Distribution of the enzymes of the salvage pathways in the different parts ofRiftia pachyptila Because the worm is unable to synthesize the pyrimidine nucleotides through the de novo pat
Trang 1R E V I E W A R T I C L E
Biochemical and enzymological aspects of the symbiosis between the
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 in an
intimate symbiosis with a sulfur-oxidizing
chemoauto-trophic bacterium This bacterial endosymbiont is localized
in the cells of a richly vascularized organ of the worm: the
trophosome These organisms are adapted to their extreme
environment and take advantage of the particular
compo-sition of the mixed volcanic and sea waters to extract and
assimilate inorganic metabolites, especially carbon, nitrogen,
oxygen and sulfur The high molecular mass hemoglobin of
the worm is the transporter for both oxygen and sulfide This
last compound is delivered to the bacterium which possesses
the sulfur oxidizing respiratory system, which produces the
metabolic energy for the two partners CO2is also delivered
to the bacterium where it enters the Calvin–Benson cycle
Some of the resulting small carbonated organic molecules are thus provided to the worm for its own metabolism As far
as nitrogen assimilation is concerned, NH3can be used by the two partners but nitrate can be used only by the bac-terium This very intimate symbiosis applies also to the organization of metabolic pathways such as those of 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 constitutes a very interesting system to study 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 depth 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 mid-oceanic ridges, 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 the biosystems of the planet [3,4] In the
vent environment, these living organisms face physical and
chemical obstacles, such as elevated pressure (up to 300 atm),
high and rapidly changing temperature (from 4C to
350C), 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 biological 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 unique chemistry of these environments Sea 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 (350–450C) 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 H2S, percolates up to the sea floor where it mixes with the cold surrounding ocean water (4C) 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 the temperature oscillates around 20C, 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)
Trang 2metabolic energy is transferred to a series of animal species
which live in an obligate 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 acceptor [12–14] The electrons extracted are used
for the synthesis of ATP (Fig 1) This ATP feeds the
Calvin–Benson cycle for the fixation of CO2for 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 resemble
the previously described Pogonophora The first one to 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 large amount of the vestimentiferan body is occupied
by the trophosome, a specialized tissue whose cells contain a large population of intracellular sulfide-oxidizing gamma proteobacteria, up to about 1011bacteria per gram [17] These symbioses are very species 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 cytochrome oxidase I and symbiont 16S ribosomal gene (16S) DNA sequences were used to explore evolutionary relationships among the vestimentiferans and their symbionts and a detailed phylo-genetic caracterization of the bacterial symbiont via 16S rRNA was recently reviewed by McMulalin et al [16] The highly specific and obligate nature of the symbiosis between vestimentiferans and their bacterial endosymbiont raises the question of the transmission of the bacteria from one worm generation to the following one In some cases the bacteria is present in the ovule and thus, is directly transmitted to the next generation This process was observed in some bivalves [21] Despite the obvious benefit
of a direct transmission, no evidence supports this mode of symbiont transmission between generations in vestimenti-ferans No bacteria have been 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 and 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 (2C and 250 atm) the larvae of
R pachyptilahas 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 exchange
of metabolites and waste products between the environment and the animal (Fig 2) The other tissues are within the Riftiatube 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: the trophosome [25]
The cells of the trophosome (bacteriocytes) are densely colonized by a sulfur-oxidizing chemoautotrophic endo-symbiotic bacterium (109cells per gram fresh tissue) [12,14,26] The bacterial volume is estimated to represent between 15 and 35% of the total volume of the trophosome [17] This tissue includes the 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 transport system and Calvin–Benson cycle in sulfide
oxidizing bacteria This figure illustrates the connection between the
sulfide-oxidizing pathway for energy production and the Calvin cycle.
All the enzymes indicated in this figure were characterized in the Riftia
pachyptila trophosome and/or the isolated bacterial symbiont H 2 S
and CO 2 are provided to the bacterium through the worm
circula-tory system after absorption in the branchial plume (see text).
Abbreviations: APS, adenylylphosphosulfate; RUBISCO, D
-ribulose-1,5-bisphosphate carboxylase; phosphoribulokinase, D
-ribulose-5-phosphate-1-phosphotransferase.
