REVIEW ARTICLE Biochemical and enzymological aspects of the symbiosis between the deep-sea tubeworm Riftia pachyptila and its bacterial endosymbiont Zoran Minic and Guy Herve ´ Laboratoire de Biochimie des Signaux Re ´ gulateurs Cellulaires et Mole ´ culaires, CNRS, Universite ´ Pierre et Marie Curie, Paris, France 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 i n an intimate symbiosis with a sulfur-oxidizing chemoauto- trophic bacterium. This bacterial endosymbiont is localized in the cells of a r ichly vascularized organ of t he worm: t he trophosome. These organisms are adapted to their extreme environment and take advantage o f t he particular compo- sition of the mixed volcanic and sea waters to extract and assimilate inorganic m etabolites, especially carbon, nitrogen, oxygen and sulfur. The high molecular mass hemoglobin of the w orm is the transporter f or both o xygen and sulfide. This last compound is delivered to the b acterium which possesses the sulfur oxidizing respiratory system, which produces the metabolic energy for the two partners. CO 2 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 w orm for its o wn metabolism. As far as n itrogen assimilation is concerned, NH 3 can be used by the two partners but nitrate c an be used only by t he bac- terium. T his very intimate symbiosis applies also to the organization of metabolic pathways such as those o f pyri- midine nucleotides and arginine. In particular, the worm lacks the first three enzymes of the de novo pyrimidine bio- synthetic 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 con stitutes a very interesting system t o stu dy the molecular and metabolic basis of biological adaptation. Keywords: deep-sea vent; Riftia pachiptila; symbiosis; assimilation; pyrimidines; arginine. Introduction It was in 1977 that geologists discovered an abundant deep- sea life community at a d epth of 2.5 km around a hot spring on the Galapagos volcanic Rift (spreading ridge) off the coast of Ecuador [1,2]. Geothermal vents are the active spreading centers along the m id-oceanic r idges, where magma erupts to form new oceanic crust. Around these vents rich biotopes developed which include microorgan- isms, huge clams and mussels, giant tube worms, crabs, fishes, etc., communities that are almost completely isolated from the rest of t he biosystems of the planet [3,4]. In the vent environment, these living o rganisms face physical and chemical obstacles, s uch as e levated pressure (up to 300 atm), high and rapidly changing temperature (from 4 °Cto 350 °C), chemical toxicity and complete absence of light [4–6]. The existence of these organisms living in extreme physical and chemical conditions raises numerous interesting questions concerning bio logical adaptation and evolution as well as the possible existence of similar environments in other worlds (Europa, Jupiter’s ice-covered moon, Mars…). The deep-sea hydrothermal vents The aptitude of living organisms to survive and constitute an important biomass around hydrothermal vents is linked to the u nique chemistry of these environments. S ea water penetrates into the fissures of the volcanic bed and interacts with the hot, newly formed rock in the volcanic crust. This heated sea water (350–450 °C) dissolves large amounts of minerals. The resulting acidic solution, containing metals (Fe, Mn, Zn, Cu …) and large amounts of reduced sulfur compounds such as sulfides and H 2 S, percolates up to the sea floor where it mixes with the cold s urrounding ocean water (4 °C) forming mineral deposits and different types of vents [4,8,9]. In the resulting temperature gradient, these minerals provide a source of energy and nutrients to chemoautotrophic organisms which are, thus, able to live in these extreme conditions [10,11]. Most of the organisms living in these environments adjust themselves to the region of the temperature gradient where t he temperature oscillates around 20 °C, due to the convection currents of hot and cold waters. The enzymatic equipments of these organisms must be adapted to these particular conditions of tempera- ture and pressure. Symbiosis In the total absence of photosynthesis in these environ- ments, the food chain relies entirely on the aptitude of some bacteria to extract energy from the oxidation of reduced mineral compounds present in the medium [9,10]. This Correspondence to G. Herve ´ or Z. Minic, Laboratoire de Biochimie des Signaux Re ´ gulateurs Cellulaires et Mole ´ culaires, UMR 7631, CNRS, Universite ´ Pierre et Marie Curie, 96 Boulevard Raspail, F-75006 Paris, France. Fax: +33 1 42 22 13 98, Tel.: +33 1 53 63 40 70, E-mail: gherve@ccr.jussieu.fr or Zoran.Minic@versailles.inra.fr (Received 5 April 2004, revised 25 May 2004, accepted 8 June 2004) Eur. J. Biochem. 271, 3093–3102 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04248.x metabolic energy is transferred to a series of animal species which live in an o bligate symbiosis with these bacteria (clams, mussels, gastropods and vestimentiferan tubeworms [1,2]. In many cases, it is from the oxidation of sulfides that bacteria extract the energy, using oxygen as the terminal electron a cceptor [12–14]. The e lectrons extracted are used for the synthesis of ATP (Fig. 1). This ATP feeds the Calvin–Benson cycle for the fixation of CO 2 for production of organic carbon metabolites which, finally, can be used in the animal’s metabolism. This process leads to the develop- ment of a very important biomass. Vestimentiferan tubeworms Abundant symbiotic organisms in venting regions are vestimentiferan tubeworms. These large animals r esemble the previously described Pogonophora. The first one t o be described in 1969 [15], was a Lamellibrachia collected by trawling, its habitat being unknown at that time. Numerous other species were later identified [16]. These tubeworms, like their pogonophoran relatives, lack a digestive tract, and rely on symbiosis with chemoautotrophic or methano- trophic bacteria [14]. A l arge amount of the vestimentiferan body is occupied by the trophosome, a s pecialized tissue whose cells con tain a large population of intracellular sulfide-oxidizing gamma proteobacteria, up to about 10 11 bacteria per gram [17]. These symbioses are v ery s pecies specific. A single type of bacterium is found in a given worm species [18]. An exception was reported in a cold seep vestimentiferan whose trophosome also contains a second bacterial species, an epsilon proteobacterium [ 19,20]. Host c ytochrome o xidase I and symbiont 16S ribosomal gene (16S) DNA sequences were used to explore evolutionary relation ships among the vestimentiferans and t heir symbionts and a d etailed phylo- genetic caracterization of the bacterial symbiont via 16S rRNA was recently reviewed by McMulalin et al.