Resolving host-episymbiont interaction dynamics through continuous cultivation

Minisyncoccota are an elusive lineage of "microbial dark matter" predicted to compose ~25% of total bacterial diversity. Despite near ubiquity, these organisms are challenging to cultivate, resulting from their specialized episymbiotic lifestyle. All cultivated representatives, predominantly Nanosynbacteria, depend on cognate prokaryotic hosts for growth. Studying the growth dynamics of episymbiotic bacteria and their hosts in batch cultures suggests that episymbionts typically reduce host populations and that hosts eventually adapt to episymbiont stress after serial passaging. However, discontinuous batch cultures do not reflect natural interactions between these organisms due to their drastically different growth rates, which complicates the investigation of host inhibition and adaptation. To describe these dynamics, we utilized continuous culture via small-scale bioreactors. Within a bioreactor, host bacteria can be cultivated at a consistent growth rate, providing the perfect substrate for cultivation of model Nanosynbacteria. Quantification of time until host crash, crash severity, host adaptation, and stable co-culture population provides mechanistic ways to describe episymbiont-host interactions. We used these techniques to compare infection by three episymbionts, revealing distinct infection patterns ranging from mild inhibition with rapid host adaptation to rapid host collapse followed by "arms-race" oscillation dynamics. Then, bioreactors were used to quantify the episymbiotic role played by a known host-binding type 4 pili (T4P-2), demonstrating that loss of pilus-range host binding (approximately 100-800 nm) significantly delayed the host crash without altering general crash dynamics. These experiments reveal that episymbionts can have drastically different effects on bacterial communities and provide the tools necessary to describe strain/species differences and molecular interactions.IMPORTANCEEpisymbiotic Minisyncoccota represent one of the largest branches of life on Earth, as well as one of the least understood. Furthermore, because Minisyncoccota can manipulate their hosts' growth and morphology, they have immense ecological potential to shape the communities they occupy, both environmental and microbiome-associated. Our study highlights, for the first time, the potential of small-scale continuous cultivation for studying episymbiotic interactions that cannot be captured in discontinuous cultures. Herein, we used these techniques to interrogate interspecies variation in host inhibition potential and to determine how loss of a pilus-dependent binding factor mechanistically alters the cycle of episymbiont infection; however, this cultivation platform will enable researchers to answer many new questions about these ubiquitous host-episymbiont interactions.