DNA-DNA Solution Hybridization Studies of the Bacterial Symbionts of Hydrothermal Vent Tube Worms (Riftia pachyptila and Tevnia jerichonana)

The giant tube worm, Riftia pachyptila (phylum Vestimentifera), is known only from four widely separated sulfide-rich deep-sea hydrothermal vent systems. This invertebrate is nourished by intracellular, chemoautotrophic bacterial symbionts which reside in a specialized trophosome tissue. The symbiont has not been cultured independently and is believed to be acquired de novo by host larvae of each generation. In the current study, R. pachyptila symbiont DNA was purified from the two most distant sites on the basis of its difference in density versus host DNA. These two standards were hybridized against trophosome DNAs of 13 individuals from the Guaymas Basin, Galapagos Rift, and 13 degrees N vents. This indicated that all R. pachyptila symbionts are conspecific and that the variability in DNA-DNA hybridization (relative binding ratio [RBR]) was comparable within or between widely separated vents. The symbiont of another tube worm, Tevnia jerichonana, was found to be the same as that of R. pachyptila, the first case in which distinct hosts possess the same sulfur bacterial symbiont. By contrast, Lamellibrachia sp. (same class as T. jerichonana) showed insignificant RBR with the R. pachyptila symbiont. DNA derived from solely eucaryotic tissue of R. pachyptila showed a surprisingly high RBR (20 to 50) with density-separated DNA standards. With DNAs obtained from physically separated symbionts, independent solution hybridization experiments confirmed the above-described conclusions. Possible explanations for this host-symbiont homology are discussed.     

Ultrastructural reinvestigation of the trophosome in adults of Riftia pachyptila (Annelida, Siboglinidae)

Abstract. The trophosome of adults of Riftia pachyptila (Vestimentifera) was reinvestigated using 3-dimensional ultrastructural reconstruction and quantitative morphological analysis. The symbionts make up 24.1%, the symbiont-containing cells (bacteriocytes) are 70.5% of the trophosome's volume. The trophosome is composed of lobules that have a central axial blood vessel surrounded by a myoepithelium containing bacteriocytes, in turn surrounded by an apolar tissue of bacteriocytes. Part of the splanchnic peritoneum lining the trunk coelom encases the bacteriocytes and forms a ramifying network of peripheral blood vessels. Based on the morphology and ultrastructure of the adult, we hypothesize a mesodermal rather than endodermal origin of trophosome and its constitute bacteriocytes. Some of the central bacteriocytes are part of the myoepithelium surrounding the axial blood vessel and act as stem cells for a proliferating tissue produced in the center and ultimately degraded at the periphery of each lobule. Similarly, the rod-shaped symbionts in the center act as stem cells and exhibit a simple cell cycle. Differentiation into cocci takes place in the median and peripheral zone. Lysis of cocci occurs in the degenerative zone.     

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.     

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.     

Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences.

The 16S rRNAs from the bacterial endosymbionts of six marine invertebrates from diverse environments were isolated and partially sequenced. These symbionts included the trophosome symbiont of Riftia pachyptila, the gill symbionts of Calyptogena magnifica and Bathymodiolus thermophilus (from deep-sea hydrothermal vents), and the gill symbionts of Lucinoma annulata, Lucinoma aequizonata, and Codakia orbicularis (from relatively shallow coastal environments). Only one type of bacterial 16S rRNA was detected in each symbiosis. Using nucleotide sequence comparisons, we showed that each of the bacterial symbionts is distinct from the others and that all fall within a limited domain of the gamma subdivision of the purple bacteria (one of the major eubacterial divisions previously defined by 16S rRNA analysis [C. R. Woese, Microbiol. Rev. 51: 221-271, 1987]). Two host specimens were analyzed in five of the symbioses; in each case, identical bacterial rRNA sequences were obtained from conspecific host specimens. These data indicate that the symbioses examined are species specific and that the symbiont species are unique to and invariant within their respective host species.     

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.     

Linking hydrothermal geochemistry to organismal physiology: physiological versatility in Riftia pachyptila from sedimented and basalt-hosted vents

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.     