[16]. The highly specific and obligate nature o f t he symbiosis between vestimentiferans and their bacterial endosymbiont raises the question of the transmission of the bacteria from one worm gen eration to the following one. In some c ases the bacteria is present in the ovule and thus, is directly transmitted to the next generation. This process was observed in some bivalves [21]. D espite the obvious benefit of a direct transmission, no evidence supports this mode of symbiont transmission between generations in vestimenti- ferans. No bacteria have b een found in either vestimentif- eran sperm or eggs [16]. Assays for the molecular detection of bacterial DNA by PCR and in situ hybridizations in gonadal tissue and freshly released sperm and eggs have both failed [22]. Alternatively, the larvae must be reinfected at each generation. This hypothesis is consistent with the observation that vestimentiferan larvae possess a digestive tract which regresses a nd disappears during their develop- ment [23]. Riftia pachyptila Among the vestimentiferans present in the East Pacific Ridge (or Rise) Riftia pachyptila is the most abundant one [16]. This giant worm (1–2 meters long) lives in colonies and has been studied extensively since its discovery. At in situ temperatures and pressures (2 °C and 250 atm) the larvae of R. pachyptila has a lifespan of about 40 days and thus, it can colonize new vent sites to a distance of tens to hundreds kilometres [24]. In this organism the only tissue in direct contact with the surrounding water is the branchial plume which has a large highly vascularized surface, allowing an efficient exchan ge of metabolites and waste products between the e nvironment and the animal (Fig. 2 ). The other tissues are within the Riftia tube. The vestimentum is a muscle that the animal uses to position itself in the tube. Within the large sac made by the body wall and terminated by the opisthosome, is the major tissue of the worm: t he trophosome [25]. The cells of the trophosome (bacteriocytes) are densely colonized by a sulfur-oxidizing chemoautotrophic endo- symbiotic b acterium (10 9 cells per gram f resh tissue) [12,14,26]. The bacterial volume is estimate d to represent between 15 and 35% of the total volume of the trophosome [17]. This t issue includes t he coelomic fluid and it is richly vascularized. The circulatory system includes a heart-like pump located in the vestimentum region. It promotes blood circulation in the entire body including the trophosome bacteriocytes to bring various nutrients to the bacteria. The Fig. 1. The electron t ransport system and Calvin–Benson cycle in sulfide oxidizing bacteria. This figure illustrates the connection b etween the sulfide-oxidizing pathway for energy prod uctio n and the Calvin cycle. All the enzymes i ndicated in this figure were characterized in the Riftia pachyptila trophosome and/or the isolated bacterial symb iont. H 2 S and CO 2 are provided t o t he b acte rium th rough t he wo rm c ircula- tory system after absorption in the branchial plume (see text). Abbreviations: APS, adenylylphosphosulfate; RUBISCO, D -ribulose- 1,5-bisphosphate carboxylase; phosphoribulokin ase, D -ribulose-5- phosphate-1-phosphotransfe rase. 3094 Z. Minic and G. Herve ´ (Eur. J. Biochem. 271) Ó FEBS 2004 branchial p lume is the equivalent of a gill system for the exchanges with the external medium. For this purpose, it is gorged with blood which confers to this tissue its intense red colour (Fig. 2 ). All metabolite exchanges between the tubeworm and the sea water are mediated via this vascular system [27–29]. Uptake and transportation of oxygen and sulfide in Riftia pachyptila An important feature of Riftia hemoglobin is that this protein is not only the transporter of oxygen but also that of H 2 S, in order to provide it to the bacteria for energy production [30,31]. This function is exerted through t he presence of a multihemoglobin system possessing h ighly r eactive c ysteine r esidues [32]. The multihemoglobin system of R. pachyptila is composed of three different extracellular hemoglobins: two dissolved in the vascular blood, and one in the coelomic fluid. All those hemoglobins are able to bind oxygen and sulfide simultaneously and reversibly at distant sites, the heme and some reactive cysteine residues that may or may not be involved in disulfide bridges, respectively. In the case of the disulfide bridges the production of hemoglobin- persulfide groups (R-SS-H) results from the cleavage of the disulfide bond by sulfide, according to the reaction: R-SS-R+ H 2 S « RSSH + R SH [35]. Moreover, H 2 S is toxic to the living organisms that display aerobic metabolism, particularly by reacting with metalloproteins such as cytochrome c oxidase and hemoglobin [33]. The cysteine residues of Riftia hemoglobin might contribute to protect the heme from H 2 S [28,33–38]. In the bacterial symbiont, sulfide is oxidized into su lfite by an electron transport system which involves some cytochromes. The reaction of SO 3 2– with AMP is then catalyzed by adenylphosphosulfate reductase, a reaction which furnishes adenylylphosphosulfate which, in turn, is phosphorylated into ATP by ATP-sulfurylase (Fig. 1 ) [13,14,39–45]. The bacterial symbiont can use this ATP for the assimilation of carbon through the Calvin– Benson cycle (Fig. 1). A detailed description of electron transport systems of sulfur oxidizing bacteria was given by Nelson & Fischer [14]. Assimilation of carbon and nitrogen in Riftia pachyptila R. pachyptila has developed a very efficient metabolism for the assimilation of inorganic CO 2 and nitrogen from nitrate and ammonia that are provided by the external environ- ment. This metabolism relies on the obligate symbiotic relationship, which e nsures an effi cient assimilation, adap- ted to the very peculiar environment in which Riftia live s. The results obtained c oncerning these a ssimilation processes are summarized below. Assimilation of carbon CO 2 is absorbed by Riftia at the level of the branchial plume. This worm is exposed to wide variations in the environmental CO 2 concentration, from about 2–11 m M , the typical vent concentration being around 5 m M [46–48]. This absorbed CO 2 can be u sed in several ways. It appears that part of it can be transported by the circulatory systems to the b acteria-containing trophosome. The CO 2 blood concentration w as found t o be 2 0–46 m M [47]. In a ddition, 14 C pulse label experiments showed that, i n the plume, immediate carboxylation provides malate [49,50]. This malate is transported immediately to the trophosome by the blood circulation. The