseudopropionibacterium sp. (blue), and common oral bacteria (black). Attempts were made to infect each bacterium with each of the 4 isolated Saccharibacteria species. Those that supported and did not support the growth of each Saccharibacteria are denoted by plus (+) and minus (–) signs, respectively. The scale equals 0.04 substitutions per site. TM7x is a strain of Saccharibacteria HMT-952; BB001 is a strain of Saccharibacteria HMT-957; AC001 is a strain of Saccharibacteria HMT-488; and PM004 is a strain of Saccharibacteria HMT-955.