Phylogenetic characterization of endosymbionts of the hydrothermal vent mussel Bathymodiolus azoricus by analysis of the 16S rRNA, cbbL, and pmoA genes

In order to assess the phylogenetic diversity of the endosymbiotic microbial community of the gills of marine bivalve Bathymodiolus azoricus, total DNA was extracted from the gills. The PCR fragments corresponding to the genes encoding 16S rRNA, ribulose-bisphosphate carboxylase (cbbL), and particulate methane monooxygenase (pmoA) were amplified, cloned, and sequenced. For the 16S rDNA genes, only one phylotype was revealed; it belonged to the cluster of thiotrophic mytilid’s symbionts within the Gammaproteobacteria. For the RuBisCO genes, two phylotypes were found, both belonging to Gammaproteobacteria. One of them was closely related to the previously known mytilid symbiont, the other, to a pogonophore symbiont, presumably a methanotrophic bacterium. One phylotype of particulate methane oxygenase genes was also revealed; this finding indicated the presence of a methanotrophic symbiont. Phylogenetic analysis of the pmoA placed this endosymbiont within the Gammaproteobacteria, in a cluster including the methanotrophic bacterial genus Methylobacter and other methanotrophic Bathymodiolus gill symbionts. These results provide evidence for the existence of two types of endosymbionts (thioautotrophic and methanotrophic) in the gills of B. azoricus and demonstrate that, apart from the phylogenetic analysis of 16S rRNA genes, parallel analysis of functional genes is essential.     

Shell nacre ultrastructure and depressurisation dissolution in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study describes the micro-morphological features of the shell nacre in the vent mytilid Bathymodiolus azoricus collected along a bathymetric gradient of deep-sea hydrothermal vents of the mid-Atlantic ridge (MAR). Pressure-dependent crystallisation patterns were detected in animals subjected to post-capture hydrostatic simulations. We provide evidence for the following: (1) shell micro morphology in B. azoricus is similar to that of several vent and cold-seep species, but the prismatic shell layers may vary among bathymodiolids; (2) nacre micro-morphology of mussels from three vent sites of the MAR did not differ significantly; minor differences do not appear to be related to hydrostatic pressure, but rather to calcium ion availability; (3) decompression stress may cause drop off in pH of the pallial fluid that damages nascent crystals, and in a more advanced phase, the aragonite tablets as well as the continuous layer of mature nacre; and (4) adverse effects of decompression on calcium salt deposition in shells was diminished by re-pressurisation of specimens. The implications of the putative influence of hydrostatic pressure on biomineralisation processes in molluscs are discussed. An erratum to this article can be found at     

Ultrastructural and molecular evidence for potentially symbiotic bacteria within the byssal plaques of the deep-sea hydrothermal vent mussel Bathymodiolus azoricus

This study reports on the presence of a putatively symbiotic bacterial flora within the byssus plaque of the deep sea hydrothermal mussel Bathymodiolus azoricus, contributing to metal sequestration/deposition and testing positive to methane oxidizing symbiont-specific fluorescent probes. Combining an array of approaches including histology, electron microscopy, X-ray microanalysis, analytical chemistry, and microbiology we provide evidence for the frequently assumed, but rarely shown influence of prokaryotes on the biogeochemical cycling of metals as well as inorganic C sources (i.e., methane) at deep sea hydrothermal vents. Our results indicate that in spite of its antibacterial protective sheath, the byssus plaque gives access to a whole range of prokaryotic organisms which may be responsible for the extremely high concentration of metallic elements (Fe, Cu, Zn, Mn, Co, Mo, Cd, Pb and Hg) measured in this attachment organ. The very high levels of metals in byssus, together with its frequent renewal rate due to the dynamic nature of the habitat, suggest that intra-byssal bacteria may have a major influence on biomineralisation/deposition of metals. The presence of a methanotroph morphotype within the byssus plaque was confirmed by FISH and TEM. The implications of the biogeochemical cycling of metals and methane at hydrothermal vents are discussed.     