concentration of malate was found to be around 10 mmolÆL )1 [50]. Because carbonic anhydrase was found to be present in all the tissues of Riftia, including the plume [51–53], CO 2 canalsobe transformed into bicarbonate which can be used by several metabolic pathways (see below). In the bacteria the CO 2 which was either directly provided by the environment or which results from the d ecarboxylation of t he transported malate, enters the Calvin–Benson cycle through t he reaction catalysed by ribulose-1,5-biphosphate carboxylase and serves as precursor for different small organic metabolites (Fig. 1 ) [49]. Ribulose-1,5-biphosphate carboxylase and ribulose-5-phosphate kinase, another enzyme of t he Calvin– Benson cycle, were shown to be present in the bacterial Fig. 2. Riftia p achyptila . (Top) The worm in its deep-sea vent envi- ronment. On e can clearly see the branchial plume, which protrudes from th e tube (w ith permission of F. Zal and the Institut de Recherche et d’Exploitation des Mers, B.P. 70, Plouzane, France). (Bottom) Anatomical organization of R. pachyptila. Ó FEBS 2004 Riftia pachyptila and its bacterial endosymbiont (Eur. J. Biochem. 271) 3095 symbiont [13,44,54,55]. These small carbon metabolites can then be delivered to the different tissues of the worm for its own metabolism and ATP production. Assimilation of nitrogen The l arge biomass [ 56,57] and t he high g rowth rate [ 58] of the chemoautotrophic symbiotic organisms imply a high demand for n itrogen. This is matched b y t he high level of availability of environmental nitrate and, in some cases, ammonia [59–61]. Dissolved organic nitrogen is probably not a significant source because its availability appears to be very low. Several measurements o f dissolved organic nitrogen levels have been made at vents [62] showing that amino acid concentrations are generally less than 0.2 nmolÆL )1 around vent communities [59] and less than 0.1 nmol ÆL )1 in high- temperature fluids [63]. Thus, inorganic n itrogen must be the major source of nitrogen for vent symbioses. In living organisms, NH 3 either provided by the environ- ment or resulting f rom n itrate reduction by nitrate reductase and nitrite reductase [54,64–66] is used by a series of N H 3 assimilating enzymes such as glutamine synthetase, glutam- ate dehydrogenase and carbamylphosphate synthetase to produce basic metabolites such as amino acids and nucleo- tides (Fig. 3). Assimilation of inorganic nitrogen was d emonstrated in few of these symbiotic organisms. At hydrothermal vents, comparisons of in situ nitrate concentrations with those of a conservative tracer (silicate) indicated that nitrate is con- sumed by the vent communities [59]. 15 N tracer experiments showed that R. pachyptila assimilates nitrate whereas Solemya reidi preferentially assimilates ammonia [67]. The mechanisms by which these organisms assimilate ammonia and nitrate are not completely understood, but many of the reactions involved probably occur in the bacterial symbionts. Howeve r, some reactions of nitrogen assimilation can be mediated by the host, as glutamine synthetase and glutamate dehydrogenase are also found in the animals (Fig. 3) [64,68]. Glutamine synthetase and glutamate dehydrogenase catalyze the formation of glutamine and glutamate from ammonia, respectively. The enzymatic potentials for nitrate reduction and ammonia assimilation were examined in different tissues from Riftia. Nitrate is reduced by assimilatory enzymes present only in the bacteria [54,64–66]. The ammonia assimilation enzymes glutamine synthetase and glutamate dehydrogenase were d etected in both the host t issues and the symbiont [64]. Distinct forms of host and symbi- ont glutamine synthetase are present in R. pachy ptila [64,66]. The concentration of nitrate in the deep-sea vent environment is about 40 l M [59] and R. pachyptila absorbs it at a rate of 3.54 lmolÆg )1 Æh )1 . In the vent fluid, ammonia can be present at concentrations up to the l M range [60]. In the vicinity of Riftia the concentrations of nitrate and ammonia w ere found to be 18.3–37 and 0.1–2.7 lmolÆL )1 , respectively [69]. No correlation was found between nitrate uptake and inorganic carbon or sulfide fluxes. It seems now, that the product of symbiont nitrate reduction, ammonia, is probably the primar y sou rce of n itrogen f or the host and the symbiont [65]. NH 3 can a lso be assimilated via the biosynthetic p yrim- idine and arginine pathways (Fig. 3). The first step of the pyrimidine and arginine metabolic pathways involves the uptake and utilization of the inorganic compounds NH 3 and CO 2 which, in Riftia, are provided by the environment. Therefore, an examination of the metabolic aspects of t he symbiosis between Riftia and the bacteria was initiated by a study of these particular metabolic pathways. Pyrimidine metabolism All living organisms rely on two metabolic pathways for the production of pyrimidine nucleotides. The de novo pathway allows the complete synthesis of these nucleotides including the synthesis of the pyrimidine ring starting with bicarbon- ate, glutamine and ATP. Thus, the first reaction, catalyzed by carbamylphosphate synthase is involved in the assimil- ation of carbon and nitrogen. The salvage pathway ensures the production of these nucleotides from the pyrimidine nucleosides and nucleotide monophosphates provided by the intracellular degradation of nucleic acids. Distribution of the enzymes of the de novo pyrimidine nucleotide pathway in the different parts of Riftia pachyptila The distribution of the subsequent enzymes of the pyrim- idine de novo pathway in different parts of the worm was examined [66]. I nterestingly, i t appeared that t he first three Fig. 3. Pathways of inorganic nitro gen a ssimilation in Riftia pachyptila. NH 3 , e ithertaken up direc tly from the environmen t or resulting from the reduction of nitrate, can be assimilated in the pathways shown, which begin by action of glutamine syntheta se (GSase), glutamate dehydro- genase (GDHase) and carbamylphosphate synthetase (CPSase), respectively, to provide the b asic organic metabolites indicated. 3096 Z. Minic and G. Herve ´ (Eur. J. Biochem. 