Stimulatory effect of sulphide on thiotaurine synthesis in three hydrothermal-vent species from the East Pacific Rise
Observatoire Océanologique de Banyuls, Laboratoire Arago, Université Pierre et Marie Curie, CNRS UMR 7621, BP 44, F-66651 Banyuls-sur-mer Cedex 01, France
* Author for correspondence (e-mail: a_pruski{at}hotmail.com)
Accepted 21 May 2003
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Summary |
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Key words: thiotaurine, hypotaurine, sulphide, sulphur-based symbiosis, hydrothermal vent, amino acid metabolism
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Introduction |
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We previously demonstrated that sulphur-based symbioses are characterised
by the presence of thiotaurine, a free sulphur amino acid, which has never
been reported in high amounts in non-symbiotic species
(Pruski et al., 2000b). Based
on its distribution in the tissues and its chemical properties, the
involvement of thiotaurine in sulphide metabolism was proposed (Pruski et al.,
2000a
,b
).
In the present study, we investigated thiotaurine biosynthesis by measuring
changes in tissue free amino acid composition in response to exposure of the
symbiont-bearing species to sulphide and thiosulphate.
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Materials and methods |
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Preparation of stock solutions of
Na2S and
Na2S2O3
Sulphide stock solutions (0.5, 1 and 2 mmol l-1, pH 7.5) were
made up with Na2S.9H2O in deoxygenated bidistilled water
inside a glove bag filled with helium. The anaerobic solutions were
distributed in 20 ml vials, sealed with Teflon caps and refrigerated until
utilisation (storage time was 2 weeks). Stock solutions of thiosulphate (2
mmol l-1) were freshly prepared before each experiment by
dissolving crystals of
Na2S2O3.5H2O in deoxygenated
bidistilled water.
Incubation of tissue homogenates with reduced sulphur
Prior to the experiments, small pieces of selected tissues (gill and mantle
of bivalves, plume and trophosome of tubeworms) were isolated from each
specimen and frozen for high performance liquid chromatography (HPLC)
determination of the free amino acid (FAA) composition (see below). Since
thiotaurine occurrence was restricted to the symbiont-containing tissues, only
the gills of bivalves and the trophosome of vestimentiferans were used in the
experiments described below.
Adult specimens of similar size were used for the experiments (clams of 15.2±1.6 cm shell length, mussels of 13.2±1.3 cm shell length, and tubeworms of approximately 80 cm length). One complete demibranch was excised from each bivalve, rinsed in filtered seawater to remove excess mucus and rapidly chopped. Because we could not weigh the tissues on board, 1 volume (10 ml) of chopped gills or trophosome was homogenized on ice in 3 volumes of a cold deoxygenated buffer (10 mmol l-1 Hepes, 5 mmol l-1 MgCl2, 0.25 mmol l-1 saccharose, pH 7.5) using a glass homogenizer. Tissue homogenates were then distributed in incubation vials with a screw top and Teflon septum (1.5 ml of homogenate per vial). At time zero, 1.5 ml of sulphide stock solution or thiosulphate solution was injected with a hypodermic syringe through the septum of the incubation vials. Throughout the experiment, vials were gently agitated. After 0, 5, 10 and 25 min, homogenates were transferred into cryovial tubes and frozen in liquid nitrogen until FAA analysis. Controls were prepared by incubating gill and trophosome homogenates with buffer. These experiments were repeated 4-6 times, each time with a different animal.
Amino acid quantification
Samples were lyophilized in the laboratory and the free amino acid pool
extracted in cold 70% ethanol (for a detailed description of the protocol, see
Pruski et al., 1998). Amino
acids were separated and quantified by reverse-phase HPLC as previously
described (Pruski et al.,
1998
). All chemicals were purchased from Sigma-Aldrich
(Saint-Quentin Fallavier, France) except for thiotaurine, which was prepared
as described by Albéric and Boulègue
(1990
).
Data presentation and statistical analyses
Data are presented as means ± S.D. and values of
N (number of repetitions) are given in the figure legends and the
tables. Simple regressions were used to show linear relationships. Two-sample
comparisons were made using paired one-tailed t-tests assuming equal
variance (Microsoft Excel). Multiple comparisons were made using analysis of
variance (ANOVA) with a Bonferroni post-hoc test (Statview).
Significance was accepted at the 5% level.
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Results |
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Gill homogenates from six clams were exposed to various concentrations of Na2S for up to 25 min and the concentration of 26 FAAs followed over time. Apart for thiotaurine and hypotaurine concentrations, no significant changes in the FAA composition were observed (not shown). Thiotaurine concentration increased considerably in gills incubated with Na2S (Fig. 1A), but no significant difference was observed between the different treatments (ANOVA, P<0.05, Fig. 1A), which suggests that the mechanism responsible for thiotaurine synthesis was saturated at concentrations above 0.25 mmol l-1 Na2S.