Trang 3branchial plume 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
inRiftia pachyptila
An important feature of Riftia hemoglobin is that this
protein is not only the transporter of oxygen but also
that of H2S, in order to provide it to the bacteria for
energy production [30,31] This function is exerted
through the presence of a multihemoglobin system
possessing highly reactive cysteine residues [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+ H2S« RSSH + RSH [35] Moreover, H2S
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 H2S [28,33–38]
In the bacterial symbiont, sulfide is oxidized into sulfite
by an electron transport system which involves some cytochromes The reaction of SO32– 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 pachyptilahas developed a very efficient metabolism for the assimilation of inorganic CO2and nitrogen from nitrate and ammonia that are provided by the external environ-ment This metabolism relies on the obligate symbiotic relationship, which ensures an efficient assimilation, adap-ted to the very peculiar environment in which Riftia lives The results obtained concerning these assimilation processes are summarized below
Assimilation of carbon
CO2 is absorbed by Riftia at the level of the branchial plume This worm is exposed to wide variations in the environmental CO2 concentration, from about 2–11 mM, the typical vent concentration being around 5 mM
[46–48] This absorbed CO2 can be used in several ways
It appears that part of it can be transported by the circulatory systems to the bacteria-containing trophosome The CO2blood concentration was found to be 20–46 mM
[47] In addition,14C pulse label experiments showed that, in 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], CO2 can also be transformed into bicarbonate which can be used by several metabolic pathways (see below) In the bacteria the CO2 which was either directly provided by the environment or which results from the decarboxylation of the transported malate, enters the Calvin–Benson cycle through the 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 the Calvin– Benson cycle, were shown to be present in the bacterial
Fig 2 Riftia pachyptila (Top) The worm in its deep-sea vent
envi-ronment One can clearly see the branchial plume, which protrudes
from the tube (with 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.
Trang 4symbiont [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 large biomass [56,57] and the high growth rate [58] of
the chemoautotrophic symbiotic organisms imply a high
demand for nitrogen This is matched by the 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 of dissolved organic nitrogen levels
have been made at vents [62] showing that amino acid
concentrations are generally less than 0.2 nmolÆL)1around
vent communities [59] and less than 0.1 nmolÆL)1in
high-temperature fluids [63] Thus, inorganic nitrogen must be
the major source of nitrogen for vent symbioses
In living organisms, NH3either provided by the
environ-ment or resulting from nitrate reduction by nitrate reductase
and nitrite reductase [54,64–66] is used by a series of NH3
assimilating enzymes such as glutamine synthetase,
glutam-ate dehydrogenase and carbamylphosphglutam-ate synthetase to
produce basic metabolites such as amino acids and
nucleo-tides (Fig 3)
Assimilation of inorganic nitrogen was demonstrated 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].15N tracer experiments
showed that R pachyptila assimilates nitrate whereas
Solemya reidipreferentially 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 However, 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 detected in both the host tissues and
the symbiont [64] Distinct forms of host and
symbi-ont glutamine synthetase are present in R pachyptila
[64,66]
The concentration of nitrate in the deep-sea vent
environment is about 40 lM[59] and R pachyptila absorbs
it at a rate of 3.54 lmolÆg)1Æh)1 In the vent fluid, ammonia
can be present at concentrations up to the lMrange [60] In
the vicinity of Riftia the concentrations of nitrate and
ammonia were 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 primary source of nitrogen for the host and the
symbiont [65]
NH3can also be assimilated via the biosynthetic pyrim-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 NH3 and CO2which, in Riftia, are provided by the environment Therefore, an examination of the metabolic aspects of the 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 thede novo pyrimidine nucleotide pathway in the different parts ofRiftia pachyptila
The distribution of the subsequent enzymes of the pyrim-idine de novo pathway in different parts of the worm was examined [66] Interestingly, it appeared that the first three
Fig 3 Pathways of inorganic nitrogen assimilation in Riftia pachyptila.