271) Ó FEBS 2004 enzymes of this pathway, carbamylphosphate synthetase, aspartate transcarbamylase and dihydroorotase are present only in the trophosome, the symbiont-harbouring tissue. In contrast, the next two enzymes dihydroorotate dehydro- genase and orotatephosphoribosyl transferase as well as the last enzyme of the pathway, CTP synthase, are present in all the organs o f the animal and the bacterium. The fact that the fi rst three enzymes are present only in the t rophosome raised the question of whether these enzymes belong to the bacterium. Therefore, the same enzymatic determinations were made on extracts from the bacterium isolated on b oard the ship, immediately after collection of the animals. In the bacterial extract all the enzyme activities of the de novo pathway were detected [66]. The presence of these enzymes, their catalytic and r egulatory properties, as well as t he fact that they are not organized into a m ultifunctional p rotein confirmed their bacterial origin [66]. Thus, in contrast to the worm, the bacterium possesses all the enzymatic equipment for the de novo pyrimidine biosynthesis. Distribution of the enzymes of the salvage pathways in the different parts of Riftia pachyptila Because the worm is unable to synthesize the pyrimidine nucleotides through the de novo p athw ay, it m ust re ly o n th e salvage pathway . Indeed, e nzymes of this pathway such as cytidine deaminase, uridine kinase a nd uracilphosphoribo- syl transferase are present in all the tissues of the worm [71]. Unexpectedly, the isolated bacterium does not exhibit a ny activity of the enzymes of this salvage pathway. Comple- mentary biochemical and kinetic analyses were performed in order to obtain information about the origin of the enzymes of the salvage pathways in the trophosome. The r esults obtained showed that these e nzymes belong to the host [71]. Distribution of the enzymes of pyrimidine catabolism in the different tissues of Riftia pachyptila Instead o f being used in the salvage pathway these nucleic acid degradation products (nucleotide monophosphates and nucleosides) can be further degraded by enzymes of catabolic pathways which liberate CO 2 and NH 3 [70]. Consequently, these products of degradation of pyrimidine nucleotides can represent a possible source of carbon and nitrogen for the organism. The analysis of t he distribution of 5¢-nucleotidase, uracil reductase and uridine phosphorylase, enzymes responsible for the catabolism of pyrimidine nucleotides, showed that they are p resent in all the tissues o f the worm. Unexpectedly, the isolated bacterium does not exhibit any activity of these enzymes, a result which was confirmed by complementary biochemical and kinetic determinations [71]. Pyrimidine metabolism and symbiosis Figure 4 assembles th e r esults reported above a nd empha- sizes the multiple metabolic exchanges involved in the symbiosis between the worm and the bacterium. This bacterium possesses the enzymatic equipment for the biosynthesis of pyrimidine nucleotides t hrough the de novo pathway, but lacks the enzymes of the salvage and catabolic pathways [66,71,72]. In contrast, the host cells (including the bacteriocytes) possess the enzymes catalyzing the final steps of the de novo pathway as well as the enzymatic equipment for the salvage pathway allowing the synthesis of pyrimi- dines from nucleic acid degradation p roducts. A s the host cells do not have the first three enzymes of the de novo Fig. 4. Integrated scheme of the metabolic pathways of pyrimidine nucleotides in R. pachyptila and its bacterial endosymbiont. The first three enzymes o f the de novo pyrimidine b iosynthetic pathway are present only in the bacterium synthesizing dihydroorotate, which can be provid ed to the worm bact eriocytes and to its other t issues throu g h the circulatory system. The first reaction catalysed by t he glutamine dependent carbamylpho sphate synthetase uses glutamine provided by glutamine synthetase, whose substrate NH 3 is either directly furnished by the e xtern al m edium o r derives from th e r eduction o f nitrate by the bacterial nitrate reductase. Both the worm and its bacterium possess the f ollowing en zymes o f the pathway (dihydroorotate de hydrogenase, etc) for the production of the pyrimidine n ucleot ide triphosphates. T he salvage pathway is present only i n the worm tissues . These tissues a lso contain the enzymes of pyrimidine catabolism which can provide carbonandnitrogentotheworm.Alltheenzymesindicatedinthe figurewerecharacterizedintheworm and/or its bacterial symbiont. The scheme describes the e xch anges between the endosymbiont, the trophosomal host cells, and th e cells of o ther host t issues. Question marks indicate steps that have not been completely elucidated. Thin arrows refer to metabolic pathways . T hick arrows refer t o transport of metabolites in compartments, tissues or body parts. Abbreviations: CPSase-P, carbamylphosphate synthetase specific to the pyrimidine biosynthetic pathway; ATCase, a spartate transcarbamylase; DHOase, dihydroorotase; GSase, glutamine synthetase. Ó FEBS 2004 Riftia pachyptila and its bacterial endosymbiont (Eur. J. Biochem. 271) 3097 pathway (carbamylphosphate synthetase, aspartate transcarbamylase and dihydroorotase), the necessary meta- bolic precursors, orotate and/or dihydroorotate, must be provided by the b acterium. Thus, R. pachyptila is absolute ly dependent on the symbiotic bacterium for the de novo biosynthesis of the pyrimidine nucleotides. The results obtained show also t hat R. p achyptila pos- sesses the activities of at least three enzymes participating in the catabolism of pyrimidine nucleotides, 5¢-nucleotidase, uridine phosphorylase and uracil reductase, in all its tissues. Notably, these enzymes do not exist in the bacterial endosymbiont. Catabolism of pyrimidine nucleotides leads to the production of CO 2 ,NH 3 , malonyl-CoA and succinyl- CoA; subsequently malonyl-CoA can be used for the biosynthesis of fatty acids while succinyl-CoA e nters i nto the citric a cid cycle [70]. I n this manner t he degradation of pyrimidine nucleotides can represent an alternative nutri- tional source of nitrogen and carbon, besides t he external environment of the worm, and can also feed other biosyn- thetic pathways. This degradation can also r esult from the reported bacterial lysis in the trophosome [73]. A study of the localization o f t hese anabolic and catabolic enzymes in t he trophosome shows that they are not homogenously distributed. The level of anabolic activities decreases from the centre of the trophosome to its periphery, an d the level of catabolic activities varies in the opposite direction. This observation s uggests some kind of structural and physiological organization of this tissue [71]. Arginine metabolism The arginine metabolic pathway is also initiated by a carbamylphosphate synthetase . In eukaryotes this reaction is catalyzed by a specific carbamylphosphate synthetase, distinct from that of the pyrimidine pathway, using NH 3 as substrate instead of glutamine. Thus, this metabolic path- way is also involved in the assimilation of carbon and nitrogen. Distribution of the enzymes of the arginine biosynthetic pathway in the different