Host-Microbe Interactions in the Chemosynthetic Riftia pachyptila Symbiosis.
ABSTRACT: The deep-sea tubeworm Riftia pachyptila lacks a digestive system but completely relies on bacterial endosymbionts for nutrition. Although the symbiont has been studied in detail on the molecular level, such analyses were unavailable for the animal host, because sequence information was lacking. To identify host-symbiont interaction mechanisms, we therefore sequenced the Riftia transcriptome, which served as a basis for comparative metaproteomic analyses of symbiont-containing versus symbiont-free tissues, both under energy-rich and energy-limited conditions. Our results suggest that metabolic interactions include nutrient allocation from symbiont to host by symbiont digestion and substrate transfer to the symbiont by abundant host proteins. We furthermore propose that Riftia maintains its symbiont by protecting the bacteria from oxidative damage while also exerting symbiont population control. Eukaryote-like symbiont proteins might facilitate intracellular symbiont persistence. Energy limitation apparently leads to reduced symbiont biomass and increased symbiont digestion. Our study provides unprecedented insights into host-microbe interactions that shape this highly efficient symbiosis.IMPORTANCE All animals are associated with microorganisms; hence, host-microbe interactions are of fundamental importance for life on earth. However, we know little about the molecular basis of these interactions. Therefore, we studied the deep-sea Riftia pachyptila symbiosis, a model association in which the tubeworm host is associated with only one phylotype of endosymbiotic bacteria and completely depends on this sulfur-oxidizing symbiont for nutrition. Using a metaproteomics approach, we identified both metabolic interaction processes, such as substrate transfer between the two partners, and interactions that serve to maintain the symbiotic balance, e.g., host efforts to control the symbiont population or symbiont strategies to modulate these host efforts. We suggest that these interactions are essential principles of mutualistic animal-microbe associations.
Project description:The uncultivated bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a central role in providing their host with fixed carbon. While this intimate association between host and symbiont indicates tight integration and coordination of function via cellular communication mechanisms, no such systems have been identified. To elucidate potential signal transduction pathways in symbionts that may mediate symbiont-host communication, we cloned and characterized a gene encoding a histidine protein kinase homolog isolated from a symbiont fosmid library. The gene, designated rssA (for Riftia symbiont signal kinase), resembles known sensor kinases and encodes a protein capable of phosphorylating response regulators in Escherichia coli. A second open reading frame, rssB (for Riftia symbiont signal regulator), encodes a protein similar to known response regulators. These results suggest that the symbionts utilize a phosphotransfer signal transduction mechanism to communicate external signals that may mediate recognition of or survival within the host. The specific signals eliciting a response by the signal transduction proteins of the symbiont remain to be elucidated.
Project description:Abstract Symbiotic relationships between vestimentiferan tubeworms and chemosynthetic Gammaproteobacteria build the foundations of many hydrothermal vent and hydrocarbon seep ecosystems in the deep sea. The association between the vent tubeworm Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone has become a model system for symbiosis research in deep?sea vestimentiferans, while markedly fewer studies have investigated symbiotic relationships in other tubeworm species, especially at cold seeps. Here we sequenced the endosymbiont genome of the tubeworm Lamellibrachia barhami from a cold seep in the Gulf of California, using short? and long?read sequencing technologies in combination with Hi?C and Dovetail Chicago libraries. Our final assembly had a size of ~4.17 MB, a GC content of 54.54%, 137X coverage, 4153 coding sequences, and a checkm completeness score of 97.19%. A single scaffold contained 99.51% of the genome. Comparative genomic analyses indicated that the L. barhami symbiont shares a set of core genes and many metabolic pathways with other vestimentiferan symbionts, while containing 433 unique gene clusters that comprised a variety of transposases, defence?related genes and a lineage?specific CRISPR/Cas3 system. This assembly represents the most contiguous tubeworm symbiont genome resource to date and will be particularly valuable for future comparative genomic studies investigating structural genome evolution, physiological adaptations and host?symbiont communication in chemosynthetic animal?microbe symbioses.
Project description:The hydrothermal vent tubeworm <i>Riftia pachyptila</i> hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that <i>Riftia</i> symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.