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The time courses of thiotaurine and hypotaurine variations in homogenates exposed to 1 mmol l-1 Na2S are shown in Fig. 2A. Thiotaurine concentration increased rapidly during the first 5 min of incubation, whereas hypotaurine concentration declined. Both concentrations remained relatively constant after this initial stage. Overall, an approximately 1:1 stoichiometric conversion of hypotaurine to thiotaurine was observed.
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The individual response was quite variable (e.g. thiotaurine synthesis ranged from 3 to 30 µmol g-1 dry mass after 25 min of incubation with 1 mmol l-1 Na2S; Table 1) with lower rates of thiotaurine synthesis in tissues with high initial thiotaurine content. A negative linear relationship between the initial thiotaurine:(thiotaurine+hypotaurine) ratio and the amount of thiotaurine synthesized was indeed apparent (r=0.83, N=6, P<0.01). This suggests that thiotaurine synthesis is strongly dependent on the initial equilibrium between hypotaurine and thiotaurine in the gill.
In the absence of Na2S, a linear, but lower synthesis of thiotaurine was evident throughout the experiment, with a concomitant decrease of hypotaurine (Fig. 2A). No significant increase in synthesis of thiotaurine compared to controls was observed when gills were incubated with 1 mmol l-1 Na2S2O3 (Fig. 1A).
Synthesis of thiotaurine in Bathymodiolus thermophilus
Thiotaurine concentration was lower in B. thermophilus than in
C. magnifica, whereas the opposite trend was observed with
hypotaurine (Table 1). The
resulting thiotaurine:(thiotaurine+hypotaurine) ratios were therefore lower
(0.41-0.73; Table 2), which
suggests the mussels were exposed to a more diluted hydrothermal fluid. This
is consistent with the location of these specimens at the periphery of the
vent sites.
As observed with the clams, Na2S exposure (at 0.25 and 0.5 mmol l-1) induced the formation of thiotaurine in B. thermophilus gills (Fig. 1B). However, thiotaurine concentration did not reach a plateau after the first 5 min of incubation, but continued to increase at a lower rate (Fig. 2B). Hypotaurine concentration decreased concomitantly throughout the incubation (Fig. 2B). Although the individual response was highly variable (Table 1), a clear inhibition of thiotaurine synthesis was observed at 1 mmol l-1 Na2S (Fig. 1B), the final thiotaurine concentration being even lower than in controls. A maximum thiotaurine synthesis rate of approximately 25 µmol g-1 dry mass was observed in homogenates incubated for 25 min with 0.25 mmol l-1 Na2S (Fig. 1B). Values of the thiotaurine:(thiotaurine+hypotaurine) ratio remained lower at the end of the incubation time than in the clams, ranging from 0.61 to 0.83 after 25 min of incubation with 0.25 mmol l-1 Na2S (Table 2).
Thiotaurine synthesis was also stimulated by 1 mmol l-1 Na2S2O3 (Fig. 1B). The amounts of thiotaurine synthesized were of the same order of magnitude as in gills incubated with 0.25 mmol l-1 Na2S (approximately 19 µmol g-1 dry mass after 25 min of incubation; Fig. 1B).
No significant synthesis of thiotaurine was observed in the absence of Na2S (Fig. 2B).
In order to determine if the ability to produce thiotaurine is a specific
adaptation to thiotrophy, gills of the methanotrophic mussel B.
childressi were also incubated with Na2S (data not shown).
This mussel, like all non-thiotrophic species, is characterized by very low
levels of thiotaurine (<0.5 µmol g-1 dry mass; for a
description of its FAA composition, see
Pruski et al., 2000a).
Hypotaurine was found in its gill tissue, but only trace amounts of
thiotaurine were synthesized (<4 µmol g-1 dry mass after 25
min of incubation with 0.5 mmol l-1 Na2S).
Synthesis of thiotaurine in Riftia pachyptila
Taurine derivatives (i.e. taurine, hypotaurine and thiotaurine) were
particularly abundant in the trophosome of R. pachyptila and
accounted for up to two thirds of the FAA pool. These compounds contained
approximately 14% of the total sulphur content of the trophosome (on the basis
of a sulphur content of 11.1% dry mass; A. M. Pruski and A.
