Comparative single-cell genomics of two uncultivated species harboring cobionts.

Amoeboflagellates of the genus are free-living protists ubiquitously found in soil and freshwater habitats worldwide. They include the "brain-eating amoeba" , an opportunistic pathogen that causes primary amoebic meningoencephalitis, a rare but fatal infection of humans. Beyond their direct pathogenicity, protists can also act as environmental reservoirs for intracellular bacterial pathogens, such as spp., to persist and multiply in the environment. In this study, we carried out single-cell genome sequencing of two uncultivated species isolated from the River Leam in England. From single cells, we generated two highly complete genomes. Phylogenetic analyses placed these species as close relatives of and . Exploring evolutionary genomics, we identified gene families encoding antistasin-like domains, which have been characterized as factors that inhibit coagulation in blood-feeding leeches. Antistasin-like domains were identified in all sequenced species and their close relative yet are otherwise largely restricted to animal genomes. Significantly, we recovered highly complete bacterial genomes from each single-cell sample. Phylogenomic analysis revealed that both bacteria belong to the Legionellaceae family. Both bacterial genomes encode comprehensive sets of secretion systems and effector arsenals. We identified putative effectors that resemble TAL (Transcription activator-like) effectors from plant pathogenic spp. in terms of protein sequence and predicted structure, representing a potentially novel class of effectors. Our study highlights the advantages of single-cell environmental genomics approaches, which enable direct association of intracellular pathogens with their hosts to better understand the evolution of host-pathogen interactions.IMPORTANCEBeyond their direct pathogenic potential, amoebae and other protists found in the environment can indirectly threaten human health by serving as reservoirs for intracellular bacterial pathogens to persist, evolve, and multiply in the environment. Despite their importance, protist-bacterial interactions remain poorly understood. In this study, we employed single-cell genomics to sequence the genomes of two uncultivated amoebae, both harboring novel bacteria. From individual cells, we recovered highly complete eukaryotic and bacterial cobiont genome assemblies. Our work demonstrates the power of single-cell sequencing approaches in directly linking intracellular pathogens to their hosts to better understand the evolution of protist-bacterial interactions and the role that protists play in facilitating bacterial pathogens to persist long term in the environment.