Project description:The bacterial endosymbionts of the hydrothermal vent tubeworm Riftia pachyptila play a key role in providing their host with fixed carbon. Results of prior research suggest that the symbionts are selected from an environmental bacterial population, although a free-living form has been neither cultured from nor identified in the hydrothermal vent environment. To begin to assess the free-living potential of the symbiont, we cloned and characterized a flagellin gene from a symbiont fosmid library. The symbiont fliC gene has a high degree of homology with other bacterial flagellin genes in the amino- and carboxy-terminal regions, while the central region was found to be nonconserved. A sequence that was homologous to that of a consensus sigma28 RNA polymerase recognition site lay upstream of the proposed translational start site. The symbiont protein was expressed in Escherichia coli, and flagella were observed by electron microscopy. A 30,000-Mr protein subunit was identified in whole-cell extracts by Western blot analysis. These results provide the first direct evidence of a motile free-living stage of a chemoautotrophic symbiont and support the hypothesis that the symbiont of R. pachyptila is acquired with each new host generation.
Project description:The mutualism between the giant tubeworm Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone has been extensively researched over the past 40 years. However, the lack of the host whole-genome information has impeded the full comprehension of the genotype/phenotype interface in Riftia. Here, we described the high-quality draft genome of Riftia, its complete mitogenome, and tissue-specific transcriptomic data. The Riftia genome presents signs of reductive evolution, with gene family contractions exceeding expansions. Expanded gene families are related to sulfur metabolism, detoxification, antioxidative stress, oxygen transport, immune system, and lysosomal digestion, reflecting evolutionary adaptations to the vent environment and endosymbiosis. Despite the derived body plan, the developmental gene repertoire in the gutless tubeworm is extremely conserved with the presence of a near intact and complete Hox cluster. Gene expression analyses establish that the trophosome is a multifunctional organ marked by intracellular digestion of endosymbionts, storage of excretory products, and hematopoietic functions. Overall, the plume and gonad tissues both in contact to the environment harbor highly expressed genes involved with cell cycle, programed cell death, and immunity indicating a high cell turnover and defense mechanisms against pathogens. We posit that the innate immune system plays a more prominent role into the establishment of the symbiosis during the infection in the larval stage, rather than maintaining the symbiostasis in the trophosome. This genome bridges four decades of physiological research in Riftia, whereas it simultaneously provides new insights into the development, whole organism functions, and evolution in the giant tubeworm.
Project description:The deep-sea tubeworm Riftia pachyptila is a model system for a mutualistic association: The adult worm has no digestive system, but completely relies on one phylotype of endosymbiotic chemosynthetic bacteria for nutrition. The bacteria, in turn, are provisioned by the host. Metabolism and physiology of this symbiosis, particularly of the uncultured symbiont, have been subject to various studies. Yet, how both partners interact on the molecular level remains largely unknown. To study these host-symbiont interactions in detail, we sequenced the R. pachyptila host transcriptome de novo, and conducted comprehensive metaproteomic comparisons of symbiont-containing and symbiont-free R. pachyptila tissues under energy-rich and energy-limiting conditions. Our results demonstrate that R. pachyptila invests a considerable part of its proteome to provision the symbionts with inorganic compounds. It acquires symbiont-derived biomass primarily by digesting parts of the symbiont population. The R. pachyptila immune system apparently not only protects the holobiont from pathogens, but is also involved in symbiont population control. The symbiont expresses a repertoire of proteins dedicated to communication with the host, including eukaryote-like proteins that may counteract phagocytosis. During energy limitation, i.e., when reduced sulfur compounds are lacking, the host apparently increases symbiont digestion. We show here an intricate network of interaction pathways that shapes the R. pachyptila holobiont. Together with the metabolic flexibility of the association under varying energy conditions, this probably forms the basis for the success of this tight association under the highly challenging deep-sea conditions.
Project description:Horizontally transmitted symbioses usually house multiple and variable symbiont genotypes that are acquired from a much more diverse environmental pool via partner choice mechanisms. However, in the deep-sea hydrothermal vent tubeworm Riftia pachyptila (Vestimentifera, Siboglinidae), it has been suggested that the Candidatus Endoriftia persephone symbiont is monoclonal. Here, we show with high-coverage metagenomics that adult R. pachyptila house a polyclonal symbiont population consisting of one dominant and several low-frequency variants. This dominance of one genotype is confirmed by multilocus gene sequencing of amplified housekeeping genes in a broad range of host individuals where three out of four loci ( atpA, uvrD and recA) revealed no genomic differences, while one locus ( gyrB) was more diverse in adults than in juveniles. We also analysed a metagenome of free-living Endoriftia and found that the free-living population showed greater sequence variability than the host-associated population. Most juveniles and adults shared a specific dominant genotype, while other genotypes can dominate in few individuals. We suggest that although generally permissive, partner choice is selective enough to restrict uptake of some genotypes present in the environment.