parts of Riftia pachyptila Concerning the a rginine biosynthetic p athway, it appeared that the ammonium dependent carbamylphosphate synthe- tase, the ornithine transcarbamylase and the argininosucci- nate synthetase are present in all the body parts of R. pachyptila as well as in the bacterial symbiont (Fig. 5) [74]. Lack of arginine catabolism via the catabolic ornithine transcarbamylase of the arginine deiminase pathway in Riftia pachyptila There are two types of ornithin e transcarbamylases, w hich participate in either the anabolism, or the catabolism of arginine. The anabolic ornithine transcarbamylase b elongs to the biosynthetic arginine pathway and catalyses citrulline formation from ornithine [75]. A number of prokaryotes also possess a catabolic ornithine transcarbamylase, which belongs to the arginine deiminase c atabolic pathway leading to the anaerobic degradation of arginine to produce NH 3 , CO 2 and ATP [75–78]. In this pathway, ornithine trans- carbamylase catalyzes the transformation of citrulline to ornithine. In view of the limiting supply of NH 3 and CO 2 to Riftia from its environment [46–48,59,60], this arginine catabolic pathway could constitute an interesting source of these inorganic metabolites. The kinetic propertie s of t he ornithine t ranscarbamylase found in Riftia strongly suggest that neither the worm n or the bacterium possess the catabolic form of this enzyme belonging to the arginine deiminase pathway. This conclu- sion was c onfirmed by the lack of arginine deiminase i n both the worm and the bacterium [74]. Arginine catabolism via the arginine and ornithine decarboxylases Although R. pa chyptila and its endosymbiont appear not to possess the enzymes of the arginine deiminase pathway, there exist several other routes for the catabolism of this amino acid. Among them, arginine decarboxylase and ornithine decarboxylase can play an important role leading to the synthesis of putrescine, precursor of polyamines. Besides t heir important physiological role, polyamines can Fig. 5. Arginine metabolism in R. pachyptila. Both the worm and the bacterium possess the enzymes for the biosynthesis of arginine. In the worm (including the bacteriocytes) t his biosynthesis involves ammo- nium dependent carbamylphosphate synthetase (CPSase- A) specific for this pathway. In the bacterium, a unique carbamylphosphate synthetase provides this metabolite for b oth the pyrimydine and the arginine pathways. The worm CPSase-A uses NH 3 and HCO 3 – pro- vided by t he external medium. Arginase a nd ur ease i nvolved in the catabolism of arginine are present in both organisms. The arginine deiminase pathway is absent. Two en zyme s o f the p olyam ines b io- synthetic pathway, ornitine decarboxylase and arginine decarboxylase are present only in the bacteria. The question marks indicate enzymes whose existence in Riftia is still hypothetical. Abbreviations: CPSase- A,carbamylphosphatesynthetasespecifictotheargininebiosynthetic pathway; ADase, arginine decarboxylase; ADIase, a rginine deiminase; ASSase, argininoguccinate synthetase; ODase, ornithine decarboxy- lase; OTCase, ornithine transcarbamylase. 3098 Z. Minic and G. Herve ´ (Eur. J. Biochem. 271) Ó FEBS 2004 be degraded and constitute an alternative source of inorganic carbon and nitrogen [79,80]. Consequently, the existence and distribution of arginine decarboxylase and ornithine decarboxylase were investigated i n Riftia and i ts bacterial endosymbiont. Interestingly, it appeared that arginine decarboxylase and ornithine decarboxylase are present only in the trophosome, the symbiont-harbouring tissue and in the isolated bacterium. The specific activities of these e nzymes are higher in the isolated bacterium than in the bacterium-containing trophosome, indicating that these enzymes are present only in t he bacterium [74]. Arginine metabolism and symbiosis Figure 5 assembles the results obtained concerning the metabolism of arginine in Riftia. The first three enzymes involved in th e arginine b iosynthetic pathway (am monium dependent carbamylphosphate synthetase, ornithine trans- carbamylase, argininosuccinate synthetase) are present in both t he host and the bacterium. The ammonium dependent carbamylphosphate synthetase that uses ATP to catalyze the conversion of the inorganic molecules HCO 3 – and NH 3 into carbamylphosphate, initiates the biosynthesis. The existence of the enzymatic equipment for this biosynthesis in all the tissues of Riftia indicates that these tissues might assimilate inorganic nitrogen and carbon through this process. It also suggests that arginine is a nonessential aminoacidforRiftia. In this way, although the symbiont is the obligatory primary site of carbon and nitrogen fixation the host tissues participate to this process [13,14]. The unusual presence of the enzymes of t his pathway in all the tissues of R. pachyptila might contribute to i ts adaptation to the extreme environment of the hydrothermal vent. Arginase and urease are also present i n all the tissues of Riftia, including the trophosome [81]. Accordingly, one observes high c oncentrations of ornithine and urea and a low concentration of arginine in this tissue [81]. Arginine can also be catabolized through the arginine succinyl pathway, which leads to the production of NH 3 ,CO 2 , glutamate and succinate. This last metabolite t hen enters t he citric acid cycle [82]. The presence of argininosuccinate synthetase in all the tissues of Riftia raises the possibility that this catabolic pathway is operative in the worm (Fig. 5 ). A basic metabolic utilization of arginine and of its derivate ornithine is t he synthesis of polyamines t hrough t he production of agmatine and putrescine by arginine decarb- oxylase and ornit hine decarboxylase. I n all living organisms, including vir uses, polyamines play key roles in the biosyn- thesis and structure of nucleic acids and are reported to b e involved in many biological processes such as membrane stability, growth and develo pment [83]. In R. pachyptila it appears that arginine decarboxylase is present only in the bacterial endosymbiont (Fig. 5). The absence of these enzymes, which i nitiate the biosynthesis of polyamines in the host t issues, strongly suggests that Riftia is depen dent on the bacterium for this pathway. The bacterial production of agmatine and putrescine in the trophosome would be followed by transportation of these compounds to the other tissues of the worm. Agmatine, putrescine and polyamine transport systems were described in many organisms [84–86]. Furthermore, t he degradation of these polyamines can also provide an additional source of carbon and nitrogen for the worm [87,88]. Conclusion The