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Gardebrecht, A, Markert S, Sievert SM, Felbeck H, Thurmer A, Albrecht D, Wollherr A, Kabisch J, Le Bris N, Lehmann R, Daniel R, Liesegang H, Hecker M, Schweder T.  2012.  Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics. ISME Journal. 6:766-776.   10.1038/ismej.2011.137   AbstractWebsite

The two closely related deep-sea tubeworms Riftia pachyptila and Tevnia jerichonana both rely exclusively on a single species of sulfide-oxidizing endosymbiotic bacteria for their nutrition. They do, however, thrive in markedly different geochemical conditions. A detailed proteogenomic comparison of the endosymbionts coupled with an in situ characterization of the geochemical environment was performed to investigate their roles and expression profiles in the two respective hosts. The metagenomes indicated that the endosymbionts are genotypically highly homogeneous. Gene sequences coding for enzymes of selected key metabolic functions were found to be 99.9% identical. On the proteomic level, the symbionts showed very consistent metabolic profiles, despite distinctly different geochemical conditions at the plume level of the respective hosts. Only a few minor variations were observed in the expression of symbiont enzymes involved in sulfur metabolism, carbon fixation and in the response to oxidative stress. Although these changes correspond to the prevailing environmental situation experienced by each host, our data strongly suggest that the two tubeworm species are able to effectively attenuate differences in habitat conditions, and thus to provide their symbionts with similar micro-environments. The ISME Journal (2012) 6, 766-776; doi: 10.1038/ismej.2011.137; published online 20 October 2011

Markert, S, Gardebrecht A, Felbeck H, Sievert SM, Klose J, Becher D, Albrecht D, Thurmer A, Daniel R, Kleiner M, Hecker M, Schweder T.  2011.  Status quo in physiological proteomics of the uncultured Riftia pachyptila endosymbiont. Proteomics. 11:3106-3117.   10.1002/pmic.201100059   AbstractWebsite

Riftia pachyptila, the giant deep-sea tube worm, inhabits hydrothermal vents in the Eastern Pacific ocean. The worms are nourished by a dense population of chemoautotrophic bacterial endosymbionts. Using the energy derived from sulfide oxidation, the symbionts fix CO(2) and produce organic carbon, which provides the nutrition of the host. Although the endosymbionts have never been cultured, cultivation-independent techniques based on density gradient centrifugation and the sequencing of their (meta-) genome enabled a detailed physiological examination on the proteomic level. In this study, the Riftia symbionts' soluble proteome map was extended to a total of 493 identified proteins, which allowed for an explicit description of vital metabolic processes such as the energy-generating sulfide oxidation pathway or the Calvin cycle, which seems to involve a reversible pyrophosphate-dependent phosphofructokinase. Furthermore, the proteomic view supports the hypothesis that the symbiont uses nitrate as an alternative electron acceptor. Finally, the membrane-associated proteome of the Riftia symbiont was selectively enriched and analyzed. As a result, 275 additional proteins were identified, most of which have putative functions in electron transfer, transport processes, secretion, signal transduction and other cell surface-related functions. Integrating this information into complex pathway models a comprehensive survey of the symbiotic physiology was established.

Markert, S, Arndt C, Felbeck H, Becher D, Sievert SM, Hugler M, Albrecht D, Robidart J, Bench S, Feldman RA, Hecker M, Schweder T.  2007.  Physiological proteomics of the uncultured endosymbiont of Riftia pachyptila. Science. 315:247-250.   10.1126/science.1132913   AbstractWebsite

The bacterial endosymbiont of the deep-sea tube worm Riftia pachyptila has never been successfully cultivated outside its host. In the absence of cultivation data, we have taken a proteomic approach based on the metagenome sequence to study the metabolism of this peculiar microorganism in detail. As one result, we found that three major sulfide oxidation proteins constitute similar to 12% of the total cytosolic proteome, which highlights the essential role of these enzymes for the symbiont's energy metabolism. Unexpectedly, the symbiont uses the reductive tricarboxylic acid cycle in addition to the previously identified Calvin cycle for CO2 fixation.