Fiala-Médioni, unpublished data). Thiotaurine was restricted to the
trophosome and its concentration was highly variable (5-140 µmol
g-1 dry mass; Table
1), as was the thiotaurine:(thiotaurine+hypotaurine) ratio
(0.03-0.53; Table 2).
A dose-dependent increase of the amount of thiotaurine synthesized was observed in trophosomes incubated with Na2S at concentrations ranging from 0 to 1 mmol l-1 (Fig. 1C). Although the individual response was again quite variable, a clear displacement of the hypotaurine-thiotaurine equilibrium was observed in each sample, with values of the thiotaurine:(thiotaurine+hypotaurine) ratio reaching 0.64-0.99 at the end of the incubation time (Table 2). This indicates that vestimentiferan tubeworms have robust ability for thiotaurine synthesis. The magnitude of thiotaurine synthesis was actually far greater in R. pachyptila than in the two bivalve species and reached a maximum of 324 µmol g-1 dry mass. Assuming 80% of the vestimentiferan's mass was water, this would allow the fixation of up to 65 µmol sulphide g-1 fresh trophosome.
The time courses of thiotaurine, hypotaurine and taurine variations in homogenates exposed to 1 mmol l-1 Na2S are shown in Fig. 3. A rapid and elevated synthesis of thiotaurine was observed during the first 5 min of incubation (mean synthesis = 150 µmol g-1 dry mass), while hypotaurine concentration decreased concomitantly. In contrast to the results obtained in the bivalve experiments, taurine concentration also declined remarkably within 5 min. The amount of thiotaurine synthesized was equivalent to the combined decrease of the taurine and hypotaurine concentrations. After 5 min of incubation, the initial reserves in hypotaurine and taurine were completely depleted in three of the four individuals tested, which explains the lack of further thiotaurine synthesis (data not shown).
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A low synthesis of thiotaurine was also evident in the absence of Na2S (approximately 30 µmol g-1 dry mass), but no significant synthesis of thiotaurine was observed in the presence of Na2S2O3 (Fig. 1C).
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Discussion |
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Hypotaurine is the only precursor in bivalves, while taurine is also
involved in the synthesis of thiotaurine in vestimentiferan tubeworms. This
suggests that different enzymatic pathways are used. Although no direct
reaction from taurine to thiotaurine has been described to date, one
hypothesis is that taurine is first reduced to hypotaurine, which subsequently
reacts with sulphide. The reverse reaction, the oxidation of hypotaurine to
taurine, occurs in most animals during the catabolism of sulphur amino acids,
whose last end-product is taurine
(Huxtable, 1992).
Thiosulphate also stimulates thiotaurine synthesis, although only in
mytilids. From the literature, two mechanisms may be proposed to explain this
result: (1) the conversion of thiosulphate to sulphide before thiotaurine
synthesis, and (2) a direct formation of thiotaurine with sulphite as a
byproduct (De Marco et al.,
1961, 1972). The symbionts of
Bathymodiolus thermophilus are known to use thiosulphate
preferentially (Nelson et al.,
1995
), in contrast to those of vesicomyids and vestimentiferans,
which rely only on sulphide. Therefore, one may expect that specific metabolic
pathways for the utilisation and storage of thiosulphate occur in B.
thermophilus.
Potential involvement of thiotaurine in the transfer of sulphide from
the blood to the bacteriocytes
One surprising finding of this work was the linear synthesis of thiotaurine
observed in tissues of C. magnifica
(Fig. 1) and R.
pachyptila after incubation with buffer alone. Those two species share
one specific adaptation to the thiotrophic mode of life, namely the occurrence
of sulphide-binding proteins in the blood (i.e. the haemoglobins in R.
pachyptila and a lipoprotein in C. magnifica;
Arp et al., 1987;
Childress et al., 1993
). In the
absence of any external source of sulphur, one may hypothesize that the
sulphur incorporated into thiotaurine originated from these sulphide-binding
proteins. Sulphide binds to the haemoglobins of R. pachyptila at the
level of free cysteine residues and disulphide bridges, with the formation of
persulphide groups (Zal et al.,
1998
). The transfer of reduced sulphur from protein persulphides
(R-SSH) was previously shown to be rapid and spontaneous in the presence of
hypotaurine (less than 10 min; Cavallini et
al., 1970
), and would explain such a result:
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In the blood of Calyptogena magnifica, sulphide is bound to a
transport lipoprotein via zinc ions
(Zal et al., 2000) and could
equally well be transferred to hypotaurine. As those sulphide-binding proteins
are too large to penetrate the bacteriocytes, thiotaurine synthesis would
indeed facilitate the transfer of sulphide from blood components to the
bacteriocytes, just as myoglobin facilitates the transfer of oxygen from blood
to muscular cells. This hypothesis is further supported by the fact that no
synthesis of thiotaurine was observed in the absence of an external source of
reduced sulphur in B. thermophilus (a species that is devoid of any
sulphide-binding protein).
