A histidine protein kinase homolog from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

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.     

Crystal structure of ATP sulfurylase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila.

In sulfur chemolithotrophic bacteria, the enzyme ATP sulfurylase functions to produce ATP and inorganic sulfate from APS and inorganic pyrophosphate, which is the final step in the biological oxidation of hydrogen sulfide to sulfate. The giant tubeworm, Riftia pachyptila, which lives near hydrothermal vents on the ocean floor, harbors a sulfur chemolithotroph as an endosymbiont in its trophosome tissue. This yet-to-be-named bacterium was found to contain high levels of ATP sulfurylase that may provide a substantial fraction of the organisms ATP. We present here, the crystal structure of ATP sulfurylase from this bacterium at 1.7 A resolution. As predicted from sequence homology, the enzyme folds into distinct N-terminal and catalytic domains, but lacks the APS kinase-like C-terminal domain that is present in fungal ATP sulfurylase. The enzyme crystallizes as a dimer with one subunit in the crystallographic asymmetric unit. Many buried solvent molecules mediate subunit contacts at the interface. Despite the high concentration of sulfate needed for crystallization, no ordered sulfate was observed in the sulfate-binding pocket. The structure reveals a mobile loop positioned over the active site. This loop is in a "closed" or "down" position in the reported crystal structures of fungal ATP sulfurylases, which contained bound substrates, but it is in an "open" or "up" position in the ligand-free Riftia symbiont enzyme. Thus, closure of the loop correlates with occupancy of the active site, although the loop itself does not interact directly with bound ligands. Rather, it appears to assist in the orientation of residues that do interact with active-site ligands. Amino acid differences between the mobile loops of the enzymes from sulfate assimilators and sulfur chemolithotrophs may account for the significant kinetic differences between the two classes of ATP sulfurylase.     

Development and characterization of 12 microsatellite markers from the deep-sea hydrothermal vent siboglinid Riftia pachyptila

Ecological processes at deep‐sea hydrothermal vents on fast‐spreading mid‐ocean ridges are punctuated by frequent physical disturbance, often accompanied by a high occurrence of population turnover. To persist through local extinction events, sessile invertebrate species living in these geologically and chemically dynamic habitats depend on larval dispersal. We characterized 12 polymorphic microsatellite loci from one such species, the siboglinid tubeworm Riftia pachyptila. All loci conformed to Hardy–Weinberg expectations without linkage (mean H_E = 0.9405, mean N_A = 20.25). These microsatellites are being employed in the investigation of spatial and temporal population genetic structure in the eastern Pacific Ocean.     

Identification and characterization of a flagellin gene from the endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

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.     

Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila.

ATP sulfurylase is a key enzyme in the energy-generating sulfur oxidation pathways of many chemoautotrophic bacteria. The utilization of reduced sulfur compounds to fuel CO2 fixation by the still-uncultured bacterial endosymbionts provides the basis of nutrition in invertebrates, such as the tubeworm Riftia pachyptila, found at deep-sea hydrothermal vents. The symbiont-containing trophosome tissue contains high levels of ATP sulfurylase activity, facilitating the recent purification of the enzyme. The gene encoding the ATP sulfurylase from the Riftia symbiont (sopT) has now been cloned and sequenced by using the partial amino acid sequence of the purified protein. Characterization of the sopT gene has unequivocally shown its bacterial origin. This is the first ATP sulfurylase gene to be cloned and sequenced from a sulfur-oxidizing bacterium. The deduced amino acid sequence was compared to those of ATP sulfurylases reported from organisms which assimilate sulfate, resulting in the discovery that there is substantial homology with the Saccharomyces cerevisiae MET3 gene product but none with the products of the cysDN genes from Escherichia coli nor with the nodP and nodQ genes from Rhizobium meliloti. This and emerging evidence from other sources suggests that E. coli may be atypical, even among prokaryotic sulfate assimilators, in the enzyme it employs for adenosine 5'-phosphosulfate formation. The sopT gene probe also was shown to specifically identify chemoautotrophic bacteria which utilize ATP sulfurylase to oxidize sulfur compounds.     