Dufour, SC, Felbeck H.  2006.  Symbiont abundance in thyasirids (Bivalvia) is related to particulate food and sulphide availability. Marine Ecology-Progress Series. 320:185-194.   10.3354/meps320185   AbstractWebsite

Many bivalve species with chemoautotrophic symbionts have mixotrophic diets and some of their nutritional requirements are met by particulate feeding. The symbionts require reduced compounds (such as sulphide) for their autotrophic production. As the concentration of both particulate food and sulphide can vary in their habitat, it has been suggested that symbiont numbers may vary in response. To address whether symbiont abundance can be influenced by the external medium, we compared symbiont. abundance (1) in specimens kept with or without particulate food, and (2) in specimens kept in sediments with low or high dissolved sulphide content. The relative surface area occupied by symbionts on semi-thin gill sections was determined for Thyasira flexuosa (and, in the sulfide experiment, for Parathyasira equalis and T sarsi) kept for up to 3 wk in experimental microcosms, where the quantity of particulate food or dissolved sulphide was manipulated. Symbiont abundance declined with time; the decline was greater in particle-starved thyasirids and in specimens kept in low sulphide. In the latter conditions, T flexuosa may have also had a greater symbiont digestion rate. The observed patterns may be due to a combination of increased symbiont uptake by the bivalve host, and reduced symbiont vigour under the imposed conditions. The flexible feeding mode of chemosymbiotic thyasirids may help them to survive in fluctuating environments.

Felbeck, H, Arndt C, Hentschel U, Childress JJ.  2004.  Experimental application of vascular and coelomic catheterization to identify vascular transport mechanisms for inorganic carbon in the vent tubeworm, Riftia pachyptila. Deep-Sea Research Part I-Oceanographic Research Papers. 51:401-411.   10.1016/j.dsr.2003.10.012   AbstractWebsite

Maintaining deep sea animals in in situ conditions has always been technically difficult because of the high-pressure requirements. Even more difficult are any attempts in manipulating or sampling these organisms while keeping them alive in high-pressure aquaria. We present a technique to withdraw blood samples by vascular catheterization which allows withdrawal of samples of during maintenance of specimens under high-pressure conditions. We have developed this technique to answer a long debated question, how carbon dioxide is transported from the ambient sea water to the bacterial symbionts inside the trophosome of the hydrothermal vent tubeworm. Riftia pachyptila. Our results indicate that the carbon supply to the symbionts is mainly through inorganic CO2 while its incorporation into malate and succinate may serve storage functions at periods Of low CO2 availability in the environment. (C) 2003 Elsevier Ltd. All rights reserved.

Hentschel, U, Millikan DS, Arndt C, Cary SC, Felbeck H.  2000.  Phenotypic variations in the gills of the symbiont-containing bivalve Lucinoma aequizonata. Marine Biology. 136:633-643.   10.1007/s002270050723   AbstractWebsite

The marine bivalve Lucinoma aequizonata (Lucinidae) maintains a population of sulfide-oxidizing chemoautotrophic bacteria in its gill tissue. These are housed in large numbers intracellularly in specialized host cells, termed bacteriocytes. In a natural population of L. aequizonata, striking variations of the gill colors occur, ranging from yellow to grey, brown and black. The aim of the present study was to investigate how this phenomenon relates to the physiology and numbers of the symbiont population, Our results show that in aquarium-maintained animals, black gills contained fewer numbers of bacteria as well as lower concentrations of sulfur and total protein. Nitrate respiration was stimulated by sulfide (but not by thiosulfate) 33-fold in homogenates of black gills and threefold in yellow gill homogenates. The total rates of sulfide-stimulated nitrate respiration were the same. Oxygen respiration could be measured in animals with yellow gills but not in animals with black gills. The cumulative data suggest that black-gilled clams maintained in the aquarium represent a starvation state. When collected from their natural habitat black gills contain the same number of bacteria as yellow gills. Also, no significant difference in glycogen concentrations of the host tissues was observed. Therefore, starvation is unlikely the cause of black gill color in a natural population. Alternative sources of nutrition to sulfur-based metabolism are discussed. Denaturing gradient gel electrophoresis (DGGE) performed on the different gill tissues, as well as on isolated symbionts, resulted in a single gill symbiont amplification product, the sequence of which is identical to published data. These findings provide molecular evidence that one dominant phylotype is present in the morphologically different gill tissues. Nevertheless, the presence of other phylotypes cannot formally be excluded. The implications of this study are that the gill of L. aequizonata is a highly dynamic organ which lends itself to more detailed studies regarding the molecular and cellular processes underlying nutrient transfer, regulation of bacterial numbers and host-symbiont communication.

Girguis, PR, Lee RW, Desaulniers N, Childress JJ, Pospesel M, Felbeck H, Zal F.  2000.  Fate of nitrate acquired by the tubeworm Riftia pachyptila. Applied and Environmental Microbiology. 66:2783-2790.   10.1128/aem.66.7.2783-2790.2000   AbstractWebsite

