High-throughput sequencing uncovered a plethora of new microorganisms, that are often referred to as microbial dark matter due to their difficult culture conditions and intractable genetics. One such example of microbial dark matter is DPANN archaea; DPANN is a widespread and highly diverse group of archaea characterised by their small size, reduced genome, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for their survival and proliferation. Despite their importance, the structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown.
Cryotomography (cryoET) combined with sub tomogram averaging STA, is capable of determining macromolecular structures in situ without recombinant expression systems, making it an ideal method to study problematic microbial communities. We used electron cryo-ET to observe the interaction of DPANN Candidatus Micrarchaeota with its host, Metallosphaera javensis, in near-native conditions at nanometer resolutions. We showed that the interaction is co-ordinated in part by intercellular proteinaceous nanotubes. Tomogram reconstructions combined with 3D segmentation showed that these tubes originate in the host, extend all the way to the DPANN cytoplasm and act like tunnels for intercellular exchange. Combining cryo-ET and sub-tomogram averaging, we revealed the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states, providing mechanistic insights into substrate exchange.
Our work demonstrates the application of cryo-ET to observe intercellular interactions in microbial dark matter. Our results provided unprecedented insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships.