1 Laboratoire d'Océanographie Biologique, CNRS-UMR 5805 Université Bordeaux 1, 2 rue du Professeur Jolyet, F-33120 Arcachon, France
2 Department of Chemistry, University of York, Heslington, York YO10 5DD, UK
Correspondence
Rutger de Wit
rde-wit{at}univ-montp2.fr
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ABSTRACT |
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Present address: UMR 5119 CNRS-Université Montpellier II Ecosystèmes lagunaires, Université Montpellier II, Case 093, 34095 Montpellier Cedex 05, France.
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INTRODUCTION |
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Bacteriochlorophyll homologues from cultures of the green sulfur bacterium Chlorobium limicola were first recognized in 1978 and farnesol was identified as the major esterifying alcohol (Caple et al., 1978). Several subsequent studies have shown that other esterifying alcohols occur in natural populations of green sulfur bacteria (Caple et al., 1978
; Otte et al., 1993
; Repeta et al., 1989
). Thereafter, the application of HPLC coupled with photodiode array (PDA) detection confirmed that larger numbers of homologues are present in several species of Chlorobiaceae (Borrego & Garcia-Gil, 1994
). These authors observed minor proportions of the so-called secondary homologues, components with longer retention times, reflecting a more lipophilic character, suggesting that alcohols other than farnesol were esterified via the C-17 propionic acid. These observations led those workers to focus their studies on the pigment composition of green sulfur bacteria both in natural habitats and in culture experiments. The first studies examining natural populations of Chlorobiaceae clearly identified the effects of light quality on the pigment composition of these micro-organisms in lakes (Borrego et al., 1993
, 1997
). Concomitantly, culture experiments with various strains of Chlorobiaceae revealed modification of the pigment composition and distribution as a response to the prevailing light conditions (Borrego & Garcia-Gil, 1995
; Borrego et al., 1999a
, Guyoneaud et al., 2001
). However, because the bacteriochlorophyll homologues have very similar in vitro absorption spectra, on-line UV/visible spectroscopy could not permit detailed assignment of pigment structures. To solve this problem, Airs et al. (2001a
, b)
applied HPLC coupled to tandem mass spectrometry (LC-MS/MS) to analyse and identify the bacteriochlorophyll homologues of Chlorobium phaeobacteroides grown at a low light intensity. This methodology revealed Chl. phaeobacteroides UdG 6053 to contain a wider range of distinct bacteriochlorophyll homologues than has been recognized previously in chlorobiaceae (Airs et al., 2001b
).
The aim of the present study was to identify the structures of the pigments from a green and a brown member of the Chlorobiaceae, i.e. Prosthecochloris aestuarii and Chlorobium phaeobacteroides, respectively, and to study the quantitative response of the pigment distributions to growth of the organisms under a range of light regimes representative of those found in deep lake ecosystems. The light climate in deep lakes is characterized by low light that is depleted in blue and red wavelengths and, therefore, dominated by green wavelengths, with a maximum around 550 nm (Fischer et al., 1996; Vila & Abella, 1999
). Whereas Chl. phaeobacteroides is strictly pelagic and is commonly found in the water column of deep lakes, Ptc. aestuarii is able to develop both in the pelagos, as has been reported for lagoon environments (Guyoneaud et al., 1998
), and in the benthos, particularly in the anoxic layers of sediments. In order to simulate the range of light conditions occurring at different depths in the water column, both strains were grown under green light under a range of intensities. The pigments from these cultures have been described and quantified by combining HPLC-PDA and LC-MS/MS following the method described by Airs et al. (2001a)
.
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METHODS |
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Ptc. aestuarii CE 2404 is a spherical and non-motile bacterium that possesses extrusions and contains different forms of bacteriochlorophyll c (BChl c) and carotenoids of the chlorobactene series. Chl. phaeobacteroides UdG 6030 is a brown rod-shaped bacterium, the major pigments of which comprise different forms of bacteriochlorophyll e (BChl e), and carotenoids of the isorenieratene series. Both strains also contain bacteriochlorophyll a (BChl a) as a component of the soluble FMO, protein in the reaction centre, and associated with the baseplate of the chlorosome.
Both Ptc. aestuarii CE 2404 and Chl. phaeobacteroides UdG 6030 are exclusively photolithotrophic, utilizing sulfide and/or elemental sulfur as major electron donors. Nevertheless, acetate and other organic compounds can be assimilated in the presence of sulfide and light. Neither thiosulfate nor sulfite is used as electron donor by these species, and the latter compound is even inhibitory for growth of Ptc. aestuarii.
Culture conditions.
Both species were grown in a synthetic medium (Biebl & Pfennig, 1978; Eichler & Pfennig, 1988
) in completely filled 125 ml screw-capped flat bottles. The basic medium composition per litre is as follows: 0·35 g KH2PO4, 0·5 g NH4Cl, 0·05 g CaCl2.2H2O, 1 g MgCl2.6H2O, 1 ml 2 M H2SO4 and 1 ml SL-12B solution (Eichler & Pfennig, 1986
). The medium was amended with 20 g NaCl, plus 0·5 g MgSO4.7H2O for Ptc. aestuarii and 0·4 g MgSO4.7H2O for Chl. phaeobacteroides. After sterilization, NaHCO3 and Na2S were added from concentrated autoclaved solutions; 1 ml vitamin solution V7 (Pfennig & Trüper, 1992
) was also added. The medium was aseptically dispensed into flat bottles, which were inoculated with 12 ml of the corresponding bacterial cultures in exponential phase. The bottles were filled to the rim, leaving a pea-sized air bubble. The cultures were gently mixed using a balancing table shaker (Bioblock Scientific).
The cultures were illuminated from above by a 450 W horticolar light source (SON-T Agro, Philips), using a 16 h light/8 h dark regime. Different light intensities were obtained by placing the culture bottles at the appropriate distance from the light source and by placing a neutral-density gelatin filter (Kodak Wratten; Tiffen) at the surface of each bottle. A green gelatin filter (CAT 149 7395 no. 58, Kodak Wratten; Tiffen) was placed in the light-path to filter out blue, red and infrared wavelengths. As a result, the light spectrum reaching the surface of the bottles was limited to the range 480615 nm (see Fig. 1). The incident light was measured at the surface of each bottle using an optic sensor OL 4000 Q (DELTA Lys & Optik) coupled to an amplifier (1000 mV/1000 µE m2 s1). The range of intensities comprised 0·2, 0·8, 1·2, 3·5, 4·3, 6, 9·2, 40 and 55·7 µmol photons m2 s1. Cultures were grown to low densities (OD600
0·1) to minimize the effect of self-shading.
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Pigment and protein analysis.
After the incubation period, 10 ml and 50 ml portions of each culture were centrifuged (Heraeus) at 4000 g for 20 min for pigment and protein analysis, respectively. The cell pellets were stored at 80 °C prior to analysis.
Protein concentration was measured from the pigment-free and sulfur-free pellets by the Lowry method. For the pigment analysis, thawed samples were extracted by sonication in acetone (100 %, 4 °C). Following centrifugation (5 min at 3000 g, Jouan), the supernatant was filtered through a solvent-extracted cotton-wool plug. The extraction procedure was repeated twice and the extracts were combined and reduced to a minimum volume in vacuo. Samples were protected from the light and stored in a freezer at 18 °C after extraction.
HPLC, liquid chromatography (LC-MS) and tandem mass spectrometry (LC-MS/MS).
Reversed-phase HPLC was accomplished using a Waters system comprising a 717 autosampler, 600 MS system controller and 996 photodiode array detector. Instrument control, data processing and analysis were performed using Waters Millennium 2010 software. All solvents were degassed using helium. The intact chlorophylls were separated by HPLC, thus maintaining retention times and in vitro absorption spectra, and permitting recognition of the presence of bacteriophaeophytins and bacteriophaeophorbides in the cultures. LC-MS was performed using a Finnigan system comprising a Thermo Separations AS3000 autosampler, P4000 gradient pump, UV2000 UV-vis detector (Thermo Quest) and a Finnigan MAT LCQ ion trap mass spectrometer equipped with an APCI source operated in the positive mode. The HPLC conditions used were as described above. LC-MS settings were as follows: capillary temperature 150 °C, APCI vaporizer temperature 450 °C, discharge current 5 µA, sheath gas flow 60 (arbitrary units). During LC-MS analysis, the chlorophylls were demetallated on-line as they eluted from the HPLC column prior to entering the mass spectrometer. The demetallation was achieved by the addition of a small amount of methanoic acid to the solvent flow (Airs & Keely, 2000). As a result of the on-line demetallation, the mass spectra were dominated by the protonated molecules (MH+) of the corresponding bacteriophaeophytins. Tandem mass spectrometry (LC-MS/MS) was used to assign bacteriochlorophyll structures from their fragmentation during MS/MS analysis (Airs et al., 2001b
, Airs & Keely, 2002
).