Lipid composition of deep-sea hydrothermal vent tubeworm Riftia pachyptila, crabs Munidopsis subsquamosa and Bythograea thermydron, mussels Bathymodiolus sp. and limpets Lepetodrilus spp

Lipid composition was determined for hydrothermal vent species collected by the Deep Submergence Vehicle ALVIN from chimneys at 2,500 m depth on the East Pacific Rise. These are the first lipid biomarker studies for most of these species. Lipid content was low and dominated by polar lipid in the vestimentiferan tubeworm Riftia pachyptila, mussels Bathymodiolus sp. and limpets Lepetodrilus spp. The galatheid (Munidopsis subsquamosa) and most brachyuran adult (Bythograea thermydron) crabs were characterized by higher storage lipid (triacylglycerol). Total polyunsaturated fatty acids were similar in R. pachyptila plume and body, but higher in the posterior part of the soft body, which had more docosahexaenoic acid (2-5% of total FA) compared to the anterior and plume (< or =0.3%). Two sulphur-oxidizing bacterial markers, 16:1(n-7)c and 18:1(n-7)c, were high in R. pachyptila and mussel (up to 23%), but lower in both crab species (4-17%). R. pachyptila had greater nonmethylene interrupted diunsaturated fatty acids (8-13%) than all other species (2-8%). R. pachyptila may desaturate and elongate 18:1(n-7)c to obtain essential polyunsaturated fatty acids 20:5(n-3) and 20:4(n-6). The sterol composition of R. pachyptila included similar amounts of cholesterol and desmosterol, whereas the other species had a more diverse sterol composition. These differences in lipids, fatty acids and sterols reflect diverse nutritional strategies and possibly temperature regimes in these species.     

Fate of nitrate acquired by the tubeworm Riftia pachyptila

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 microM, nitrate is acquired by R. pachyptila at a rate of 3.54 micromol g(-1) h(-1), while elimination of nitrite and elimination of ammonia occur at much lower rates (0. 017 and 0.21 micromol 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.     

ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube worm)

ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) was extensively purified from trophosome tissue of Riftia pachyptila, a tube worm that thrives in deep ocean hydrothermal vent communities. The enzyme is probably derived from the sulfide-oxidizing bacteria that densely colonize the tissue. Glycerol (20% v/v) protected the enzyme against inactivation during purification and storage. The native enzyme appears to be a dimer (MW 90 kDa +/- 10%) composed of identical size subunits (MW 48 kDa +/- 5%). At pH 8.0, 30 degrees C, the specific activities (units x mg protein-1) of the most highly purified sample are as follows: ATP synthesis, 370; APS synthesis, 23; molybdolysis, 65; APSe synthesis or selenolysis, 1.9. The Km values for APS and PPi at 5 mM Mg2+ are 6.3 and 14 microM, respectively. In the APS synthesis direction, the Km values for MgATP and SO4(2-) are 1.7 and 27 mM, respectively. The Km values for MgATP and MoO4(2-) in the molybdolysis reaction are 80 and 150 microM, respectively. The Kia for MgATP is 0.65 mM. APS is a potent inhibitor of molybdolysis, competitive with both MgATP and MoO4(2-) (Kiq = 2.2 microM). However, PPi (+ Mg2+) is virtually inactive as a molybdolysis inhibitor. Oxyanion dead end inhibitors competitive with SO4(2-) include (in order of decreasing potency) ClO4- greater than FSO3- (Ki = 22 microM) greater than ClO3- greater than NO3- greater than S2O3(2-) (Ki's = 5 and 43 mM). FSO3- is uncompetitive with MgATP, but S2O3(2-) is noncompetitive. Each subunit contains two free SH groups, at least one of which is functionally essential. ATP, MgATP, SO4(2-), MoO4(2-), and APS each protect against inactivation by excess 5,5'-dithiobis-(2-nitrobenzoate). FSO3- is ineffective as a protector unless MgATP is present. PPi (+Mg2+) does not protect against inactivation. Riftia trophosome contains little or no "ADP sulfurylase." The high trophosome level of ATP sulfurylase (67-176 ATP synthesis units x g fresh wt tissue-1 from four different specimens, corresponding to 4-10 microM enzyme sites), the high kcat of the enzyme for ATP synthesis (296 s-1), and the high Km's for MgATP and SO4(2-) are consistent with a role in ATP formation during sulfide oxidation, i.e., the physiological reaction is APS + MgPPi in equilibrium SO4(2-) + MgATP.     

Enzymatic defenses against oxygen toxicity in the hydrothermal vent animals Riftia pachyptila and Calyptogena magnifica

Two deep-sea hydrothermal vent organisms, the tube worm Riftia pachyptila and the clam Calyptogena magnifica, contain superoxide dismutase, dianisidine peroxidase, and glutathione peroxidase. The tube worm trophosome exhibits an iron-containing superoxide dismutase, ordinarily associated with prokaryotes and not previously seen in an animal tissue, in accord with the presence of symbiotic bacteria in this tissue. The enzymes which provide a defense against oxygen toxicity are thus present in these animals.     