An alternative explanation for the observed synthesis of thiotaurine in
controls from C. magnifica and R. pachyptila is provided by
the recent results from Arndt et al.
(2001). This study showed that
sulphide is produced under anoxic conditions in several thiotrophic symbioses
including C. magnifica, B. thermophilus and R. pachyptila.
Elemental sulphur (S°) is proposed to be the substrate responsible for
this anaerobic generation of sulphide, which started a few hours after anoxia
had set in (Arndt et al.,
2001
). Since S° was never found in B. thermophilus,
one expects this species to be unable to generate sulphide under anoxic
conditions, which is consistent with the lack of thiotaurine synthesis that we
observed in mussel gills. In contrast, the symbiont-bearing tissues of C.
magnifica and R. pachyptila contain large amounts of S°
(Somero et al., 1989
). If the
anaerobic production of sulphide is responsible for the synthesis of
thiotaurine observed in the absence of an external input of reduced sulphur,
large amounts of sulphide must be generated within minutes (up to 4 µmol
g-1 and 8 µmol g-1 fresh mass after 25 min of
incubation in C. magnifica and R. pachyptila, respectively).
These rates of sulphide production are higher than the values reported by
Arndt et al. (2001
). For
example, in R. pachyptila, less than 0.5 µmol g-1
sulphide were released in 15 min under anoxic and hypoxic conditions. This
suggests that if some of the sulphide incorporated to thiotaurine in our
controls was produced anaerobically, there must be another source of sulphide
(i.e. sulphide bound to proteins).
Function(s) of thiotaurine in sulphur-based symbioses: thiotaurine as
a carrier of reduced sulphur
Living in association with sulphur-oxidising symbionts has forced the hosts
to evolve specific adaptations to survive the toxic sulphide, such as
molecules that can reversibly bind sulphide until it is oxidised by the
symbionts. Thiosulphonates such as thiotaurine meet the requirements for a
biologically perfect carrier of reduced sulphur. They exhibit higher lipid
solubility than inorganic thiosulphate and have been shown to penetrate cell
membranes readily (Petrikovics et al.,
1994). One may thus expect that thiotaurine can easily enter the
bacteriocytes to deliver the energy-containing substrate to the symbionts.
Another requisite satisfied by thiotaurine is that it can accumulate in the
cytoplasm in high concentrations without perturbing protein function. In
tissues containing high amounts of thiotaurine, we have previously shown a
compensatory loss of other free amino acids such as glycine, enabling
maintenance of a steady intra-osmotic pressure and thus the integrity of the
cell (Pruski et al., 2000a
).
Thiotaurine appears to have other positive effects on the cell, such as
protection of DNA from damage by sulphur compounds (i.e. mustard gas;
Baskin et al., 2000
).
Furthermore, thiotaurine has no other known metabolic function in the cell and
is synthesized from non-essential compounds. The energetic cost of thiotaurine
synthesis is thus relatively little.
To be efficient, a carrier has to be able to bind and release reduced
sulphur rapidly. We have shown that reduced sulphur can be incorporated to
thiotaurine and have provided arguments in favour of the possible involvement
of this amino acid in the transfer of sulphide from the blood to the
bacteriocytes. The release of reduced sulphur from thiotaurine has previously
been demonstrated (Pruski et al.,
2001) by a catalytic factor present in the trophosome of
vestimentiferans that enables the release of reduced sulphur from thiotaurine
prior to its oxidation by sulphur-oxidising bacteria
(Pruski et al., 2001
).
Although the incorporation of reduced sulphur into thiotaurine can occur
spontaneously (De Marco et al.,
1961
), the differences observed between the three species and the
lack of significant thiotaurine synthesis in symbiont-free tissues (data not
shown) and in methanotrophic mussels suggest some form of enzymatic control.
Whether the host or its symbionts produce this enzyme is a key question for
understanding how energy delivery (as sulphur) is controlled in the
symbiosis.
Conclusion
Thiotaurine synthesis appears as a general adaptation supporting life with
sulphur-oxidising symbionts (Pruski et
al., 2000b). The incorporation of reduced sulphur to thiotaurine
provides the host with an efficient and relatively cost-free mechanism to
bind, transport and/or store sulphide in its host tissues. As thiotaurine is
not the only compound involved in the transport of reduced sulphur in
sulphide-based symbioses, future studies will have to determine how these
different binding systems interact.
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Acknowledgments |
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