@phdthesis{Ponnudurai2018, author = {Ponnudurai, Ruby}, title = {Chemosymbiosis in marine bivalves - unravelling host-symbiont interactions and symbiotic adaptions}, institution = {Institut f{\"u}r Pharmazie}, pages = {1}, year = {2018}, abstract = {Symbiosis essentially forms the cornerstone of complex life on earth. Spearheading symbiosis research in the last few decades include the exploration of diverse mutualistic animal-bacterial associations from marine habitats. Yet, many facets of symbiotic associations remain under-examined. Here we investigated marine bivalves of the genera Bathymodiolus and Codakia, inhabiting hydrothermal vents and shallow water ecosystems, respectively, and their bacterial symbionts. The symbionts reside intracellularly within gill epithelia and supply their host with chemoautotrophically fixed carbon. They oxidize reduced substrates like sulfide (thiotrophic symbionts) and methane (methanotrophic symbionts) from surrounding fluids for energy generation. The nature of interactions between host and symbiont at the metabolic and physical level, as well as between the holobiont and its environment remain poorly understood. In vitro cultivations of both symbiont and host are difficult till date, hampering the feasibility of targeted molecular investigations. We bypassed culture-based experiments by proteogenomically investigating physically separated fractions of host and symbiont cell components for the bivalves Bathymodiolus azoricus, Bathymodiolus thermophilus and Codakia orbicularis. Using these enrichments, we sequenced the symbionts' genomes and established semi-quantitative host-symbiont (meta-) proteomic profiles. This combined approach enabled us to resolve symbiosis-relevant metabolic pathways and adaptations, detect molecular factors mediating physical interactions amongst partners and to understand the association of symbiotic traits with the environmental factors prevailing within habitats of the respective bivalve. Our results revealed intricate metabolic interdependence between the symbiotic partners. In Bathymodiolus, these metabolic interactions included (1) the concentration of essential substrates like CO2 and thiosulfate by the host for the thiotrophic symbiont, and (2) the host's replenishment of essential TCA cycle intermediates for the thiotroph that lacks biosynthetic enzymes for these metabolites. In exchange (3), the thiotroph compensates the host's putative deficiency in amino acid and cofactor biosynthesis by cycling aminoacids derived from imported precursors back to the host. In case of Codakia orbicularis, the symbionts may metabolically supplement their host with N-compounds derived from fixation of molecular nitrogen, a trait that was hitherto unknown in chemosynthetic thiotrophic symbionts. Individual proteogenomic investigations of the bivalves Bathymodiolus azoricus and Bathymodiolus thermophilus showed that their symbionts are able to exploit a multitude of energy sources like sulfide, thiosulfate, methane and hydrogen to fuel chemosynthesis. The bivalves and their thiotrophic symbionts, however, are particularly adapted to thiosulfate-utilization, as indicated by mitochondrial production and concentration of thiosulfate by host and dominant expression of thiosulfate oxidation enzymes in the symbiont. This may be advantageous, because thiosulfate is less toxic to the host than sulfide. The central metabolic pathways for energy generation, carbon and nitrogen assimilation and amino acid biosynthesis in thiotrophic symbionts of both Bathymodiolus host species are highly conserved. Expression levels of these pathways do, however, vary between symbionts of both species, indicating differential regulation of enzyme synthesis, possibly to accommodate differences in host morphology and environmental factors. Systematic comparison of symbiont-containing and symbiont-free sample types within and between B. azoricus and B. thermophilus revealed the presence of 'symbiosisspecific' features allowing direct host-symbiont physical interactions. Host proteins engaged in symbiosis-specific functions include 1) a large repertoire of host digestive enzymes predominant in the gill, possibly facilitating symbiont population control and carbon acquisition via direct enzymatic digestion of symbiont cells and 2) a set of host pattern-recognition receptors, which may enable the host to selectively recognize pathogens or even symbionts "ripe" for consumption. Symbiont proteins engaged in symbiosis-specific interactions included 3) an enormous set of adhesins and toxins, putatively involved in symbiont colonization, persistence and host-feeding. Bathymodiolus symbionts also possess repertoires of CRISPR-Cas and restrictionmodification genes for phage defense that are unusually large for intracellular symbionts. Genomic and proteomic comparisons of thiotrophic symbionts of distinct Bathymodiolus host species from different vent sites revealed a conserved core genome but divergent accessory genomes. The B. thermophilus thiotroph's accessory genome was notably more enriched in genes encoding adhesins, toxins and phage defense proteins than that of other Bathymodiolus symbionts. Phylogenetic analyses suggest that this enrichment possibly resulted from horizontal gene acquisition followed by multiple internal gene duplication events. In others symbionts, these gene functions may be substituted by alternate mechanisms or may not be required at all: The methanotrophic symbionts of B. azoricus, for example, has the genetic potential to supplement phage defense functions. Thus, the accessory genomes of Bathymodiolus symbionts are species- or habitat-associated, possibly facilitating adaptation of the bivalves to their respective micro- and macroenvironments. In support of this, we show that symbiont biomass in B. thermophilus, which hosts only one thiotrophic symbiont phylotype, is considerably higher than in B. azoricus that hosts thiotrophic and methanotrophic symbionts. This suggests that different symbiont compositions in each species produce distinct microenvironments within the holobiont. Our study presents an exhaustive assessment of the genes and proteins involved in this bivalve-microbe interaction, hinting at intimate host-symbiont interdependencies and symbiotic crosstalk between partners. The findings open novel prospects for microbiologists with regard to mechanisms of host-symbiont interplay within highly specialized niches, origin and distribution of prokaryote-eukaryote interaction factors across both mutualistic and pathogenic associations.}, subject = {Bathymodiolus symbiosis}, language = {en} }