Biochemical and enzymological aspects of the symbiosis between the deep-sea tubeworm Riftia pachyptila and its bacterial endosymbiont.

Riftia pachyptila (Vestimentifera) is a giant tubeworm living around the volcanic deep-sea vents of the East Pacific Rise. This animal is devoid of a digestive tract and lives in an intimate symbiosis with a sulfur-oxidizing chemoautotrophic bacterium. This bacterial endosymbiont is localized in the cells of a richly vascularized organ of the worm: the trophosome. These organisms are adapted to their extreme environment and take advantage of the particular composition of the mixed volcanic and sea waters to extract and assimilate inorganic metabolites, especially carbon, nitrogen, oxygen and sulfur. The high molecular mass hemoglobin of the worm is the transporter for both oxygen and sulfide. This last compound is delivered to the bacterium which possesses the sulfur oxidizing respiratory system, which produces the metabolic energy for the two partners. CO2 is also delivered to the bacterium where it enters the Calvin-Benson cycle. Some of the resulting small carbonated organic molecules are thus provided to the worm for its own metabolism. As far as nitrogen assimilation is concerned, NH3 can be used by the two partners but nitrate can be used only by the bacterium. This very intimate symbiosis applies also to the organization of metabolic pathways such as those of pyrimidine nucleotides and arginine. In particular, the worm lacks the first three enzymes of the de novo pyrimidine biosynthetic pathways as well as some enzymes involved in the biosynthesis of polyamines. The bacterium lacks the enzymes of the pyrimidine salvage pathway. This symbiotic organization constitutes a very interesting system to study the molecular and metabolic basis of biological adaptation.     

Characterization of carbonic anhydrases from Riftia pachyptila,a symbiotic invertebrate from deep-sea hydrothermal vents

The symbiotic hydrothermal vent tubeworm Riftia pachyptila needs to supply its internal bacterial symbionts with carbon dioxide, their inorganic carbon source. Our aim in this study was to characterize the carbonic anhydrase (CA) involved in CO(2) transport and conversion at various steps in the plume and the symbiotic tissue, the trophosome. A complete 1209 kb cDNA has been sequenced from the trophosome and identified as a putative alpha-CA based on BLAST analysis and the similarities of total deduced amino-acid sequence with those from the GenBank database. In the plume, the putative CA sequence obtained from cDNA library screening was 90% identical to the trophosome CA, except in the first 77 nucleotides downstream from the initiation site identified on trophosome CA. A phylogenetic analysis showed that the annelidan Riftia CA (CARp) emerges clustered with invertebrate CAs, the arthropodan Drosophila CA and the cnidarian Anthopleura CA. This invertebrate cluster appeared as a sister group of the cluster comprising mitochondrial and cytosolic isoforms in vertebrates: CAV, CAI II and III, and CAVII. However, amino acid sequence alignment showed that Riftia CA was closer to cytosolic CA than to mitochondrial CA. Combined biochemical approaches revealed two cytosolic CAs with different molecular weights and pI's in the plume and the trophosome, and the occurrence of a membrane-bound CA isoform in addition to the cytosolic one in the trophosome. The physiologic roles of cytosolic CA in both tissues and supplementary membrane-bound CA isoform in the trophosome in the optimization of CO(2) transport and conversion are discussed.     

Molecular characteristics of the tubeworm, Ridgeia piscesae, from the deep-sea hydrothermal vent

Ridgeia piscesae, living around the extremely harsh hydrothermal vent in deep sea, is an ideal model for studying the adaptative mechanism to extreme environment. For insights of its molecular characteristics, a cDNA library of R. piscesae was constructed. A total of 879 expressed sequence tags (ESTs) were sequenced and 199 genes were identified for the first time. They were found to be involved in basal metabolism, adaptation and defense, or signal transduction. Among them, we found 23 various chitin-binding proteins, which are the major component of the chitinous tube that prevents the tubeworms from predators and surrounding extreme environment. Additionally, high polymorphism also exists in other genes, such as myohemerythrin, lysozyme. The gene-expression profile might help to further understand the molecular basis of tubeworm physiology. It will also lay a good foundation for functional studies on the adaptation to extreme environments.     