Metaproteogenomic analysis of a community of sponge symbionts

Sponges harbour complex communities of diverse microorganisms, which have been postulated to form intimate symbiotic relationships with their host. Here we unravel some of these interactions by characterising the functional features of the microbial community of the sponge Cymbastela concentrica through a combined metagenomic and metaproteomic approach. We discover the expression of specific transport functions for typical sponge metabolites (for example, halogenated aromatics, dipeptides), which indicates metabolic interactions between the community and the host. We also uncover the simultaneous performance of aerobic nitrification and anaerobic denitrification, which would aid to remove ammonium secreted by the sponge. Our analysis also highlights the requirement for the microbial community to respond to variable environmental conditions and hence express an array of stress protection proteins. Molecular interactions between symbionts and their host might also be mediated by a set of expressed eukaryotic-like proteins and cell–cell mediators. Finally, some sponge-associated bacteria (for example, a Phyllobacteriaceae phylotype) appear to undergo an evolutionary adaptation process to the sponge environment as evidenced by active mobile genetic elements. Our data clearly show that a combined metaproteogenomic approach can provide novel information on the activities, physiology and interactions of sponge-associated microbial communities.     

Deep-sea hydrothermal vent Epsilonproteobacteria encode a conserved and widespread nitrate reduction pathway (Nap)

Despite the frequent isolation of nitrate-respiring Epsilonproteobacteria from deep-sea hydrothermal vents, the genes coding for the nitrate reduction pathway in these organisms have not been investigated in depth. In this study we have shown that the gene cluster coding for the periplasmic nitrate reductase complex (nap) is highly conserved in chemolithoautotrophic, nitrate-reducing Epsilonproteobacteria from deep-sea hydrothermal vents. Furthermore, we have shown that the napA gene is expressed in pure cultures of vent Epsilonproteobacteria and it is highly conserved in microbial communities collected from deep-sea vents characterized by different temperature and redox regimes. The diversity of nitrate-reducing Epsilonproteobacteria was found to be higher in moderate temperature, diffuse flow vents than in high temperature black smokers or in low temperatures, substrate-associated communities. As NapA has a high affinity for nitrate compared with the membrane-bound enzyme, its occurrence in vent Epsilonproteobacteria may represent an adaptation of these organisms to the low nitrate concentrations typically found in vent fluids. Taken together, our findings indicate that nitrate reduction is widespread in vent Epsilonproteobacteria and provide insight on alternative energy metabolism in vent microorganisms. The occurrence of the nap cluster in vent, commensal and pathogenic Epsilonproteobacteria suggests that the ability of these bacteria to respire nitrate is important in habitats as different as the deep-sea vents and the human body.     

Bacteriocyte-associated gammaproteobacterial symbionts of the Adelges nordmannianae/piceae complex (Hemiptera: Adelgidae)

Adelgids (Insecta: Hemiptera: Adelgidae) are known as severe pests of various conifers in North America, Canada, Europe and Asia. Here, we present the first molecular identification of bacteriocyte-associated symbionts in these plant sap-sucking insects. Three geographically distant populations of members of the Adelges nordmannianae/piceae complex, identified based on coI and ef1alpha gene sequences, were investigated. Electron and light microscopy revealed two morphologically different endosymbionts, coccoid or polymorphic, which are located in distinct bacteriocytes. Phylogenetic analyses of their 16S and 23S rRNA gene sequences assigned both symbionts to novel lineages within the Gammaproteobacteria sharing <92% 16S rRNA sequence similarity with each other and showing no close relationship with known symbionts of insects. Their identity and intracellular location were confirmed by fluorescence in situ hybridization, and the names ‘Candidatus Steffania adelgidicola'' and ‘Candidatus Ecksteinia adelgidicola'' are proposed for tentative classification. Both symbionts were present in all individuals of all investigated populations and in different adelgid life stages including eggs, suggesting vertical transmission from mother to offspring. An 85 kb genome fragment of ‘Candidatus S. adelgidicola'' was reconstructed based on a metagenomic library created from purified symbionts. Genomic features including the frequency of pseudogenes, the average length of intergenic regions and the presence of several genes which are absent in other long-term obligate symbionts, suggested that ‘Candidatus S. adelgidicola'' is an evolutionarily young bacteriocyte-associated symbiont, which has been acquired after diversification of adelgids from their aphid sister group.     