The hydrothermal vent tubeworm Riftia pachyptila lacks a mouth and gut and lives in association with intracellular, sulfide-oxidizing chemoautotrophic bacteria. Growth of this tubeworm requires an exogenous source of nitrogen for biosynthesis, and, as determined in previous studies, environmental ammonia and free amino acids appear to be unlikely sources of nitrogen. Nitrate, however, is present in situ (K, Johnson, J. Childress, R. Hessler, C. Sakamoto-Arnold, and C. Beehler, Deep-Sea Res. 35:1723-1744, 1988), is taken up by the host, and can be chemically reduced by the symbionts (U. Hentschel and H. Felbeck, Nature 366:338-340, 1993), Here we report that at an in situ concentration of 40 mu M, nitrate is acquired by R, pachyptila at a rate of 3.54 mu mol g(-1) h(-1), while elimination of nitrite and elimination of ammonia occur at much lower rates (0.017 and 0.21 mu mol g(-1) h(-1), respectively), We also observed reduction of nitrite (and accordingly nitrate) to ammonia in the trophosome tissue. When R. pachyptila tubeworms are exposed to constant in situ conditions for 60 h, there is a difference between the amount of nitrogen acquired via nitrate uptake and the amount of nitrogen lost via nitrite and ammonia elimination, which indicates that there is a nitrogen "sink" Our results demonstrate that storage of nitrate does not account for the observed stoichiometric differences in the amounts of nitrogen, Nitrate uptake was not correlated with sulfide or inorganic carbon flux, suggesting that nitrate is probably not an important oxidant in metabolism of the symbionts, Accordingly, we describe a nitrogen flux model for this association, in which the product of symbiont nitrate reduction, ammonia, is the primary source of nitrogen for the host and the symbionts and fulfills the association's nitrogen needs via incorporation of ammonia into amino acids.

Gros, O, Frenkiel L, Felbeck H.  2000.  Sulfur-oxidizing endosymbiosis in Divaricella quadrisulcata (Bivalvia : Lucinidae): Morphological, ultrastructural, and phylogenetic analysis. Symbiosis. 29:293-317. AbstractWebsite

Based on light and electron microscopy, the cellular organization of the gill filament of Divaricella quadrisulcata is described and compared with other gill filaments of lucinids examined to date. TEM observations revealed a dense population of Gram-negative bacteria located within bacteriocytes in the lateral zone of the gill filament which looks similar to that of Codakia orbicularis with typical "granule cells". The digestive tract of this shallow-water lucinid species is less modified than in other lucinid species. The stomach has a well developed gastric shield, a cristalline style protruding in the stomach from a typical style sac, and active digestive diverticula. The mid gut is coiled through the visceral mass. Therefore, D. quadrisulcata appears to be at least partially dependent on filter-feeding for nutrition. Only one type of bacterial 16S rRNA gene was FCR-amplified from symbiont-containing gill tissue of two specimens, indicating a symbiont population composed of a single species. Phylogenetic analysis showed that sequences of D. quadrisulcata- and C. orbicularis-symbiont were 100% identical at all nucleotide positions determined, suggesting that this other tropical lucinid species harbors the same bacterial symbiont species as the previously analyzed C. orbicularis. Thus, D. quadrisulcata appears as the fifth tropical bivalve colonized by the same symbiont species even though it lives in a different habitat as the four other ones. The symbiont transmission mode was investigated by PCR amplifications from mature ovaries and testes. The C. orbicularis-specific 16S rDNA primer set was unsuccesful in amplifying DNA target for all individuals investigated suggesting that the gill-endosymbionts are environmentally transmitted to the new host generation as for all lucinids examined to date.

Hentschel, U, Berger EC, Bright M, Felbeck H, Ott JA.  1999.  Metabolism of nitrogen and sulfur in ectosymbiotic bacteria of marine nematodes (Nematoda, Stilbonematinae). Marine Ecology-Progress Series. 183:149-158.   10.3354/meps183149   AbstractWebsite

Nematodes of the family Stilbonematinae are known for their highly specific association with ectosymbiotic bacteria. These worms are members of the meiofauna in marine, sulfide-rich sediments, where they migrate around the redox boundary layer. In this study, bacterial ectosymbionts of 2 species of marine nematodes, Stilbonema sp. and Laxus oneistus, were shown to be capable of the respiratory reduction of nitrate and nitrite (denitrification). The use of these alternative electron accepters to oxygen by the bacteria allows the animals to migrate into the deeper, anoxic sediments, where they can exploit the sulfide-rich patches of the deeper sediment layers. The accumulation of thiols (sulfide, thiosulfate, sulfate and glutathione) in body tissues of the worms was determined following incubation in the presence of various electron donors (sulfide, thiosulfate) and accepters (nitrate). In their chemoautotrophic metabolic potential, the ectosymbionts of the 2 nematode species were found to resemble the phylogenetically related, intracellular symbionts of macrofaunal hosts of deep-sea hydrothermal vents and other sulfide-rich habitats.

