Báo cáo khoa học: Biochemical and enzymological aspects of the symbiosis between the deep-sea tubeworm Riftia pachyptila and its bacterial endosymbiont pptx

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:
or
(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.

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