Several deep-sea mussels and their associated symbionts are able to live both on wood and on whale falls

Bathymodiolin mussels occur at hydrothermal vents and cold seeps, where they thrive thanks to symbiotic associations with chemotrophic bacteria. Closely related genera Idas and Adipicola are associated with organic falls, ecosystems that have been suggested as potential evolutionary 'stepping stones' in the colonization of deeper and more sulphide-rich environments. Such a scenario should result from specializations to given environments from species with larger ecological niches. This study provides molecular-based evidence for the existence of two mussel species found both on sunken wood and bones. Each species specifically harbours one bacterial phylotype corresponding to thioautotrophic bacteria related to other bathymodiolin symbionts. Phylogenetic patterns between hosts and symbionts are partially congruent. However, active endocytosis and occurrences of minor symbiont lineages within species which are not their usual host suggest an environmental or horizontal rather than strictly vertical transmission of symbionts. Although the bacteria are close relatives, their localization is intracellular in one mussel species and extracellular in the other, suggesting that habitat choice is independent of the symbiont localization. The variation of bacterial densities in host tissues is related to the substrate on which specimens were sampled and could explain the abilities of host species to adapt to various substrates.     

Draft genome sequence of Caminibacter mediatlanticus strain TB-2, an epsilonproteobacterium isolated from a deep-sea hydrothermal vent

Caminibacter mediatlanticus strain TB-2(T) [1], is a thermophilic, anaerobic, chemolithoautotrophic bacterium, isolated from the walls of an active deep-sea hydrothermal vent chimney on the Mid-Atlantic Ridge and the type strain of the species. C. mediatlanticus is a Gram-negative member of the Epsilonproteobacteria (order Nautiliales) that grows chemolithoautotrophically with H(2) as the energy source and CO(2) as the carbon source. Nitrate or sulfur is used as the terminal electron acceptor, with resulting production of ammonium and hydrogen sulfide, respectively. In view of the widespread distribution, importance and physiological characteristics of thermophilic Epsilonproteobacteria in deep-sea geothermal environments, it is likely that these organisms provide a relevant contribution to both primary productivity and the biogeochemical cycling of carbon, nitrogen and sulfur at hydrothermal vents. Here we report the main features of the genome of C. mediatlanticus strain TB-2(T).     

Inorganic carbon fixation by chemosynthetic ectosymbionts and nutritional transfers to the hydrothermal vent host-shrimp Rimicaris exoculata

The shrimp Rimicaris exoculata dominates several hydrothermal vent ecosystems of the Mid-Atlantic Ridge and is thought to be a primary consumer harbouring a chemoautotrophic bacterial community in its gill chamber. The aim of the present study was to test current hypotheses concerning the epibiont''s chemoautotrophy, and the mutualistic character of this association. In-vivo experiments were carried out in a pressurised aquarium with isotope-labelled inorganic carbon (NaH¹³CO₃ and NaH¹⁴CO₃) in the presence of two different electron donors (Na₂S₂O₃ and Fe²⁺) and with radiolabelled organic compounds (¹⁴C-acetate and ³H-lysine) chosen as potential bacterial substrates and/or metabolic by-products in experiments mimicking transfer of small biomolecules from epibionts to host. The bacterial epibionts were found to assimilate inorganic carbon by chemoautotrophy, but many of them (thick filaments of epsilonproteobacteria) appeared versatile and able to switch between electron donors, including organic compounds (heterotrophic acetate and lysine uptake). At least some of them (thin filamentous gammaproteobacteria) also seem capable of internal energy storage that could supply chemosynthetic metabolism for hours under conditions of electron donor deprivation. As direct nutritional transfer from bacteria to host was detected, the association appears as true mutualism. Import of soluble bacterial products occurs by permeation across the gill chamber integument, rather than via the digestive tract. This first demonstration of such capabilities in a decapod crustacean supports the previously discarded hypothesis of transtegumental absorption of dissolved organic matter or carbon as a common nutritional pathway.     