symbiosis between Riftia pachyptila and its chemo- autotrophic bacterial endosymbiont relies on a very particular metabolic organization and a nutritional strat- egy involving numerous interactions and metabolic exchanges. This association is especially aimed at the assimilation of the mineral metabolites present in the environment. This is true especially for sulfide which is used by the bacterium for the production of metabolic energy for the two partners. These exchanges are also involved in the assimilation of carbon, nitrogen and oxygen. In addition, they extend to the organization of entire metabolic pathways such as those of pyrimidine, arginine and probably polyamines. As reported above, the worm does not possess any arginine decar boxylase or o rnithine decarboxylase a ctivity. This absence h as also been reported in t he case of human and animal filarial worm parasites Dirofilaria immitis, Brugia patei and Litomosoides [89]. In a similar way, in Riftia the first three enzymes of the pyrimidine nucle otide biosynthetic pathway are present only in the bacterium but not in the w orm [74]. The absence of these enzymes is also characteristic of protozoan parasites s uch as Gia rdia lamb- lia, Trichomonas vaginalis and Tritrichomonas foetus. Thus, it appears t hat Riftia has developed a metabolism for the biosynthesis of pyrimidines and polyamines which is reminiscent of w hat is observed i n some parasites, s uggest- ing some similarity in the adaptation of metabolic pathways in symbiosis and parasitism. This complex metabolic organization is the b asis of the adaptation of Riftia pachyptila to the extreme hydrothermal vent environment a nd to the a bsence of a r eadily available source of organic carbon through photosynthesis. Acknowledgements This work was supported by the Centre N ational de la Recherche Scientifique and by the Universite ´ Pierre et Marie Curie, Paris. References 1. Lonsdale, P. (1977) C lustering of s uspensio n-feeding macroben- thos near abyssal hydrothermal vents at oceanic spreading ce nte rs. Deep Sea Res. 24, 857–863. 2. Corliss, J.B., Dymond, J., Gordon, L.I., Edmond, J.M., Herzen, R.P.V., Ballard, R .D., Green, K., W illiams, D., Bai nbridge, A., Crane, K. & Andel, T.H. (1979) Submarine thermal s prings on the Galapagos Rift. Science 203, 1073–1083. 3. MacDonald, I.R., Boland, G.S., Baker, J.S., Brooks, J.M., Kennicutt, M.C.I.I. & Bidigare, R.R. (1989) Gulf of Mexico hydrocarbon seep communities. II. Spatial distribution of seep organisms and hydro carbons at Bush Hill. Mar. Biol. 101, 235–247. 4. MacDonald, I.R., Guinasso, N.L., Reilly, J.F., Brooks, J.M., Callender, W.R. & Gabrielle, S.G. (1990) Gulf of Mexico hydrocarbon seep communities. IV. Patterns in community structure and habitat. Geo-Mar. Lett. 10, 244–252. 5. Tunnicliffe, V. (1991) The b iology of hydrothermal vents: ecology and evolution. Oceanogr. Mar. Biol. Annu. Rev. 29, 319–407. Ó FEBS 2004 Riftia pachyptila and its bacterial endosymbiont (Eur. J. Biochem. 271) 3099 6. Blum, J. & Fridovich, I. (1984) Enzymatic defenses against oxygen toxicity in the hydrothermal v ent animals Riftia pachyptila and Calyptogena magnifica. Arch. Biochem. Biophys. 228, 617–620. 7. Sicot, F.X., Mesnage, M., Masselot, M., Exposito, J.Y., Garrone, R., Deutsch, J. & Gaill, F. (2000) Molecular a daptation to an extreme environment: origin of the thermal stability of the pompeii worm collagen. J. Mol. Biol. 302, 811–820. 8. Tunnicliffe, V., M cArthur, A. & McHugh, D. (1998) A biogeo- graphical perspective of the deep-sea hydrothermal vent fauna. Adv. Mar. Biol. 34, 353–442. 9. Zierenberg, R.A., Adams, M.W. & Arp, A.J. (2000) Life in extreme environments: hydrothermal vents. Proc. Natl Acad. Sci. USA 97, 12961–12962. 10. Fisher, C.R. (1996) Chemoautotrophic and methanotrophic symbioses in marine inverterbrates. Aquat. Sci. 2, 399–436. 11. Arndt, C., Gaill, F. & Felbeck, H. (2001) Anaerobic sulfur metabolism in thiotrophic symbioses. J. Exp. Biol. 204, 741–750. 12. Felbeck, H. (1981) Chemoautotrophic pote ntial of the hyd ro- thermal vent tube worm Riftia pachyptila Jones ( Venstimentifera). Science 213, 336–338. 13. Felbeck, H., C hildress, J.J. & Somero, G .N. (1981) Cal vin–Benson cycle and sulfide oxidation enzymes in animals from sulfide rich environment habitats. Nature 293, 291–293. 14.Nelson,D.C.&Fisher,C.R.(1995)Chemoautotrophicand methanotrophic endosymbiotic bacteria at deep-sea vents and seeps. In Microbiology of Deep Sea Hydrothermal Vents (K arl, D.M., ed.), pp. 125–167. CRC Press Inc., Boca Raton, FL. 15. Webb, M. (1969) Lamellibrachia barhami, General nov., sp. nov. (Pogonophora), from the northeast Pacific. Bull. Mar. Sci. 19, 18–47. 16.McMullin,E.R.,Hourdez,S.,Schaeffer,S.W.&Fisher,C.R. (2003) Phylogeny a nd biogeography of deep sea vestimentiferan tubeworms and their bacte rial symbiont s. Symbiosis 34, 1–41. 17. Powel, M.A. & Somero, G.N. (1986) Adaptations to sulfide by hydrothermal vent animals: sites and mechanisms of detoxification and metabolism. Biol. Bull. 1971, 274–290. 18. Distel, D.L., Lane, D.J., Olsen, G.J., Giovannoni, S.J., Pace, B., Pace, N.R., Stahl, D.A. & Felbeck, H. (1988) Sulfur-oxidizing bacterial endosymbionts: analysis of phylogeny and specificity by 16S rRNA sequences. J. Bacteriol. 170, 2506–2510. 19. Naganuma, T., Ka to, C., Hirayama, H., Moriyama, N., Hashi- moto, J. & Horikoshi, K. (1997) Intracellular occurrence of e-proteobacterial 16S rDNA sequences in the vestimentiferan trophosome. J. Oceanogr. 53, 193–197. 20. Naganuma, T., Naka, J., Okayama, Y., Minami, A. & Horikoshi, K. (1997) Morphological diversity of the microbial population in a vestimentiferan tubeworm. J. Mar. Biotechnol. 5, 119–123. 21. Peek, A., Gustafson, R., Lutz, R. & Vrijenhoek, R. (1997) Evo- lutionary relationships of deep sea h ydro thermal vent and cold- water seep clams (Bivalvia: Vesicomyidae): Results from the mitochondrial cytochrome oxidase subunit I. Mar. Biol. 130, 151–161. 22. Carry, S.C., Warren, W., A nderson, E. & Giovannoni, S.J. (1993) Identification and localization of bacterial endosymbionts in hydrothermal vent taxa with symbion s pecific polymerase chain reaction amplification and in situ hybridization techniques. Mol. Mar. Biol. Biotechnol. 2, 51–62. 23. Jones, M.L. & Gardiner, S.L. (1988) Evidence for a transient digestive tract in vestimentifera. Proc. Biol. Soc. Wash. 101, 423– 433. 24. Marsh, A.G., Mullineaux, L.S., Young, C.M. & Manahan, D.T. (2001) Larval dispe rsal potential of th e tubeworm Riftia pachyptila at deep-sea hydrothermal vents. Nature 411, 77–80. 25. Gaill, F. (1993) Aspects o f life development at deep sea hydro- thermal vents. FASEB J. 7, 558–565. 