Ribulose bisphosphate carboxylase of the procaryotic symbiont of a hydrothermal vent tube worm: kinetics, activity and gene hybridization

Abstract The giant tube worm, Riftia pachyptila , which is abundant at deep-sea hydrothermal vents, contains an extremely high density of bacterial symbionts in a specialized 'trophosome' tissue. Although the symbiont has not been cultured, enzymatic studies by others indicate that the symbiont is capable of hydrogen-sulfide- or sulfur-based lithoautotrophy and fixes CO₂ via the Calvin-Benson cycle. Here we report additional findings for a specimen from the Guaymas Basin vent site (Gulf of California, 2000 m). Under assay conditions where activity was proportional to cell-free extract concentration, ribulose bisphosphate carboxylase/oxygenase (RuBisCO) activity was 6.3 nmol CO₂/mg protein per min (30°C). This is within the range observed for non-CO₂ limited cultures of sulfur bacteria. The activity vs. temperature profile suggests that the symbiont is a mesophile and not a thermophile. A substrate saturation curve shows an apparent K _m (with respect to ribulose 1,5-bisphosphate) of 65 μM which is considerably lower than the single previous report for a sulfur bacterial symbiont. Strong hybridization was detected between a gene probe derived from the RuBisCO large subunit gene of Anacystis nidulans and Riftia trophosome DNA. A Rhodospirillum rubrum -derived probe also showed hybridization with the same restriction fragments of symbiont DNA.     

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.     

The anatomy of the blood vascular system of the giant vestimentiferan tubeworm Riftia pachyptila (Siboglinidae,Annelida)

The giant dimensions of vestimentiferan Riftia pachyptila (Jones, 1981 ) are achieved thanks to the well‐developed vascular system. In the vestimentum, there is a complicated net of lacunae, including the brain blood supply and the ventral lacuna underlying the ciliary field. The trunk region has an extensive network of blood vessels feeding the gonads («rete mirabile»). The thick muscular lining of the mesenterial vessels in the trunk and the dorsal vessel in the opisthosome serves as an additional pump, pushing blood into numerous vessels in the segments. It was hypothesized that the blood envelope of the ventral blood vessel in the trunk provides the blood supply to the trophosome. The 3D reconstruction has revealed that there are two vascular systems of the tentacular crown of R. pachyptila . Blood runs into the tentacles via axial afferent vessels, as described earlier only for Riftia , and also via basal ones, as described for other vestimentiferans except Riftia . The basal ones are poorly developed, and the number of lamellar blood vessels is small, indicating a lack of demand for these within huge R. pachyptila . It appears that the presence of these vessels is the preserved ancestral state of Vestimentifera. In different portions of the dorsal vessel, the morphology of the intravasal body varies, depending on function.     

Molecular characterization of bacteria associated with the trophosome and the tube of Lamellibrachia sp., a siboglinid annelid from cold seeps in the eastern Mediterranean

Specimens of Lamellibrachia ( Annelida: Siboglinidae ) were recently discovered at cold seeps in the eastern Mediterranean. In this study, we have investigated the phylogeny and function of intracellular bacterial symbionts inhabiting the trophosome of specimens of Lamellibrachia sp. from the Amon mud volcano, as well as the bacterial assemblages associated with their tube. The dominant intracellular symbiont of Lamellibrachia sp. is a gammaproteobacterium closely related to other sulfide-oxidizing tubeworm symbionts. In vivo uptake experiments show that the tubeworm relies on sulfide for its metabolism, and does not utilize methane. Bacterial communities associated with the tube form biofilms and occur from the anterior to the posterior end of the tube. The diversity of 16S rRNA gene phylotypes includes representatives from the same divisions previously identified from the tube of the vent species Riftia pachyptila , and others commonly found at seeps and vents.     