Pigment quantification.
Since authentic standards were not available, pigment contents were quantified from peak areas assuming an absorption coefficient of 75·4 mM1 cm1 at 662·5 for BChl c (Oelze, 1985), 84 mM1 cm1 at 768 nm for BChl a (Korthals & Steenbergen, 1985
) and 48·1 mM1 cm1 at 649 nm for BChl e (Borrego et al., 1999b
).
Trends of pigment proportions versus light intensities were tested by linear regression analyses and the P value of the regression was taken as a criterion for significance of trends (Sokal & Rohlf, 1995).
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RESULTS |
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Ptc. aestuarii CE 2404
Pigments were identified (Table 1) from the HPLC-PDA spectra, protonated molecules and fragment ions in LC-MS/MS (Airs et al., 2001a
, b
; Airs & Keely, 2002
). The chromatogram of the pigment extracts from Ptc. aestuarii grown at 1·2 µmol photons m2 s1 is shown in Fig. 2
; it comprised mainly farnesyl-esterified forms of BChl c, chlorobactene and other carotenoids. The chromatogram depicts a group of six major BChl c homologues esterified with farnesol, eluting between 20 and 25 min (peaks 6 to 11). The mass spectra of these components revealed protonated molecules differing by 14 Da, corresponding to three different alkylated states of the BChl c molecule, at positions C-8 and C-12 (see Table 1
). The protonated molecules observed during LC-MS analysis correspond to BChl c homologues comprising a tetrapyrrole macrocycle (TMC) of (i) 34 C atoms with ethyl and methyl [Et, Me] groups at positions C-8 and C-12, respectively (peaks 6 and 7 referred to as TMC34), (ii) 35 C atoms with [Et, Et] or [n-Pr, Me] at positions C-8 and C-12, respectively (peaks 8 and 9 referred to as TMC35) and (iii) 36 C atoms with [n-Pr, Et] or [i-Bu, Me] at positions C-8 and C-12, respectively (peaks 10 and 11 referred to as TMC36). A single stage of tandem mass spectrometry (LC-MS/MS) gives unambiguous information of the molecular mass of bacteriochlorophyll homologues and of the tetrapyrrole macrocycle. Unfortunately, however, if homologues exist it is not possible to distinguish between the two possible combinations of alkyl substituents at position C-8 and C-12, e.g. for peaks 10 and 11 no distinction is made between [n-Pr, Et] and [i-Bu, Me]. The pattern is further complicated by the fact that forms can exist as diastereoisomers. This phenomenon gives rise to peak broadening due to partial separation of the two diastereoisomers (Wilson et al., 2003
). A number of components (peaks 15) eluted prior to the farnesyl-esterified forms of BChl c. Their UV/visible spectra exhibit absorbance bands at approximately 390, 460 and 680 nm, clearly different from those of reported bacteriochlorophylls. The protonated molecules of these components fragmented in a similar way to the bacteriochlorophylls during LC-MS/MS analysis, specifically losing 204 as a neutral species, indicating the esterifying alcohol to be farnesol. The precise nature of these components is, as yet, unknown. A further series of homologous components (peaks 12, 13 and 15) eluted later (tR 3032 min). These components also gave unusual UV/visible spectra (Table 1
) and fragmented in a similar manner to bacteriochlorophylls. Peaks 14, 16, 17, 18, 20, 22 and 23 gave UV/visible spectra typical for bacteriophaeophytins. The latest-eluting components were the carotenoids
-carotene and chlorobactene and various geometric isomers together with several unidentified carotenoids.
