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.

Isolation and cultivation of candidate phyla radiation Saccharibacteria (TM7) bacteria in coculture with bacterial hosts

“…However, microbiome analysis by 16S rRNA sequencing of cultures is considered the gold standard of culture analysis as it can detect low-level contamination of the binary coculture with a third or additional bacterial species. Species followed by asterisk were first used in experiments we reported previously [25].…”

“…GenBank accession numbers are in curly brackets. Source of sequences marked is indicated with following symbols: this work; Previous Dewhirst lab publications [25,26,30] or GenBank deposits; Cross et al [47]; Kantor et al [18]; Marcy et al [14]; Albertson et al [19]; Kong et al [73]; Brown et al [20]; Tringe et al (GenBank). Nodes to be compared between Figures 2-4 are marked with green circles and lettered A-F.…”

Complete Genome Sequence of Strain BB001, a Novel Epibiont Bacterium from the Candidate Phylum Saccharibacteria (TM7)

“…The first member of the Saccharibacteria , Nanosynbacter lyticus strain TM7x (GenBank accession number GCA_000803625.1 ), was cultivated from the human oral cavity ( 4 – 7 ). Consequently, additional Saccharibacteria strains were cultivated on their bacterial hosts ( 8 , 9 ). Strain BB001 was isolated on its bacterial host Actinomyces sp.…”

“…Strain BB001 was isolated on its bacterial host Actinomyces sp. strain F0337 from the healthy human oral cavity, as previously described ( 8 ). The saliva was filtered through a 0.45-μm filter to separate ultrasmall bacteria.…”

“…Ultrasmall bacteria that did not grow were cleaned by sequential passaging and agar plating. A detailed procedure can be found in reference 8 . BB001 is the first isolate of human microbiome taxon 957 (HMT-957).…”