Annual spawning of the hydrothermal vent mussel, Bathymodiolus azoricus, under controlled aquarium, conditions at atmospheric pressure

Dense macrofaunal communities of modioliform mussels are a major component of many hydrothermal vent and cold seep ecosystems. The hydrothermal vent mussel Bathymodiolus azoricus, that dominates hydrothermal vent communities near the Azores Triple Junction, can be maintained in aquaria at atmospheric pressure. Cages containing these mussels were placed over diffuse vent outlets and recovered at different times. Cages recovered in January 2003 contained mussels with ripe gonads while those recovered in July, August and November 2001 and in April 2003 did not. Mussels collected post-spawning in April 2003 spawned in the aquaria in January 2004. Young mussels recruited to the cages in April 2003. The data support a main single period of spawning and of juvenile recruitment for B. azoricus.     

Acquisition of epibiotic bacteria along the life cycle of the hydrothermal shrimp Rimicaris exoculata

The caridean shrimp Rimicaris exoculata dominates the fauna at several Mid-Atlantic Ridge hydrothermal vent sites. This shrimp has an enlarged gill chamber, harboring a dense ectosymbiotic community of chemoautotrophic bacteria associated with mineral oxide deposits. Until now, their acquisition is not fully understood. At three hydrothermal vent sites, we analyzed the epibionts diversity at different moult stages and also in the first stages of the shrimp life (eggs, hatched eggs (with larvae) and juveniles). Hatched eggs associated with young larvae were collected for the first time directly from gravid females at the Logachev vent site during the Serpentine cruise. An approach using 16S rRNA clone libraries, scanning and transmission electron microscopy, and fluorescent in situ hybridization was used. Molecular results and microscope observations indicated a switch in the composition of the bacterial community between early R. exoculata life cycle stage (egg libraries dominated by the Gammaproteobacteria) and later stages (juvenile/adult libraries dominated by the Epsilonproteobacteria). We hypothesized that the epibiotic phylotype composition could vary according to the life stage of the shrimp. Our results confirmed the occurrence of a symbiosis with Gammaproteobacteria and Epsilonproteobacteria, but more complex than previously assumed. We revealed the presence of active type-I methanotrophic bacteria colonizing the cephalothorax of shrimps from the Rainbow site. They were also present on the eggs from the Logachev site. This could be the first ‘epibiotic'' association between methanotrophic bacteria and hydrothermal vent crustacean. We discuss possible transmission pathways for epibionts linked to the shrimp life cycle.     

Chlamydial symbionts in the enigmatic Xenoturbella (Deuterostomia)

Ultrastructural observations of the gastrodermal cells in the enigmatic Xenoturbella revealed numerous chlamydiae. They are related to "Candidatus Fritschea" and Simkania (Simkaniaceae) based on 16S and 23S rRNA. Their 23S rRNA gene contains an intron encoding a putative homing endonuclease. The chlamydiae were pleomorphic and formed intravacuolar colonies. They have flattened disk-shaped elementary bodies, either oval or bow tie-shaped in cross-section, and reticulate bodies that are spherical, polygonal or irregularly shaped. All stages have five-layered cell wall with rippled appearance. Bacteria were not observed in the nuclei. The association between the chlamydiae and Xenoturbella is characterized by absence of cytopathological effects; limited host cell response against the chlamydiae; the confinement of the chlamydiae to inclusions in some part of the host cell; and complete and uniform infection of all examined hosts.     

Exploring the limit of metazoan thermal tolerance via comparative proteomics: thermally induced changes in protein abundance by two hydrothermal vent polychaetes

Temperatures around hydrothermal vents are highly variable, ranging from near freezing up to 300°C. Nevertheless, animals thrive around vents, some of which live near the known limits of animal thermotolerance. Paralvinella sulfincola, an extremely thermotolerant vent polychaete, and Paralvinella palmiformis, a cooler-adapted congener, are found along the Juan de Fuca Ridge in the northwestern Pacific. We conducted shipboard high-pressure thermotolerance experiments on both species to characterize the physiological adaptations underlying P. sulfincola's pronounced thermotolerance. Quantitative proteomics, expressed sequence tag (EST) libraries and glutathione assays revealed that P. sulfincola (i) exhibited an upregulation in the synthesis and recycling of glutathione with increasing temperature, (ii) downregulated nicotinamide adenine dinucleotide (NADH) and succinate dehydrogenases (key enzymes in oxidative phosphorylation) with increasing temperature, and (iii) maintained elevated levels of heat shock proteins (HSPs) across all treatments. In contrast, P. palmiformis exhibited more typical responses to increasing temperatures (e.g. increasing HSPs at higher temperatures). These data reveal differences in how a mesotolerant and extremely thermotolerant eukaryote respond to thermal stress, and suggest that P. sulfincola's capacity to mitigate oxidative stress via increased synthesis of antioxidants and decreased flux through the mitochondrial electron transport chain enable pronounced thermotolerance. Ultimately, oxidative stress may be the key factor in limiting all metazoan thermotolerance.     