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Fig. 3 depicts the relative proportion of [Et, Me] (TMC34), [Et, Et] or [n-Pr, Me] (collectively TMC35) and [n-Pr, Et] or [i-Bu, Me] (collectively TMC36) BChl c homologues as a function of the light intensity during growth. Variations were only minor and no significant trend was observed when considering the entire dataset. Notably, however, above 1·2 µmol photons m2 s1, we observed a general trend for TMC34 to increase in proportion with increasing irradiance intensity (P=0·01), i.e. from 10 % when cultures were grown at the low light intensity (0·8 µmol photons m2 s1), to approximately 25 % at 9·2 µmol photons m2 s1. The relative proportion of TMC35 remained rather constant at the level of about 55 % of the total farnesyl-esterified BChl c homologues for the whole range of light intensities tested. In accordance, the proportion of TMC36 ranged between about 40 % and 20 % for cultures grown at 1·2 µmol photons m2 s1 and 9·2 µmol photons m2 s1, with a general trend for a decrease with increasing light intensity (P=0·01).
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Chl. phaeobacteroides UdG 6030
Qualitatively, the pigment distribution for Chl. phaeobacteroides UdG 6030 showed the same profiles for all the light intensities tested (Fig. 4, Table 2
). The distributions are comparable to the profile of a related strain cultured under 1 µmol photons m2 s1 of white light (Airs et al., 2001b
). The chromatogram (Fig. 4
) reveals the main group of six major BChl e homologues, eluting between 17 and 22 min (peaks 38). These components were identified from their LC-MS/MS spectra and corresponded to farnesyl-esterified BChl e (BChl eF) with three different alkylated states, i.e. [Et, Et] (peaks 3 and 4), [n-Pr, Et] (peaks 5 and 6) and [i-Bu, Et] (peaks 7 and 8) at positions C-8 and C-12, respectively (see Table 2
). The [Et, Me] BChl eF reported by Airs et al. (2001b)
was not detected. In the chromatogram, this group eluted between a first group of two BChl e-type pigments (peaks 1 and 2) and a second group of pigments comprising BChl e forms esterified with alcohols other than farnesol (peaks 919, tr. 2535 min) and unidentified pigments (peaks 2024). LC-MS/MS analysis (cf. Airs et al., 2001b
) revealed BChl e esterified with geranylgeraniol (peak 10) and a number of straight-chain alcohols (Table 2
). Previously, dodecanol (C-12) and tetradecanol (C-14) have been reported as esterifying alcohols in this species (Airs et al., 2001a
). These uncommon esterifying alcohols were present only in minor amounts in the previous study and have not been detected in the present study, where the samples were cultured under green light. A number of carotenoids eluted between 60 and 75 min, including isorenieratene and
-isorenieratene as the main components.
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DISCUSSION |
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In the present work, we chose to simulate the light conditions that are characteristic of the water column and the benthos of deep lakes, by using a range of green light intensities (480615 nm). Two different species of Chlorobiaceae were studied using the same incubation conditions to check the species-specificity of responses. Both Chl. phaeobacteroides and Ptc. aestuarii were able to grow under green light. The growth rates of Ptc. aestuarii under green light achieved a maximum of 0·06 h1 at light intensities exceeding 6 µmol photons m2 s1, which is lower than the maximum observed under white light (approx. 0·1 h1). Under green light, the maximal growth rates of Chl. phaeobacteroides were slightly higher (0·07 h1) than observed for Ptc. aestuarii and were achieved at 3·5 and 4·3 µmol photons m2 s1 green light.
