Genomics of Uncultivated Gracilibacteria Shows Diversity and Broad Human Presence

Objectives: The phylum Gracilibacteria/GN02 is part of the large, but poorly understood, bacterial Candidate Phylum Radiation (CPR). Possible obligate epibionts of bacteria, they are one of three CPR phylum found in the human oral cavity. We explored the diversity of this phylum by studying the phylogeny and human presence of several novel Gracilibacteria genomes from human oral samples.

Methods: Genomes were assembled from metagenomic samples using MegaBLAST to find Gracilibacteria hits to 16S rRNA genes in contigs. Raw reads were then re-assembled with SPAdes, binned into genomes, manually curated, and a phylogeny constructed based on concatenated single copy ribosomal genes.

Results: We have confirmed that these are also reduced genomes like the Saccharibacteria (TM7). The phylogeny showed the new genomes split between class level groups G1 and G2, known from previous 16S studies to be mixed oral and environmental groups. GC content supported the split with G1 genomes at 21-25% and G2 at 36-38%. Comparing orthologous genes showed high gene similarity of the oral genomes within each group and significantly lower overlap between G1 and G2 oral. The phylogenic tree indicated distinctions between the oral and environmental genomes but overall confirmed the similarities of oral and environmental genomes within G1 and also G2. Mapping sequences from human body sites to the genomes showed broad presence across the human oral cavity, as well as presence in other body sites. The oral G1 had their highest coverage in the tongue dorsum, anterior nares, and mid vagina, the oral G2 in the posterior fornix, and tongue dorsum.

Conclusions: Our study provides new insight into this mostly unknown phylum showing that Gracilibacteria from the oral cavity are distinct from environmental species even within the same group and are present across multiple human body sites, including non-oral sites.