Environmental diversity of <i>Candidatus</i> Babelota and their relationships with protists

<i>Ca</i>. Babelota is a phylum of strictly intracellular bacteria whose representatives are commonly detected in various environments through metagenomics, though their presence, ecology, and biology have never been addressed so far. As a group of strict intracellular, we hypothesize that their presence, occurrence, and abundance heavily depend on their hosts, which are known as heterotrophic protists, based on few described isolates. Here, we conducted a sampling campaign allowing to characterize protists and associated bacterial communities, using high-throughput sequencing. In parallel, a systematic enrichment of protists from samples was performed to attempt characterization and isolation of new <i>Ca</i>. Babelota within native hosts. We found that <i>Ca</i>. Babelota are among the most widespread phylum among the rare ones. Protist enrichments are allowed in certain cases to enrich as well for <i>Ca</i>. Babelota, which could be visualized <i>in vivo</i> infecting protist cells. Though cosmopolitan, <i>Ca</i>. Babelota diversity was highly site-specific. Cooccurrence analyses allowed to retrieve well-known as well as new putative associations involving numerous protists of various trophic regimes. The combination of approaches developed in this study enhances our understanding of <i>Ca</i>. Babelota ecology and biology, while paving the way for future isolation of new members of this elusive phylum, which could have huge impact on protists-and ecosystems-functioning.IMPORTANCEOur understanding of microbial diversity surrounding us and colonizing the environment has been dramatically impacted by the advent of DNA-based analyses. Such progress helped shine a new light on numerous lineages of yet-to-be-characterized microbes, whose ecology and biology are basically unknown. Among those uncharacterized clades is the <i>Candidatus</i> Babelota, a bacterial phylum for which parasitism seems to be an ancestral trait. All known <i>Ca.</i> Babelota thrive by infecting phagotrophic protist hosts, thereby impacting this basal link of the trophic chain. The <i>Ca.</i> Babelota constitutes a model that stands out, as phylum-wide conserved parasitism has only been described in one previous occurrence for Bacteria, with the Chlamydiota. Thus, exploring the intricate interplay between <i>Ca.</i> Babelota and their protist hosts will advance our knowledge of bacterial diversity, their ecology, and global impact on ecosystem functioning.