Both strains exhibited changes in pigment composition in response to the different green light intensities used for culture, but in very different ways. In extracts of Ptc. aestuarii we found farnesol (m/z 204; see Table 1) as the predominant esterifying alcohol of BChl c for all the green light intensities tested. These observations are in agreement with previous studies in which most of the BChl c of green sulfur bacteria has been reported to be esterified with farnesol (Caple et al., 1978
). Borrego & Garcia-Gil (1994)
suggested the occurrence of secondary homologues of BChl c, comprising forms that are esterified with other alcohols. The proportion of the different alkylated forms of BChl c esterified with farnesol showed very little variation, although some dependence on light was observed. Our observations are in agreement with previous findings (Borrego et al., 1999a
) suggesting that the highly alkylated form, now identified as BChl cF [n-Pr, Et] or [i-Bu, Me], proportionally increased with decreasing light intensities down to 1·2 µmol photons m2 s1. Accordingly, the less alkylated form, now identified as BChl cF [Et, Me], increased with increasing light intensity. The content of BChl cF [Et, Et] or [n-Pr, Me] remained constant at a level of 55 %. The specific content of BChl c of Ptc. aestuarii was maximal at 400±150 nmol BChl c (mg protein)1, when the bacterium was cultivated under the intermediate green light intensity of 36 µmol photons m2 s1. These observations are comparable to those made previously by Guyoneaud et al. (2001)
, where the same strain was cultivated at a light intensity of 5 µmol photons m2 s1 white light, and exhibited a BChl c content of 430 nmol BChl c (mg protein)1. Under white light, the strain also achieved a higher specific growth rate of about 0·1 h1, which was attained at 100 µmol photons m2 s1.
Under green light, the cultures of Chl. phaeobacteroides showed a slightly higher specific growth rate than Ptc. aestuarii, i.e. 0·07 h1, which was achieved at 4·3 µmol photons m2 s1, a lower saturating value than observed for Ptc. aestuarii. This confirms that the brown-coloured Chlorobiaceae are better adapted to low light than the green strains, as reported previously in many studies (Overmann et al., 1992; Borrego & Garcia-Gil, 1995
; Borrego et al., 1997
; Airs et al., 2001b
). In contrast with the observations for Ptc. aestuarii cultures, the BChl e distributions contained a number of components with different esterifying alcohols (see Fig. 3
and Table 2
). For Chl. phaeobacteroides, the bacteriochlorophylls were esterified with farnesol and other isoprenoids as well as straight-chain C15, C15 : 1, C16, C16 : 1 and C17 alcohols. Bacteriochlorophylls esterified with alcohols other than farnesol have been reported previously, but were usually found in minor proportion compared to farnesyl esters (Caple et al., 1978
; Borrego et al., 1998
). The alcohols are grouped according to the biosynthetic pathways, i.e. isoprenoid and straight chain. The results reported here show that the relative proportion of each family depends on the light intensity used for culture. The straight-chain esters increased proportionally with increasing light, with a high contribution from hexadecanyl esters at all intensities tested. Conversely, the relative proportion of isoprenoid alcohols increased at the lower intensities and comprised mainly farnesol. When Chl. phaeobacteroides was cultured at higher intensities, the isoprenoid alcohols were dominated by geranylgeraniol, suggesting that this longer isoprenoid chain (C20) was synthesized when green light intensity was increased.
The complexity and the species-specificity of the responses in pigment composition to different light intensities make it very difficult to understand their eco-physiological significance. We assume that the response is either an acclimation to optimize the functioning of the photosynthetic apparatus under the prevailing light conditions or a pragmatic response to optimize efficiency in biosynthetic pathways. Ptc. aestuarii responded to low light intensity by increasing the extent of alkylation at C-8 and C-12. While these modifications do not change the in vitro absorption spectra, a higher proportion of the more extended tetrapyrrole macrocycle (TMC36) in the BChl c aggregates results in a red shift in the absorption spectra of the chlorosomes, and the impact of this phenomenon on the whole-cell in vivo absorption spectra has been documented for this species (Guyoneaud et al., 2001). It remains questionable, however, whether this spectral modification increases light harvesting at low light, particularly under green light conditions.