Hentschel, U, Hand SC, Felbeck H.  1996.  The contribution of nitrate respiration to the energy budget of the symbiont-containing clam Lucinoma aequizonata: A calorimetric study. Journal of Experimental Biology. 199:427-433. AbstractWebsite

Heat production and nitrate respiration rates were measured simultaneously in the gill tissue of Lucinoma aequizonata. This marine bivalve contains chemoautotrophic, intracellular, bacterial symbionts in its gill tissue. The symbionts show constitutive anaerobic respiration, using nitrate instead of oxygen as a terminal electron acceptor. An immediate increase in heat production was observed after the addition of nitrate to the perfusion medium of the calorimeter and this was accompanied by the appearance of nitrite in the effluent sea water. The nitrate-stimulated heat output was similar under aerobic and anaerobic conditions, which is consistent with the constitutive nature of nitrate respiration. The amount of heat released was dependent on the concentration of nitrate in the perfusion medium. At nitrate concentrations between 0.5 and 5 mmoll(-1), the total heat production was increased over twofold relative to baseline values. A mean (+/-S.E.M.) enthalpy of -130+/-22.6 kJ mol(-1) nitrite (N=13) was measured for this concentration range.

Felbeck, H, Turner PJ.  1995.  CO2 transport in catheterized hydrothermal vent tubeworms, Riftia pachyptila (vestimentifera). Journal of Experimental Zoology. 272:95-102.   10.1002/jez.1402720203   AbstractWebsite

Isolated plumes and vestimenta of the tubeworm Riftia pachyptila were perfused to determine the form in which carbon is transported to the animal's symbionts via the circulatory system. Catheters supplying colored saline were inserted into an afferent blood vessel while samples were collected from the efferent vessel. During perfusion, the plumes were immersed in sea water containing radiolabeled CO2. The effluent showed radioactivity in inorganic carbonate (Sigma CO2, sum of all forms), malate, and succinate. When isolated vestimenta were perfused with saline containing labeled CO2, labeled malate and succinate could be detected in the effluent. Carbon transport in the blood as Sigma CO2 is estimated to be of similar importance to that transported after incorporation into organic carbon. The significance for the establishment of the carbon isotope ratio of tubeworms is discussed. (C) 1995 Wiley-Liss, Inc.

Distel, DL, Felbeck H, Cavanaugh CM.  1994.  Evidence for phylogenetic congruence among sulfur-oxidizing chemoautotrophic bacterial endosymbionts and their bivalve hosts. Journal of Molecular Evolution. 38:533-542.   10.1007/bf00178852   AbstractWebsite

Sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria are now known to occur as endosymbionts in phylogenetically diverse bivalve hosts found in a wide variety of marine environments. The evolutionary origins of these symbioses, however, have remained obscure. Comparative 16S rRNA sequence analysis was used to investigate whether thioautotrophic endosymbionts are monophyletic or polyphyletic in origin and to assess whether phylogenetic relationships inferred among these symbionts reflect those inferred among their hosts. 16S rRNA gene sequences determined for endosymbionts from nine newly examined bivalve species from three families (Vesicomyidae, Lucinidae, and Solemyidae) were compared with previously published 16S rRNA sequences of thioautotrophic symbionts and free-living bacteria. Distance and parsimony methods were used to infer phylogenetic relationships among these bacteria. All newly examined symbionts fall within the gamma subdivision of the Proteobacteria, in clusters containing previously examined symbiotic thioautotrophs. The closest free-living relatives of these symbionts are bacteria of the genus Thiomicrospira. Symbionts of the bivalve superfamily Lucinacea and the family Vesicomyidae each form distinct monophyletic lineages which are strongly supported by bootstrap analysis, demonstrating that host phylogenies inferred from morphological and fossil evidence are congruent with phylogenies inferred for their respective symbionts by molecular sequence analysis. The observed congruence between host and symbiont phylogenies indicates shared evolutionary history of hosts and symbiont lineages and suggests an ancient origin for these symbioses.

Hentschel, U, Felbeck H.  1993.  Nitrate respiration in the hydrothermal vent tubeworm Riftia pachyptila. Nature. 366:338-340.   10.1038/366338a0   AbstractWebsite

THE vestimentiferan tubeworm Riftia packyptila is found around hydrothermal vent areas in the deep sea. Intracellular bacterial chemoautotrophic symbionts use the oxidation of sulphide from the effluent of the vents as an energy source for CO2 fixation. They apparently provide most or all of the nutritional requirements for their gutless hosts1-5. This kind of symbiosis has since been found in many other species from various other phyla from other habitats6-9. Here we present results that the bacteria of R. pachyptila may cover a significant fraction of their respiratory needs by the use of nitrate in addition to oxygen. Nitrate is reduced to nitrite, which may be the end product (nitrate respiration)10 or it may be further reduced to nitrogen gas (denitrification)11. This metabolic trait may have an important role in the colonization of hypoxic habitats in general by animals with this kind of symbiosis.