NH 3 , either taken up directly from the environment or resulting from the reduction of nitrate, can be assimilated in the pathways shown, which begin by action of glutamine synthetase (GSase), glutamate dehydro-genase (GDHase) and carbamylphosphate synthetase (CPSase), respectively, to provide the basic organic metabolites indicated.
Trang 5enzymes 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 of the animal and the bacterium The fact that
the first three enzymes are present only in the trophosome
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 board
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 regulatory properties, as well as the fact
that they are not organized into a multifunctional protein
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 ofRiftia pachyptila
Because the worm is unable to synthesize the pyrimidine
nucleotides through the de novo pathway, it must rely on the
salvage pathway Indeed, enzymes of this pathway such as
cytidine deaminase, uridine kinase and
uracilphosphoribo-syl transferase are present in all the tissues of the worm [71]
Unexpectedly, the isolated bacterium does not exhibit any
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 results obtained showed that these enzymes belong to
the host [71]
Distribution of the enzymes of pyrimidine catabolism
in the different tissues ofRiftia pachyptila
Instead of 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 CO2 and NH3 [70]
Consequently, these products of degradation of pyrimidine
nucleotides can represent a possible source of carbon and
nitrogen for the organism
The analysis of the distribution of 5¢-nucleotidase, uracil
reductase and uridine phosphorylase, enzymes responsible
for the catabolism of pyrimidine nucleotides, showed that
they are present in all the tissues of 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 the results reported above and
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 through 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 products As 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 of the de novo pyrimidine biosynthetic pathway are present only in the bacterium synthesizing dihydroorotate, which can
be provided to the worm bacteriocytes and to its other tissues through the circulatory system The first reaction catalysed by the glutamine dependent carbamylphosphate synthetase uses glutamine provided by glutamine synthetase, whose substrate NH 3 is either directly furnished
by the external medium or derives from the reduction of nitrate by the bacterial nitrate reductase Both the worm and its bacterium possess the following enzymes of the pathway (dihydroorotate dehydrogenase, etc) for the production of the pyrimidine nucleotide triphosphates The salvage pathway is present only in the worm tissues These tissues also contain the enzymes of pyrimidine catabolism which can provide carbon and nitrogen to the worm All the enzymes indicated in the figure were characterized in the worm and/or its bacterial symbiont The scheme describes the exchanges between the endosymbiont, the trophosomal host cells, and the cells of other host tissues Question marks indicate steps that have not been completely elucidated Thin arrows refer to metabolic pathways Thick arrows refer to transport of metabolites in compartments, tissues or body parts Abbreviations: CPSase-P, carbamylphosphate synthetase specific to the pyrimidine biosynthetic pathway; ATCase, aspartate transcarbamylase; DHOase, dihydroorotase; GSase, glutamine synthetase.
Trang 6pathway (carbamylphosphate synthetase, aspartate
transcarbamylase and dihydroorotase), the necessary
meta-bolic precursors, orotate and/or dihydroorotate, must be
provided by the bacterium Thus, R pachyptila is absolutely
dependent on the symbiotic bacterium for the de novo
biosynthesis of the pyrimidine nucleotides
The results obtained show also that R pachyptila
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 CO2, NH3, malonyl-CoA and
succinyl-CoA; subsequently malonyl-CoA can be used for the
biosynthesis of fatty acids while succinyl-CoA enters into
the citric acid cycle [70] In this manner the degradation of
pyrimidine nucleotides can represent an alternative
nutri-tional source of nitrogen and carbon, besides the external
environment of the worm, and can also feed other
biosyn-thetic pathways This degradation can also result from the
reported bacterial lysis in the trophosome [73]
A study of the localization of these anabolic and catabolic
enzymes in the trophosome shows that they are not
homogenously distributed The level of anabolic activities
decreases from the centre of the trophosome to its
periphery, and the level of catabolic activities varies in the
opposite direction This observation suggests 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 NH3as
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 ofRiftia pachyptila
Concerning the arginine biosynthetic pathway, 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 pachyptilaas well as in the bacterial symbiont (Fig 5) [74]
Lack of arginine catabolism via the catabolic ornithine