Chl. phaeobacteroides responded to low light by increasing the relative proportion of components esterified with isoprenoid alcohols. Surprisingly, a very high proportion (80 %) of straight-chain esterifying alcohols was found at higher light intensities. It has been suggested that the different C-8 and C-12 side chains on the bacteriochlorophylls modify the aggregation and therefore the packing of structures in the chlorosomes, with an impact on the in vivo absorption spectrum (Airs et al., 2001b), which might provide a basis for a cellular response to light limitation (Borrego et al., 1998
). On the other hand, we need to consider the low specificity of bacteriochlorophyll synthase. For example, the incorporation of exogenous alcohols into bacteriochlorophylls has been shown for Chloroflexus aurantiacus and Chlorobium tepidum (Larsen et al., 1995
; Steensgaard et al., 1996
). Clearly, the use of exogenous alcohols can represent a means for the growing cell to save energy, provided that uptake does not impair light harvesting for photosynthesis. Since our experiments were performed using mineral media, the alcohol groups in BChl e were obtained mainly through direct biosynthesis from CO2 and reducing power, in part, perhaps, from reallocation, turnover and/or conversion of biomolecules. Accordingly, we compared the biosynthetic pathways for the straight-chain alcohols (1) and isoprenoid alcohols (2).
The long straight-chain alcohols are synthesized by a reductive pathway comprising fatty acid biosynthesis (Stryer, 1995; Schweizer, 1989
) and subsequent reduction of the carboxy group into the corresponding alcohol:
(1) 8 Acetyl-CoA+7 ATP+14 NADPH+6 H+palmitate+14 NADP++8 CoA+6 H2O+7 ADP+7 Pi
Two additional NADPH2 are required to convert palmitate to the corresponding C16 alcohol:
(2) 9 Acetyl-CoA+3 H2O+6 NADPH+6 H++9 ATP9 CoA+6 NADP++9 ADP+3 Pi+3CO2+farnesyl PP+2 PPi
In terms of reducing power, the formation of fatty acids such as palmitate needs more than twice as much NADPH2 as the synthesis of an isoprenoid form such as farnesyl. Additional NADPH2 is required to convert palmitate to the corresponding C16 alcohol. Thus, synthesis of reduced straight-chain alcohols has a very high demand for reducing power. In Chlorobiaceae, it was generally assumed that NADP is reduced to NADPH2 by reduced ferredoxin, which would be coupled directly to linear electron transport in photosynthesis. However, no genes encoding the enzyme involved (ferredoxin NADP+ reductase) are present in the complete genome sequence of Chlorobium tepidum (Eisen et al., 2002) and it has been proposed that NADPH2 is obtained through uphill electron flow coupled to dissipation of proton-motive force. Accordingly, both NADP+ reduction and ADP phosphorylation to ATP are driven by proton-motive force, formation of which is mainly driven by photosynthesis in Chlorobiaceae. Accordingly, from energetic constraints it can be expected that the formation of isoprenoid forms is favoured over straight-chain fatty acids at low light intensities, because the greatly decreased NADPH2 requirement largly compensates the slightly higher requirement of ATP. In contrast, at higher light intensities, more energy is available for the synthesis of straight-chain counterparts, in keeping with the overall changes in the distributions observed at different light levels (Fig. 6
). Thus, it appears that the variations in the relative proportions of isoprenoid with respect to linear esterifying alcohols of BChl e in Chl. phaeobacteroides can be understood as a consequence of the energy available for biosynthesis in the cell, rather than representing a subtle mechanism of photo-acclimation to the prevailing light conditions. Self-assembly of BChl c and probably BChl e into a rod structure is achieved by spatial arrangement, with hydrogen bonding linking the C-13 carbonyl group and the C-31 hydroxy group of different bacteriochlorophyll molecules located at different layers. The latter group ligates the Mg of another bacteriochlorophyll molecule (Balaban et al., 1995
). The alcohol esterified at position C-17 is believed not to influence this self-assembly or the energy transfer (A. R. Holzwarth, personal communication). This may explain why variations in the esterifying alcohols are mainly determined by the energetic state of the cell and probably do not represent a mechanism of light acclimation.
For this reason, we think that it is difficult to compare field observations directly with liquid cultures using mineral media. Because of the pragmatic esterification of different alcohols, which appears to be ruled by the energetic status of the cells, we can anticipate that incorporation of exogenous alcohols is important in field populations and this might obscure the interpretation when we compare laboratory experiments directly with field observations.
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ACKNOWLEDGEMENTS |
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Received 22 April 2004;
revised 27 April 2004;
accepted 3 May 2004.
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