Contribution of the bacterial endosymbiont to the biosynthesis of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyptila

The deep-sea tube worm Riftia pachyptila (Vestimentifera) from hydrothermal vents lives in an intimate symbiosis with a sulfur-oxidizing bacterium. That involves specific interactions and obligatory metabolic exchanges between the two organisms. In this work, we analyzed the contribution of the two partners to the biosynthesis of pyrimidine nucleotides through both the "de novo" and "salvage" pathways. The first three enzymes of the de novo pathway, carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, were present only in the trophosome, the symbiont-containing tissue. The study of these enzymes in terms of their catalytic and regulatory properties in both the trophosome and the isolated symbiotic bacteria provided a clear indication of the microbial origin of these enzymes. In contrast, the succeeding enzymes of this de novo pathway, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase, were present in all body parts of the worm. This finding indicates that the animal is fully dependent on the symbiont for the de novo biosynthesis of pyrimidines. In addition, it suggests that the synthesis of pyrimidines in other tissues is possible from the intermediary metabolites provided by the trophosomal tissue and from nucleic acid degradation products since the enzymes of the salvage pathway appear to be present in all tissues of the worm. Analysis of these salvage pathway enzymes in the trophosome strongly suggested that these enzymes belong to the worm. In accordance with this conclusion, none of these enzyme activities was found in the isolated bacteria. The enzymes involved in the production of the precursors of carbamyl phosphate and nitrogen assimilation, glutamine synthetase and nitrate reductase, were also investigated, and it appears that these two enzymes are present in the bacteria.     

The ionic composition of the hydrothermal vent tube worm Riftia pachyptila: evidence for the elimination of SO2-4SO and H+ and for a Cl-/HCO-3HCO shift

Riftia pachyptila is one of the most specialized invertebrate hosts of chemoautotrophic symbionts. Crucial to the functioning of this symbiosis is how these worms cope with fluctuating ion concentrations. Internal sulfate levels in R. pachyptila appear comparable with other benthic marine invertebrates, despite the production of sulfate internally by means of the bacterial oxidation of hydrogen sulfide, suggesting that these worms are able to eliminate sulfate effectively. Internal chloride levels appear comparable; however, coelomic fluid chloride levels decrease significantly as the amount of coelomic fluid bicarbonate increases, demonstrating a 1:1 stoichiometry. We believe this shift in chloride, out of the body fluids, is needed to compensate for changes in electrochemical balance caused by the large increase (up to and greater than 60 mmol L-1) in negatively charged bicarbonate. Riftia pachyptila fits the general pattern of monovalent ion concentrations that is seen in other benthic marine invertebrates, with a high [Na+] : [K+] ratio extracellularly and low [Na+] : [K+] ratio intracellularly. Extracellular pH values of 7.38+/-0.03 and 7.37+/-0. 04 for coelomic fluid and vascular blood, respectively, as well as intracellular pH values of 7.37+/-0.04 and 7.04+/-0.05 for plume and trophosome tissue, respectively, were measured. On the basis of significant decreases in extracellular pH and, in some cases, Na+ and K+, in worms exposed to carbonyl cyanide m-chlorophenylhydrazone, sodium vanadate, and N-ethylmaleimide, we suggest that high concentrations of H+-ATPases, perhaps Na+/H+- or K+/H+-ATPases, are involved in H+ elimination in these animals.     

Arginine metabolism in the deep sea tube worm Riftia pachyptila and its bacterial endosymbiont

The present study describes the distribution and properties of enzymes involved in arginine metabolism in Riftia pachyptila, a tubeworm living around deep sea hydrothermal vents and known to be engaged in a highly specific symbiotic association with a bacterium. The results obtained show that the arginine biosynthetic enzymes, carbamyl phosphate synthetase, ornithine transcarbamylase, and argininosuccinate synthetase are present in all of the tissues of the worm and in the bacteria. Thus, Riftia and its bacterial endosymbiont can assimilate nitrogen and carbon via this arginine biosynthetic pathway. The kinetic properties of ornithine transcarbamylase strongly suggest that neither Riftia nor the bacteria possess the catabolic form of this enzyme belonging to the arginine deiminase pathway, the absence of this pathway being confirmed by the lack of arginine deiminase activity. Arginine decarboxylase and ornithine decarboxylase are involved in the biosynthesis of polyamines such as putrescine and agmatine. These activities are present in the trophosome, the symbiont-harboring tissue, and are higher in the isolated bacteria than in the trophosome, indicating that these enzymes are of bacterial origin. This finding indicates that Riftia is dependent on its bacterial endosymbiont for the biosynthesis of polyamines that are important for its metabolism and physiology. These results emphasize a particular organization of the arginine metabolism and the exchanges of metabolites between the two partners of this symbiosis.     