Global depression in gene expression as a response to rapid thermal changes in vent mussels

Hydrothermal vent mussels belonging to the genus Bathymodiolus are distributed worldwide and dominate communities at shallow Atlantic hydrothermal sites. While organisms inhabiting coastal ecosystems are subjected to predictable oscillations of physical and chemical variables owing to tidal cycles, the vent mussels sustain pronounced temperature changes over short periods of time, correlated to the alternation of oxic/anoxic phases. In this context, we focused on the short-term adaptive response of mussels to temperature change at a molecular level. The mRNA expression of 23 genes involved in various cell functions of the vent mussel Bathymodiolus azoricus was followed after heat shocks for either 30 or 120 min, at 25 and 30°C over a 48 h recovery period at 5°C. Mussels were genotyped at 10 enzyme loci to explore a relationship between natural genetic variation, gene expression and temperature adaptation. Results indicate that the mussel response to increasing temperature is a depression in gene expression, such a response being genotypically correlated at least for the Pgm-1 locus. This suggests that an increase in temperature could be a signal triggering anaerobiosis for B. azoricus or this latter alternatively behaves more like a ‘cold’ stenotherm species, an attribute more related to its phylogenetic history, a cold seeps/wood fall origin.     

Possible role of a taurine transporter in the deep-sea mussel Bathymodiolus septemdierum in adaptation to hydrothermal vents

Various invertebrates inhabiting hydrothermal vents possess sulfur-oxidizing bacteria in their tissues; however, the mechanisms by which toxic sulfides are delivered to these endosymbionts remain unknown. Recently, detoxification of sulfides using thiotaurine, a sulfur-containing amino acid, has been suggested. In this study, we propose the involvement of a taurine transporter in sulfide detoxification in the deep-sea mussel Bathymodiolus septemdierum by demonstrating: (i) the abundance of its mRNA in the gill; (ii) its activity under a wide range of salinities; (iii) its low Michaelis constant value in taurine transportation; and (iv) its affinity for thiotaurine and the thiotaurine precursor, hypotaurine.     

An invasive Mimosa in India does not adopt the symbionts of its native relatives

Background and Aims

The large monophyletic genus Mimosa comprises approx. 500 species, most of which are native to the New World, with Central Brazil being the main centre of radiation. All Brazilian Mimosa spp. so far examined are nodulated by rhizobia in the betaproteobacterial genus Burkholderia. Approximately 10 Mya, transoceanic dispersal resulted in the Indian subcontinent hosting up to six endemic Mimosa spp. The nodulation ability and rhizobial symbionts of two of these, M. hamata and M. himalayana, both from north-west India, are here examined, and compared with those of M. pudica, an invasive species.

Methods

Nodules were collected from several locations, and examined by light and electron microscopy. Rhizobia isolated from them were characterized in terms of their abilities to nodulate the three Mimosa hosts. The molecular phylogenetic relationships of the rhizobia were determined by analysis of 16S rRNA, nifH and nodA gene sequences.

Key Results

Both native Indian Mimosa spp. nodulated effectively in their respective rhizosphere soils. Based on 16S rRNA, nifH and nodA sequences, their symbionts were identified as belonging to the alphaproteobacterial genus Ensifer, and were closest to the ‘Old World’ Ensifer saheli, E. kostiensis and E. arboris. In contrast, the invasive M. pudica was predominantly nodulated by Betaproteobacteria in the genera Cupriavidus and Burkholderia. All rhizobial strains tested effectively nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica.

Conclusions

The native Mimosa spp. in India are not nodulated by the Burkholderia symbionts of their South American relatives, but by a unique group of alpha-rhizobial microsymbionts that are closely related to the ‘local’ Old World Ensifer symbionts of other mimosoid legumes in north-west India. They appear not to share symbionts with the invasive M. pudica, symbionts of which are mostly beta-rhizobial.