26. Hand, S.C. (1987) Trophosome ultrastructure and the character- ization of isolated bacteriocytes from invertebrate-sulfur bacteria symbioses. Biol. Bull. 173, 260–276. 27. Zal, F., Lallier, F.H., Green, B.N., Vinogradov, S.N. & Toul- mond, A. (1996) The multi-hemoglobin system of the hydro- thermal vent tube worm Riftia pachyptila.II.Complete polypeptide chain c omposition investigated b y maxim um ent ropy analysis of mass spectra. J. Biol. Chem. 271, 8875–8881. 28. Zal, F., Lallier, F.H., Wall, J.S., Vinogradov, S.N. & Toulmond, A. (1996) The multi-hemoglobin system of the hydrothermal ve nt tu be wo rm Riftia pachyptila.I.Reexamination of the number and masses of its constituents. J. Biol. Chem. 271, 8869–8874. 29. Fisher, C .R., Childress, J .J. & Sanders, N. K. (1988) The role of vestimentiferan hemoglobin in providing an environment suitable for chemoautotrophic sulfide-oxidizing endosymbionts. Symbiosis 5, 229–246. 30. Arp, A.J., Doyle, M.L., Di Cera, E . & Gill, S.J. (1990) Oxyge- nation properties of t he two co-occurring hemoglobins of the t ube worm Riftia pachyptila. Respir. Physiol. 80, 323–334. 31. Goffredi, S.K., C hildress, J.J., Desaulniers, N.T. & Lallier, F.J. (1997) Sulfide acquisition by the vent worm Riftia pachyptila appears to be via uptake of HS – , rather than H 2 S. J. Exp. Biol. 200, 2609–2616. 32. Weber, R.E. & Vinogradov, S.N. (2001) Nonvertebrate Hemo- globins: Functions and Molecular Adaptations. Physiol. Rev. 81, 569–628. 33. Nicholls, P. (1975) The effect of sulphide on cytochrome aa3. Isosteric and allosteric shifts of the reduced alpha-peak. Biochim. Biophys. Acta 396, 24–35. 34. Suzuki, T ., Takagi, T. & Ohta, S. (1990) Prim ary structure of a constituent polypeptide chain (AIII) of the giant haemoglob in from the deep-sea tube worm Lamellibrachia. A possible H 2 S- binding site. Biochem. J. 266, 221–225. 35. Zal,F.,Leize,E.,Lallier,F.H.,Toulmond,A.,VanDorsselaer,A. & Childress, J.J. (1998) S-Sulfohemoglobin and disulfide exchange: the mechanisms of sulfide binding by Riftia pachyptila hemoglobins. Proc.NatlAcad.Sci.USA95, 8997–9002. 36. Bailly, X., Jollivet, D., Vanin, S., Deutsch, J., Zal, F., Lallier, F. & Toulmond, A. (2002) Evolution of the sulfide-binding function within the globin multigenic family of the deep-sea hydrothermal vent tub eworm Riftia pachyptila. Mol. Biol. Evol. 19, 1421–1433. 37. Zal, F., Suzuki, T., Kawasaki, Y., Childress, J.J., Lallier, F.H. & Toulmond, A. (1997) Primary structure of the common polypep- tide chain b from the m ulti-hemo globin system of the h ydro- thermal vent t ube w orm Ri ftia pachyptila: an insigh t on the s ulfid e binding-site. Proteins 29, 562–574. 38. Bailly, X., Leroy, R., Carney, S., Collin, O., Zal, F., Toulmond, A. & Jollivet, D. (2003) The loss of the hemoglobin H 2 S-binding function in annelids f rom sulfide-free habitats reveals molecular adaptation driven by Darwinian positive selection. Proc. Natl Acad. Sci. USA 100, 5885–5890. 39. Renosto,F.,Martin,R.L.,Borrell,J.L.,Nelson,D.C.&Segel, I.H. (1991) ATP sulfurylase from trophosome tissue of Riftia pachyptila (hydrothermal vent tube w orm). Arch. B iochem. Bio- phys. 290, 66–78. 40. Smith, D.W. & Strohl, W.R. (1991) Sulfur-oxidizing bacteria. In Variations in Autotrophic L ife (Shively, J .M. & Barton, L .L., eds), pp. 121–146, Academic Press, San Diego, CA. 41. Rau, G.H. (1981) Hydrothermal vent clam and tubeworm 13 C/ 12 C. Further evidence of nonphotosynthetic food sources. Science 213, 338–340. 42. Beynon, J.D., MacRae, I.J., Huston, S.L., Nelson, D.C., Segel, I.H. & Fisher, A.J. (2001) Crystal structure of ATP sulfurylase from the bacterial symbiont of the hydrothermal 3100 Z. Minic and G. Herve ´ (Eur. J. Biochem. 271) Ó FEBS 2004 vent tubeworm Riftia pachyptila. Biochemistry 40, 14509– 14517. 43. Laue, B.E. & Nelson, D.C. (1994) Characterization of the gene encoding the autotrophic ATP sulfurylase from the bacterial endosymbiont of the hydrothermal vent tubeworm Riftia pachyptila. J. Bacteriol. 176, 3723–3729. 44. Robinson, J.J., Stein, J.L. & Cavanaugh, C.M. (1998) Cloning and sequencing of a form II ribulose-1,5-biphosphate carboxylase/ oxygenase from the bacterial symbiont of the hydrothermal vent tubeworm Riftia pachyptila. J. Bacteriol. 180, 1596–1599. 45. Williams, C.A., Nelson, D.C., Farah, B.A., Jannasch, H.W. & Shively, J.M. (1988) Ribulose bisphosphate carboxylase of the prokaryotic symbiont o f a hydro therm al vent tu be worm: k inetics, activity and gene hybridization. FEMS Microbiol. Lett. 50,107– 112. 46. Childress, J.J. & Fis her, C.R. ( 1992) The b iology of hydrothermal vent animals: physiology, biochemistry and autotrophic symbio- sis. Oceanogr. Mar. Biol. Annu. Rev. 30, 337–441. 47. Childress, J .J., Lee, R.W., S anders, N.K., Felbeck, H ., Oros, D.R., Toulmond, A., Desbruyeres, A., Kennicutt, M.C. & Brooks, J. (1993) Inorganic carbon uptake in hydrothermal vent tubeworms facilitated by high environmental pC02. Nature 362, 147–149. 48.Scott,K.M.,Fisher,C.R.,Vodenichar,J.S.,Nix,E.R.& Minnich, E. (1994) Inorganic carbon and te mperat ure require- ments for autotrophic carbon fi xation by t he chemoautotro phic symbionts of the giant hydrothermal vent tube worm, Riftia pachyptila. Physiol. Zool. 67, 617–638. 49. Felbeck, H. & Jarchow, J. (1998) Carbon re lease f rom p urified chemoautotrophic bacterial symbionts of the h ydroth ermal vent tubeworm Riftia pachyptila. Physiol. Zool. 71, 294–302. 50. Felbeck, H. (198 5) CO 2 fixation in the hydrothermal ve nt tube worm Riftia pachyptila (Jones). Physiol. Zool. 58, 272–281. 51. Kochevar, R.E., G ovind, N.S. & Childress, J.J. (1993) Identifi- cation and characterization of two carbonic anhydrases from the hydrothermal vent tube-worm Riftia pachyptila Jones. Mol. Mar. Biol. Biotechnol. 2, 10–19. 52. De Cian, M.C., Bail ly, X., Morales, J., Strub, J .M., Van D ors- selaer, A. & Lallier, F .H. (2003) Characterization of carbonic anhydrases from Riftia pachyptila, a symbiotic invertebrate from deep-sea hydrothermal vents. Proteins 51, 327–339. 53. De Cian, M.C., Andersen, A.C., Bailly, X. & Lallier, F.H. (2003) Expression and localization of carbonic anhydrase and ATPases in the symbiotic tubeworm Riftia pachyptila. J. Exp. Biol. 206, 399–409. 54. Hentschel, U. & Felbeck, H. (1993) Nitrate respiration in the hydrothermal vent tubeworm Riftia pachyptila. Nature 366,338– 340. 55. Cavanaugh,C.M.,Gardiner,S.L.,Jones,M.L.,Jannasch,H.W.& Waterbury, J.B. (1981) Procaryotic cells in the hydrothermal vent tube worm Riftia pachyptila Jones: Possible chemoautotrophic symbionts. Science 213, 340. 56. Nix, E.R., Fisher, C.R., Vodenichar, J. & Scott, K.M. (1995) Physiological ecology of a mussel with m etha notroph ic endosymbionts at three hydrocarbon seep sites in the Gulf of Mexico. Mar. Biol. 122, 605–617. 