transcarbamylase of the arginine deiminase pathway
inRiftia pachyptila
There are two types of ornithine transcarbamylases, which
participate in either the anabolism, or the catabolism of
arginine The anabolic ornithine transcarbamylase belongs
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 catabolic pathway leading
to the anaerobic degradation of arginine to produce NH3,
CO2 and ATP [75–78] In this pathway, ornithine
trans-carbamylase catalyzes the transformation of citrulline to
ornithine In view of the limiting supply of NH3and CO2to Riftia from its environment [46–48,59,60], this arginine catabolic pathway could constitute an interesting source of these inorganic metabolites
The kinetic properties of the ornithine transcarbamylase found in Riftia strongly suggest that neither the worm nor the bacterium possess the catabolic form of this enzyme belonging to the arginine deiminase pathway This conclu-sion was confirmed by the lack of arginine deiminase in both the worm and the bacterium [74]
Arginine catabolism via the arginine and ornithine decarboxylases
Although R pachyptila 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 their 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) this biosynthesis involves ammo-nium dependent carbamylphosphate synthetase (CPSase-A) specific for this pathway In the bacterium, a unique carbamylphosphate synthetase provides this metabolite for both the pyrimydine and the arginine pathways The worm CPSase-A uses NH 3 and HCO 3 pro-vided by the external medium Arginase and urease involved in the catabolism of arginine are present in both organisms The arginine deiminase pathway is absent Two enzymes of the polyamines bio-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, carbamylphosphate synthetase specific to the arginine biosynthetic pathway; ADase, arginine decarboxylase; ADIase, arginine deiminase; ASSase, argininoguccinate synthetase; ODase, ornithine decarboxy-lase; OTCase, ornithine transcarbamylase.
Trang 7be 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 in Riftia and its
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 enzymes are higher in the isolated bacterium than in
the bacterium-containing trophosome, indicating that these
enzymes are present only in the 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 the arginine biosynthetic pathway (ammonium
dependent carbamylphosphate synthetase, ornithine
trans-carbamylase, argininosuccinate synthetase) are present in
both the host and the bacterium The ammonium dependent
carbamylphosphate synthetase that uses ATP to catalyze
the conversion of the inorganic molecules HCO3 and NH3
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
amino acid for Riftia 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 this pathway in all the
tissues of R pachyptila might contribute to its adaptation to
the extreme environment of the hydrothermal vent
Arginase and urease are also present in all the tissues of
Riftia, including the trophosome [81] Accordingly, one
observes high concentrations 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 NH3, CO2,
glutamate and succinate This last metabolite then enters the
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 the synthesis of polyamines through the
production of agmatine and putrescine by arginine
decarb-oxylase and ornithine decarbdecarb-oxylase In all living organisms,
including viruses, polyamines play key roles in the
biosyn-thesis and structure of nucleic acids and are reported to be
involved in many biological processes such as membrane
stability, growth and development [83] In R pachyptila it
appears that arginine decarboxylase is present only in the
bacterial endosymbiont (Fig 5) The absence of these
enzymes, which initiate the biosynthesis of polyamines in
the host tissues, strongly suggests that Riftia is dependent 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, the 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 decarboxylase or ornithine decarboxylase activity This absence has also been reported in the case of human and animal filarial worm parasites Dirofilaria immitis, Brugia patei and Litomosoides [89] In a similar way, in Riftiathe first three enzymes of the pyrimidine nucleotide biosynthetic pathway are present only in the bacterium but not in the worm [74] The absence of these enzymes is also characteristic of protozoan parasites such as Giardia lamb-lia, Trichomonas vaginalis and Tritrichomonas foetus Thus,
it appears that Riftia has developed a metabolism for the biosynthesis of pyrimidines and polyamines which is reminiscent of what is observed in some parasites, suggest-ing some similarity in the adaptation of metabolic pathways
in symbiosis and parasitism
This complex metabolic organization is the basis of the adaptation of Riftia pachyptila to the extreme hydrothermal vent environment and to the absence of a readily available source of organic carbon through photosynthesis
Acknowledgements
This work was supported by the Centre National de la Recherche Scientifique and by the Universite´ Pierre et Marie Curie, Paris.
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