New insights into symbiont diversity in deep sea mussels

Exceptional diversity of chemosynthetic endosymbionts. Many deep-sea investigations have revealed the presence of dense colonies of large mussels belonging to the genus Bathymodiolus attached to rocks in the vicinity of hydrothermal vents and cold seeps. The gills of these mussels contain chemosynthetic bacterial symbionts within their cells. These endosymbionts use reduced inorganic compounds as a source of energy to fix CO₂ into organic compounds using the Calvin-Benson-Bassham (CBB) cycle (the most widespread mechanism of CO₂ fixation, which is also used by green plants, algae and photosynthetic protists). This fixed carbon provides a large component of the mussels’ nutrition.   Depending on the geology of the site, the vent and seep fluids may contain different proportions of gases, sulfide compounds and hydrocarbons, and this results in diverse symbiotic associations. Some mussels contain bacteria that oxidize methane (methanotrophs), some contain bacteria that use hydrogen sulfide (thiotrophs) and some contain mixtures of both types. Additional metabolic diversity is provided by bacteria that can degrade hydrocarbons or use hydrogen. These symbionts cannot currently be cultured, but genome analysis shows that they are closely related to free-living members of the class Gammaproteobacteria. The mussels are thought to acquire their symbionts from the environment at the larval stage and they colonize the gills continuously as they grow throughout the host’s lifetime (Wentrup et al., 2014).

Bathymodiolus and its symbionts have been extensively studied by the team led by Nicole Dubilier at the Max Planck Institute for Marine Microbiology, Bremen. Professor Dubilier recently gave the Ply­mouth Mar­ine Sci­ence Edu­ca­tion Fund (PlyMSEF) Mar­ine Sci­ence Medal Lec­ture, in recognition of her ex­cep­tional con­tri­bu­tions to mar­ine sci­ence. Her lecture described how meta- ‘omic approaches are revealing how symbiont diversity plays a key role in the ecology and evolution of these host-microbe associations. In particular, her talk focussed on the recent studies by Ansorge et al. (2019) in collaboration with Jillian Petersen’s team at the University of Vienna, showing that individual mussels can contain multiple strains of sulfide-oxidizing (Sox) symbionts. These displayed a variety of differences in key functions such as use of energy nutrient sources, electron acceptors and indicators of viral defence functions. Traditional evolutionary theory predicts that a variety of mechanisms exist to prevent competition between symbiont strains to ensure the stability of mutualistic symbioses. This paradigm developed largely because studies were conducted by analysis of sequences of 16S rRNA genes and a few other single markers. The development of metagenomic techniques, with their ability to compare diversity across entire genomes, means that this idea requires reevaluation. For example, the large-scale studies of the human microbiome have shown that intra-species diversity of bacterial symbionts has been greatly underestimated. We also know that microscale processes and niche partitioning (as well as infection by phages) explains the diversity of free-living ocean bacteria and their role in global processes. Rebecca Ansorge and colleagues used high resolution metagenomic and metatranscriptomic methods to examine individual mussels from four Bathymodiolus species, collected from geochemically distinct hydrothermal vents in the Mid-Atlantic Ridge. Their analysis indicated the presence of intermixed symbiont populations among co-existing hosts, rather than genetic isolation. Up to 16 versions of the of the most variable bacterial genes were found within single mussels. Could this diversity be explained by variation in the geochemistry of the habitats in which the mussels occur? By collecting mussels from different vent sites, the team were able to explain the genetic diversity of symbionts by suggesting that this is advantageous by allowing different energy sources and metabolic processes to be used according to changing environmental conditions. This could explain why Bathymodiolus mussels are so productive and widely distributed at vents and seeps. Another important conclusion put forward by Ansorge et al. to explain the apparent deviation from current evolutionary theory is that the association between Bathymodiolus and its Sox bacterial symbionts incurs a low cost, because the bacteria are obtaining their nutrients directly from the surrounding seawater, rather than being diverted from host metabolism as is the case with legume–Rhizobium and insect–Buchnera mutualisms. Wider evaluation of other symbiotic interactions using the new ‘omics tools may reveal that functional diversity of symbionts may be more prevalent than is currently known and will require rethinking of the evolutionary basis of adaptation.