57. Lutz, R .A. & K ennish, M.J. (1993) E cology of deep-sea hydro- thermal vent communities: a review. Rev. Geophys. 31, 211–242. 58. Lutz, R .A., Shank, T.M., Fornari, D .J., Haymon, R .M., Lilley, M.D., Von Damm, K .L. & Desbruy eres, D. (199 4) Rapid g rowth at deep-sea vents. Nature 371, 663–664. 59. Johnson, K .S., Childress, J.J., Hessler, R.R., Sakamoto-Arnold, C.M. & Beehler, C.L. (1988) Chemical and b iological interactions in the R ose Garden hydrothermal vent fi eld . Deep-Sea Res. 35, 1723–1744. 60. Lilley, M.D., Butterfield, D.A., Olson, E.J., Lupton, J.E., Macko, S.A. & McDuff, R.E. (1993) Anomalous CH 4 and NH4 + concentrations at an unsedimented mid-ocean-ridge hydrothermal system. Nature 364, 45–47. 61. Conway, N.M., Howes, B.L., McDowellcapuzzo, J.E., Turner, R.D. & Cavanaugh, C.M. (1992) Characterization and site description of Solemya borealis (Bivalvia; Solemyidae), another bivalve-bacteria symbiosis. Mar. Biol. 112, 601–613. 62. Karl, D.M. (1995) Ecology of free-living, hydrothermal vent microbial communities. In The Microbiology of Deep-Sea Hydrothermal Vents (Karl, D.M., ed.), pp. 35–124. CRC P ress, Boca Raton, FL. 63. Haberstroh, P.R. & Karl, D.M. (1989) D issolved free a mino a cids in hydro the rmal vent habitats of the Guaymas Basin. Geochim. Cosmochim. Acta 53, 2937–2945. 64. Lee, R.W., Robinson, J.J. & Cavanaugh, C.M. (1999) Pathways of inorganic nitrogen assimilation in chemoautotrophic bacteria– marine invertebrate symbioses: expre ssio n of host and symbiont glutamine synthetase. J. Exp. Biol. 202, 289–300. 65. Girguis, P.R., Lee, R.W., Desaulniers, N., Childress, J.J., Pospe- sel, M., Felbeck, H. & Zal, F. (2000) Fate of nitrate acquired by the tubeworm Riftia pachyptila. Appl. Environ. Microbiol. 66, 2783–2790. 66. Minic, Z., Simon, V., Penverne, B., Gaill, F. & Herve, G. (2001) Contribution of the bacterial endosymbiont to the biosynthesis of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyp- tila. J. Biol. Chem. 276, 23777–23784. 67. Lee, R.W. & Childress, J.J. (1994) Assimilation of inorganic nitrogen by c hemoautotro phic and methanotrophic symbioses. Appl. Env. Microbiol. 60, 1852–1858. 68. Bender, D.A. (1985) AminoAcidMetabolism. Wiley, Chichester, UK. 69. Lee, R.W. & Childress, J.J. (1996) Inorganic N assimilation and ammonium pools i n a de ep -sea mussel c on taining methanotrophic endosymb ion ts. Biol. Bull. 190, 367–372. 70. Borel, J.P ., Maquart, F.X., Le Pe uch, C., R andoux, A., Gillery, P ., Bellon,G.&Monboisse,J.C.(1997)Biochimie Dynamique, pp. 773–795. De Boeck Universite ´ , Paris, Bruxelles. 71. Minic,Z.,Pastra-Landis,S.,Gaill,F.&Herve ´ , G. (2002) Cata- bolism of pyrimidine nucleo tides i n t he deep-sea tube worm Riftia pachyptila. J. Biol. Chem. 277, 127–134. 72. Simon, V., Purcarea, C., Sun, K., Joseph, J., Frebourg, G., Lechaire, J.P., Gail , F. & Herve ´ , G . (2000) The enzyme involved i n synthesis and utilization of carbamylphosphate in the d eep-sea tube worm Riftia pachyptila. Mar. Biol. 136, 115–127. 73. Bright, M., Keckeis, H. & Fisher, C.R. (2000) An auto- radiographic examination o f c ar bon fi xation, t ransfer and utili- zation in th e Riftia pachyptila symbiosis. Mar. Biol. 136, 621– 632. 74. Minic, Z. & Herve ´ , G . ( 2003) Arginine metabolism i n the deep sea tube worm Riftia pachyptila and its bac terial endosymbiont. J. Biol. Chem. 278, 40527–40533. 75. Cunin, R. , Glansdorff, N., Pierard, A. & Stalon, V. (1986) Bi o- synthesis and metabolism of arginine in bacteria. Microbiol. Rev. 50, 314–352. 76. Weickmann, J.L. & Fahrney, D.E. (1977) Arginine deiminase from Mycoplasma arthritidis. E vidence for multiple forms. J. Biol. Chem. 252, 2615–2620. 77. Stalon, V., Ramos, F., Pierard, A. & Wiame, J.M. (1967) The occurrence o f a catabo lic and an anabo lic ornithine c arba- moyltransferase in Pseudomonas. Biochim. Biophys. Acta 139, 91–97. 78. Vander Wauven, C., Pierard, A., Kley-Raymann, M. & Haas, D. (1984) Pseudomo nas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J. Bacteriol. 160, 928–934. 79. Stalon, V., Vander Wauven, C., Momin, P. & L egrain, C. (1987) Catabolism of arginine, citrulline and ornithine by Ó FEBS 2004 Riftia pachyptila and its bacterial endosymbiont (Eur. J. Biochem. 271) 3101 Pseudomonas and related bacteria. J. Gen. Microbiol. 133, 2487– 2495. 80. Tabor, C.W. & Tabor, H. (1985) Polyamines in microorganisms. Microbiol. Rev. 49, 81–99. 81. De Cian, M., Regnault, M. & Lallier, F.H. (2000) Nitrogen metabolites and related enzymatic activities in the body fluids and tissues of the hydrothermal vent tubeworm Riftia pachyptila. J. Exp. Biol. 203, 2907–2920. 82. Schneider, B.L., Kiupakis, A.K. & Reitzer, L.J. ( 1998) Arginine catabolism and the arginine succinyltransferase pathway in Escherichia coli. J. Bacteriol. 180, 4278–4286. 83. Cohen, S. (1988) A Guide to the P olyamines. Oxford University Press, Oxford, UK. 84. Kandpal, M. & Tekwani, B.L. ( 1997) Polyamine t ransport systems of Leishmania donovani promastigotes. Life Sci. 60, 1793–1801. 85. Cabella, C., Gardini, G., C orpillo, D., Testore, G., Bedino, S., Solinas, S.P., Cravanzola, C., Vargiu, C., Grillo, M.A. & Colombatto, S. (2001) Transport and metabolism of agmatine in rat hepatocyte cultures. Eur. J. Biochem. 268, 940–947. 86. Satriano, J., Isome, M., Casero , R.A. Jr, Th omson, S. C. & Blantz, R.C. (2001) Polyamine transport system mediates agmatine transport in mammalian c ells. Am. J . Physiol. Cell Phys iol. 281, C329–C334. 87. Nakada, Y., Jiang, Y., Nishijyo, T., Itoh, Y. & Lu, C.D. (2001) Molecular characterization and regulation of the aguBA operon, responsible for agmatine utilization in Pseudomonas aeruginosa PAO1. J. Bacteriol. 183, 6517–6524. 88. Mercenier, A., S imon, J.P., Haas, D. & Stalon, V . (1980) Cata- bolism of 1-arginine by Pseudomonas aeruginosa. J. Gen. Micro- biol. 116, 381–389. 89. Wittich, R.M., Kilian, H.D. & Walter, R.D. (1987) Polyamine metabolism in filarial worms. Mol . Biochem. Parasitol. 24, 155 – 162. 3102 Z. Minic and G. Herve ´ (Eur. J. Biochem. 271) Ó FEBS 2004 . REVIEW ARTICLE Biochemical and enzymological aspects of the symbiosis between the deep-sea tubeworm Riftia pachyptila and its bacterial endosymbiont Zoran Minic and Guy Herve ´ Laboratoire. specific and obligate nature o f t he symbiosis between vestimentiferans and their bacterial endosymbiont raises the question of the transmission of the bacteria from one worm gen eration to the following. products between the e nvironment and the animal (Fig. 2 ). The other tissues are within the Riftia tube. The vestimentum is a muscle that the animal uses to position itself in the tube. Within the