Catabolism of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyptila

The present study describes the distribution and properties of enzymes of the catabolic pathway of pyrimidine nucleotides in Riftia pachyptila, a tubeworm living around deep-sea hydrothermal vents and known to be involved in a highly specialized symbiotic association with a bacterium. The catabolic enzymes, 5'-nucleotidase, uridine phosphorylase, and uracil reductase, are present in all tissues of the worm, whereas none of these enzymatic activities were found in the symbiotic bacteria. The 5'-nucleotidase activity was particularly high in the trophosome, the symbiont-harboring tissue. These results suggest that the production of nucleosides in the trophosome may represent an alternative source of carbon and nitrogen for R. pachyptila, because these nucleosides can be delivered to other parts of the worm. This process would complement the source of carbon and nitrogen from organic metabolites provided by the bacterial assimilatory pathways. The localization of the enzymes participating in catabolism, 5'-nucleotidase and uridine phosphorylase, and of the enzymes involved in the biosynthesis of pyrimidine nucleotides, aspartate transcarbamylase and dihydroorotase, shows a non-homogeneous distribution of these enzymes in the trophosome. The catabolic enzymes 5'-nucleotidase and uridine phosphorylase activities increase from the center of the trophosome to its periphery. In contrast, the anabolic enzymes aspartate transcarbamylase and dihydroorotase activities decrease from the center toward the periphery of the trophosome. We propose a general scheme of anatomical and physiological organization of the metabolic pathways of the pyrimidine nucleotides in R. pachyptila and its bacterial endosymbiont.     

Wide bacterial diversity associated with tubes of the vent worm Riftia pachyptila

We carried out a 16S rDNA-based molecular survey of the prokaryotic diversity associated with the chitin tubes of the giant vent tubeworm Riftia pachyptila (collected at the East Pacific Rise, 9 degrees N and 13 degrees N). Scanning electron microscopy showed dense microbial populations, particularly on the external surface of the middle and upper tube regions, which included very diverse prokaryotic morphotypes. We used archaeal- and bacterial-specific primers for polymerase chain reaction (PCR) amplification, but only bacterial amplicons were obtained. We analysed a total of 87 clones. Most belonged to the epsilon-Proteobacteria, but also to the delta-, alpha- and gamma-Proteobacteria. A broad diversity of phylotypes belonging to other bacterial divisions was detected, including Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides group and the candidate division OP8. We also retrieved a sequence, R76-B150, of uncertain phylogenetic affiliation, which could represent a novel candidate division. The sequence of the R. pachyptilagamma-proteobacterial endosymbiont was not detected. The bacterial diversity found suggests that complex metabolic interactions, particularly based on sulphur chemistry, may be occurring in different microniches of the R. pachyptila tubes.     

Partially glucose-capped oligosaccharides are found on the hemoglobins of the deep-sea tube worm Riftia pachyptila

We report here the structural determination of N-linked oligosaccharides found on extracellular hemoglobins of the hydrothermal vent tube worm Riftia pachyptila. Structures were elucidated by a combination of electrospray ionization tandem mass spectrometry, matrix- assisted laser desorption/ionization mass spectrometry, normal-phase high performance liquid chromatography, and exoglycosidase digestion. The sugar chains were found to consist mainly of high-mannose-type glycans with some structures partially capped by one or two terminal glucose residues. The present study represents the first report of the occurrence of glucose capping of N-linked carbohydrates in a secreted glycoprotein of a metazoan. Previously, glucose capping has only been described for a membrane-bound surface glycoprotein from the unicellular parasite Leishmania mexicana amazonensis.      

Environmental differences in hemoglobin gene expression in the hydrothermal vent tubeworm, Ridgeia piscesae

Ridgeia piscesae, the siboglinid tubeworm inhabiting the hydrothermal vents of the northeast Pacific Juan de Fuca Ridge, displays a wide range of microhabitat-specific, genetically indistinguishable phenotypes. Local microhabitat conditions are hypothesized to play a role in the differentiation of R. piscesae phenotypes. Extracellular hemoglobins serve to connect the tubeworm and the surrounding vent fluid, binding environmental sulfide and oxygen for transport to endosymbionts that use the chemical energy for carbon fixation. Because hemoglobin is essential for this symbiosis, we examined its expression in two of the most extreme R. piscesae phenotypes at two levels: the mRNA encoding the globin subunits and the whole molecules in coelomic and vascular fluids. Levels of gene expression were up to 12 times greater in short-fat R. piscesae from higher temperature, sulfide chimney environments compared to long-skinny animals from a low temperature, diffuse flow basalt habitat. Gene expression levels were consistent with the relative concentrations of hemoglobin molecules in the vascular and coelomic fluids. Up to a 20-fold variation in globin gene expression was detected between the same phenotype from different sites. These data demonstrate that local environmental factors influence not only phenotype but gene expression and its resulting physiological outcome within this unique species.