Project description:The symbiotic relationship between vestimentiferan tubeworms and their intracellular chemosynthetic bacteria is one of the more noteworthy examples of adaptation to deep-sea hydrothermal vent environments. The tubeworm symbionts have never been cultured in the laboratory. Nucleotide sequences from the small subunit rRNA gene suggest that the intracellular symbionts of the eastern Pacific vent tubeworms Oasisia alvinae, Riftia pachyptila, Tevnia jerichonana, and Ridgeia piscesae belong to the same phylotype of gammaproteobacteria, "Candidatus Endoriftia persephone." Comparisons of symbiont genomes between the East Pacific Rise tubeworms R. pachyptila and T. jerichonana confirmed that these two hosts share the same symbionts. Two Ridgeia symbiont genomes were assembled from trophosome metagenomes from worms collected from the Juan de Fuca Ridge (one and five individuals, respectively). We compared these assemblies to those of the sequenced Riftia and Tevnia symbionts. Pangenome composition, genome-wide comparisons of the nucleotide sequences, and pairwise comparisons of 2,313 orthologous genes indicated that "Ca Endoriftia persephone" symbionts are structured on large geographical scales but also on smaller scales and possibly through host specificity.Remarkably, the intracellular symbionts of four to six species of eastern Pacific vent tubeworms all belong to the same phylotype of gammaproteobacteria, "Candidatus Endoriftia persephone." Understanding the structure, dynamism, and interconnectivity of "Ca Endoriftia persephone" populations is important to advancing our knowledge of the ecology and evolution of their host worms, which are often keystone species in vent communities. In this paper, we present the first genomes for symbionts associated with the species R. piscesae, from the Juan de Fuca Ridge. We then combine these genomes with published symbiont genomes from the East Pacific Rise tubeworms R. pachyptila and T. jerichonana to develop a portrait of the "Ca Endoriftia persephone" pangenome and an initial outline of symbiont population structure in the different host species. Our study is the first to apply genome-wide comparisons of "Ca Endoriftia persephone" assemblies in the context of population genetics and molecular evolution.
Project description:Much of what is known regarding Riftia pachyptila physiology is based on the wealth of studies of tubeworms living at diffuse flows along the fast-spreading, basalt-hosted East Pacific Rise (EPR). These studies have collectively suggested that Riftia pachyptila and its chemoautotrophic symbionts are physiologically specialized, highly productive associations relying on hydrogen sulfide and oxygen to generate energy for carbon fixation, and the symbiont's nitrate reduction to ammonia for energy and biosynthesis. However, Riftia also flourish in sediment-hosted vents, which are markedly different in geochemistry than basalt-hosted systems. Here we present data from shipboard physiological studies and global quantitative proteomic analyses of Riftia pachyptila trophosome tissue recovered from tubeworms residing in the EPR and the Guaymas basin, a sedimented, hydrothermal vent field. We observed marked differences in symbiont nitrogen metabolism in both the respirometric and proteomic data. The proteomic data further suggest that Riftia associations in Guaymas may utilize different sulfur compounds for energy generation, may have an increased capacity for energy storage, and may play a role in degrading exogenous organic carbon. Together these data reveal that Riftia symbionts are far more physiologically plastic than previously considered, and that--contrary to previous assertions--Riftia do assimilate reduced nitrogen in some habitats. These observations raise new hypotheses regarding adaptations to the geochemical diversity of habitats occupied by Riftia, and the degree to which the environment influences symbiont physiology and evolution.
Project description:Forty years after discovery of chemosynthetic symbiosis in the tubeworm Riftia pachyptila, how organisms maintain their unique host-symbiont associations at the cellular level is still largely unknown. Previous studies primarily focus on symbionts associated with host lineages living in hydrothermal vents. To understand physiological adaptations and evolution in these holobiont systems in markedly different habitats, we characterized four novel siboglinid-symbiont genomes spanning deep-sea seep and sedimented environments. Our comparative analyses suggest that all sampled siboglinid chemoautotrophic symbionts, except for frenulate symbionts, can use both rTCA and Calvin cycle for carbon fixation. We hypothesize that over evolutionary time siboglinids have been able to utilize different bacterial lineages allowing greater metabolic flexibility of carbon fixation (e.g., rTCA) enabling tubeworms to thrive in more reducing habitats, such as vents and seeps. Moreover, we show that sulfur metabolism and molecular mechanisms related to initial infection are remarkably conserved across chemoautotrophic symbionts in different habitats. Unexpectedly, we find that the ability to use hydrogen, as an additional energy source, is potentially more widespread than previously recognized. Our comparative genomic results help elucidate potential mechanisms used to allow chemosynthetically dependent holobionts adapt to, and evolve in, different environments.