Diversity, function and oxygen relationship of free-living and flagellate-associated Opitutales (phylum: Verrucomicrobiota) in the termite gut.

The phylum Verrucomicrobiota is a relatively new bacterial lineage that belongs to the PVC superphylum. They are ubiquitous in soil and aquatic environments but can also be found in association with animal hosts. Especially the order Opitutales comprises members that are inhabiting the guts of ants, cockroaches and termites. During my doctoral studies, I investigated different aspects of Verrucomicrobiota in the termite gut. While most members of the family Opitutaceae had been described without special attention to their oxygen relationship, recent studies suggested that at least host-associated species are potential microaerobes that are specifically adapted to their suboxic environments. Therefore, I investigated the oxygen relationship of three representative family members at the genomic, transcriptomic and physiological level. Functional genome analysis of the respiratory chains revealed that Opitutaceae have high-affinity oxidases (HATOx) that potentially allow them to respire low concentrations of oxygen. Transcriptional analysis showed that all terminal oxidases of Opitutaceae are constitutively expressed but can adapt to changing oxygen conditions. Using microelectrodes and the deoxygenation of myoglobin as a measure for oxygen concentration, I determined oxygen uptake rates and affinities of Opitutaceae under different oxygen concentrations. These experiments showed that Opitutaceae can take up oxygen at nanomolar concentrations and can respond to changes in the oxygen availability in their temporarily or permanently oxygen-limited environments. A recent study that recovered metagenome-assembled genomes (MAGs) from termite gut metagenomes yielded 90 MAGs of uncultured Verrucomicrobiota, most of them in the order Opitutales. Building on these MAGs, I was able to show that they represent several deep-branching, termite-specific lineages with highly reduced genomes that are endosymbionts of termite gut flagellates. Fluorescence in situ hybridization experiments confirmed their endosymbiotic association with different flagellate species from lower termites. Functional genome analysis revealed a severe genome erosion that affected energy metabolism, central carbon metabolism, cofactor biosynthesis and amino acid biosynthesis. The acquisition of ATP/ADP translocases and sugar phosphate transporters via horizontal gene transfer underscores that the flagellate-associated Opitutales are obligately dependent on the provision of metabolites from the cytoplasm of their respective hosts and suggests a potentially parasitic nature of their relationship.