Acquisition of the CBB carbon fixation cycle by newly-discovered episymbionts

As well as the gammaproteobacterial endosymbionts living inside the gill cells. Assié et al. (2016) demonstrated the presence of episymbiotic bacteria that belong to a different phylogenetic group in different species of Bathymodiolus mussels from around the world. Assie at al. used FISH and electron microscopy to show that these grow as dense patches of filaments between the mussel’s gills, through which the mussel pumps oxygenated seawater. In a recent paper, Assié et al. (2020) proposed that these belong to the new genus “Candidatus Thiobarba” belonging to a group which includes a range of episymbionts found on deep-sea invertebrates including the Pompeii worm, Rimicaris shrimps and the scaly-foot snail. These were originally assigned to the Epsilonproteobacteria class, but they have recently been reclassified in the phylum Campylobactereota using genome-based taxonomy. Until now, all previously described sulfur-oxidizing members of this group were believed to use the reverse (reductive) TCA cycle as the mechanism for autotrophic C fixation. However, Assié et al. (2020) made the surprising discovery that “Ca. Thiorbarba” lacks most of the key genes of the rTCA cycle but possesses and expresses all of the genes required for the CBB cycle. Furthermore, a highly sensitive stable isotope method was used to detect small differences in the ¹²C/¹³C ratio between the inorganic carbon source and the generated cell material in a species of mussel that contains sulfide-oxidizing “Ca. Thiobarba” episymbionts plus methane-oxidizing gammaproteobacteria as the only type of endosymbiont and main source of nutrition. This provided evidence that “Ca. Thiobarba” uses the rTCA cycle for fixation of inorganic C from host respiration and/or methane oxidation by the endosymbionts. Adrien Assie and colleagues conclude that the “patchwork” of CBB pathway genes in these members of the Campylobacterota were most likely acquired by horizontal gene transfer (HGT) with multiple evolutionary origins, a finding that has important implications for understanding the importance of HGT in evolution of metabolic pathways during adaptation to environmental niches. Why has the more energy-efficient rTCA cycle been replaced by the less efficient CBB cycle in these bacteria? The most likely explanation seems to be that the CBB enzymes are much less affected by oxygen levels, which are very high in the niche occupied by “Ca. Thiobarba”, due to the pumping of oxygen-rich seawater across the gills. Perhaps the ancestor of “Ca. Thiobarba” might have colonized mussel gills before switching its C fixation pathway? In the gills, it came to share a niche with came to share a niche in close proximity with the gammaproteobacterial symbionts, providing strong selective pressure for gene transfer. Perhaps “Ca. Thiobarba” is in a phase of evolutionary transition to becoming fully integrated as an endosymbiont, as speculated by Assie et al. (2016)?

References

Ansorge, R., Romano, S., Sayavedra, L., Porras, M.Á.G., Kupczok, A., Tegetmeyer, H.E., … & Petersen, J. (2019). Functional diversity enables multiple symbiont strains to coexist in deep-sea mussels. Nat. Microbiol. 4: 2487-2497.

Assié, A., Borowski, C., van der Heijden, K., Raggi, L., Geier, B., Leisch, N., … & Petersen, J.M. (2016). A specific and widespread association between deep‐sea Bathymodiolus mussels and a novel family of Epsilonproteobacteria. Environ. Microbiol. Rep. 8: 805-813.

Assié, A., Leisch, N., Meier, D.V., Gruber-Vodicka, H., Tegetmeyer, H.E., Meyerdierks, A., … & Dubilier, N. (2020). Horizontal acquisition of a patchwork Calvin cycle by symbiotic and free-living Campylobacterota (formerly Epsilonproteobacteria).  ISME J., 14: 104-122.