A 31P nuclear magnetic resonance study of the hydrothermal vent tube worm Riftia pachyptila.

31P nuclear magnetic resonance (NMR) was used to study the major phosphorylated compounds visible in perchloric extracts of three body regions of the vestimentiferan worm Riftia pachyptila: winged vestimentum, trunk and segmented posterior opisthosome. Two phosphagens (PGs) were present in vestimentum and opisthosome. The major resonance corresponded to those of phosphoarginine and phosphotaurocyamine, which cannot be discriminated on 31P NMR spectra. We have identified four distinct phosphodiesters (PDEs) in these tissues: glycerophosphorylethanolamine (GPE), serine ethanolamine phosphodiester (SEP), glycero-phosphorylcholine (GPC) and threonine ethanolamine phosphodiester (TEP). Three phosphonates or derivates (PAs) were observed in the three body regions. The minor one was identified as 2-aminoethyl phosphonate (2-AEP). The phosphorus profile of the trunk was appreciably different: one additional resonance in the PDE region and only one phosphagen peak were observed.     

Organization and microanatomy of the Sclerolinum contortum trophosome (Polychaeta, Siboglinidae)

The trophosome-an organ especially evolved to accommodate symbiotic bacteria-is a key character of the polychaete family Siboglinidae. Astonishingly, the trophosomes vary in organization and origin between the different siboglinid taxa. The trophosome of the small genus Sclerolinum was nearly unknown until now. Here we investigated the trophosome of S. contortum from the Gulf of Mexico, using light and electron microscopy. We show that this organ derives from the visceral mesoderm and propose that the trophosome of the sister clade Vestimentifera and Sclerolinum is a homologous character. Like that of juvenile vestimentiferans, the trophosome of Sclerolinum trophosome is simply organized. This study reveals that the Sclerolinum trophosome exhibits two regions that differ in the organization of host tissue and the size and shape of the symbionts. We suggest that a specific cell cycle within the symbiont-housing organ is directed along the longitudinal body axis, with a region of proliferation anteriorly and a region of degradation posteriorly. Using Raman microspectroscopy we demonstrate that the endosymbionts of S. contortum from the Gulf of Mexico contain sulfur vesicles, and we argue for a chemoautotrophic sulfur-oxidizing metabolism.     

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.     

Hypotaurine, N-methyltaurine, taurine, and glycine betaine as dominant osmolytes of vestimentiferan tubeworms from hydrothermal vents and cold seeps

Organic osmolytes, solutes that regulate cell volume, occur at high levels in marine invertebrates. These are mostly free amino acids such as taurine, which are "compatible" with cell macromolecules, and methylamines such as trimethylamine oxide, which may have a nonosmotic role as a protein stabilizer, and which is higher in many deep-sea animals. To better understand nonosmotic roles of osmolytes, we used high-performance liquid chromatography and (1)H-nuclear magnetic resonance (NMR) to analyze vestimentiferans (vestimentum tissue) from unusual marine habitats. Species from deep hydrothermal vents were Riftia pachyptila of the East Pacific Rise (2,636 m) and Ridgeia piscesae of the Juan de Fuca Ridge (2,200 m). Species from cold hydrocarbon seeps were Lamellibrachia sp. and an unnamed escarpid species from subtidal sediment seeps (540 m) off Louisiana and Lamellibrachia barhami from bathyal tectonic seeps (1,800-2,000 m) off Oregon. Riftia were dominated by hypotaurine (152 mmol/kg wet wt), an antioxidant, and an unidentified solute with an NMR spectrum consistent with a methylamine. Ridgeia were dominated by betaine (N-trimethylglycine; 109 mmol/kg), hypotaurine (64 mmol/kg), and taurine (61 mmol/kg). The escarpids were dominated by taurine (138 mmol/kg) and hypotaurine (69 mmol/kg). Both Lamellibrachia populations were dominated by N-methyltaurine (209-252 mmol/kg), not previously reported as a major osmolyte, which may be involved in methane and sulfate metabolism. Trunk and plume tissue of the Oregon Lamellibrachia were nearly identical to vestimentum in osmolyte composition. The methylamines may also stabilize proteins against pressure; they were significantly higher in the three deeper-dwelling groups.     

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.