Ammonium metabolism in the green hydra symbiosis.
Invertebrate-microalgal symbioses are able not only to reassimilate catabolically produced ammonium but also to take up and assimilate ammonium from the environment (Kawaguti, 1953; Cates and McLaughlin, 1976; Szmant-Froelich and Pilson, 1977; Muscatine and D'Elia, 1978; Muscatine et al., 1979; Wilkerson and Muscatine, 1984; Wilkerson and Trench, 1986; Rees, 1986; McAuley, 1990). However, there is some controversy over whether ammonium assimilation is due to the symbiotic algae, or to the animal host, or to the combined activities of both (Miller and Yellowlees, 1989). The possibility that animal rather than algal enzymes may be responsible, in part or in whole, for assimilation of ammonium has important implications for the mechanisms by which animal hosts regulate cell division in populations of symbiotic algae, since a number of workers have suggested that the growth of populations of symbiotic algae may be nitrogen-limited (Rees, 1986; Cook and D'Elia, 1987; McAuley, 1987a).
Miller and Yellowlees (1989) pointed out that in corals symbiotic with dinoflagellates (zooxanthellae), levels of the ammonium assimilatory enzyme NADPH-glutamate dehydrogenase (NADPH-GDH) are higher in host tissue than in symbionts, although the catabolic production of ammonium probably exceeds the capacity of host NADPH-GDH (Rahav et al., 1989; Falkowski et al., 1993; Spencer-Davies, 1992), which has a low specificity for ammonium (Catmull et al., 1987). More recently, glutamine synthetase (GS) activity was reported in giant clam (Tridacna gigas) and coral (Pocillopora damicornis) host tissues (Fitt et al., 1993; Yellowlees et al., 1994). Because there are relatively high levels of ammonium-assimilating enzyme in host tissues, it is possible that much of the ammonium present in natural seawater concentrations is assimilated before it reaches the symbiotic zooxanthellae (Yellowlees et al., 1994).
However, several lines of evidence suggest that some if not all ammonium assimilation is due to the zooxanthellae. Assimilation is light dependent and does not occur in aposymbiotic animals (for review, see Rees, 1986). Rates of uptake by intact associations are similar to those of freshly isolated symbionts (D'Elia et al., 1983; Wilkerson and Trench, 1986), and intact associations exhibit uptake kinetics which suggest that symbionts assimilate ammonium from seawater by depleting ammonium in host tissue and causing a diffusion gradient to form (D'Elia and Cook, 1988). Finally, addition of ammonium to sea-water causes increases in zooxanthellar mitosis, biomass, and amino acid levels (Cook et al., 1988; Muscatine et al., 1989; Hoegh-Guldberg and Smith, 1989; Dubinsky et al., 1990; Fitt and Cook, 1990; Stambler et al., 1991; McAuley, 1994; Muller-Parker et al., 1994; McAuley and Cook, 1994), and reduces ammonium uptake rates (Yellowlees et al., 1994). Ammonium assimilation is assumed to involve the coupled glutamine synthetase/2-oxoglutarate amido transferase (GS/GOGAT) pathway. At present, GS but not GOGAT has been detected in zooxathellae (Summons and Osmond, 1981; Wilkerson and Muscatine, 1984), although azaserine, a potent inhibitor of GOGAT, prevents ammonium uptake in intact corals (Rahav et al., 1989), and the presence of GS/GOGAT activity is supported by the data of Summons et al. (1986).
In contrast to marine symbioses, in the symbiosis between the freshwater cnidarian Hydra viridissima (green hydra) and Chlorella algae, the host rather than the algal symbionts may be responsible for assimilation of ammonium. Ammonium assimilation by maltose-releasing Chlorella is inhibited at the low pH that stimulates maltose release (Rees, 1989). Recent evidence suggests that in symbiosis the algae are carbon- rather than nitrogen-limited, in that in vivo release of maltose consumes considerable amounts of photosynthetically fixed carbon that would otherwise be used to assimilate ammonium into amino acids (McAuley, 1992). Treatment of the intact symbiosis with the photosynthetic inhibitor DCMU does not cause ammonium excretion into the medium, and rates of ammonium uptake by freshly isolated symbionts are only 40% of those of the intact symbiosis (Rees, 1986; McAuley, 1990). Finally, the hosts possess GS, and levels of GS activity are higher in symbiotic than aposymbiotic animals (Rees, 1986).
Although green hydra do not normally excrete ammonium into the medium, release can be induced by treatment with methionine sulphoximine (MSX), which inhibits GS in intact hydra (Rees, 1986). However, the effect of MSX on algal GS has not been tested, because of the difficulty in isolating sufficient numbers of symbionts uncontaminated by host material. This paper describes the effects of inhibitors of enzymes of ammonium assimilation on perturbation of animal and algal internal pools of glutamate and glutamine, amino acids closely associated with ammonium assimilation (Miflin and Lea, 1976). Recovery of symbiotic algae after SDS washing, which is necessary to remove host contamination, is low (McAuley, 1986a), but unlike assays for enzyme activity, measurement of free amino acid pools by HPLC requires only small numbers of algae (the equivalent of [10.sup.4] cells or fewer per sample injection). Uptake of MSX by algae in vitro and in the intact symbiosis was also measured using HPLC.
Materials and Methods
Maintenance of organisms
Green hydra of the European strain (EE hydra) were grown in unbuffered 'M' solution (Muscatine and Lenhoff, 1965) at 15 [degrees] C in constant light (60 [[micro]mol] photons [m.sup.-2] [s.sup.-1] PAR). Cultures were fed each Monday, Wednesday, and Friday with freshly hatched brine shrimp. All hydra used in experiments had not been fed for the previous 3 days to allow complete digestion of food. Cultures of the 3N813A strain of maltose-releasing Chlorella, grown in Kessler's medium (Kessler et al., 1963), pH 6.3, were maintained in a shaking, illuminated incubator in growth conditions similar to those of hydra cultures.
Isolation of symbiotic algae from hydra
The SDS-washing technique (McAuley, 1986a) was used to isolate symbiotic Chlorella algae from hydra.
Determination of numbers of algae per digestive cell
Five gastric regions of hydra were isolated in a drop of macerating fluid (David, 1973). After 10 min, pieces of hydra were teased apart into individual cells and examined using phase contrast microscopy. Numbers of algae were determined in 100 randomly selected digestive cells in each preparation.
Measurement of ammonium uptake
Ammonium uptake was measured as depletion from the medium of either hydra or algae. Duplicate samples (200 [[micro]liter]) were taken at the beginning and end of experiments, and amounts of ammonium were determined with a scaled-down hypochlorite-nitroprusside colometric assay (Rees, 1986). After reagents were added, samples were incubated for 20 min in darkness, then absorbence was read at 630 nm and compared to that of ammonium sulphate standards (reagents and standards from Sigma Chemical Company).
Methionine sulphoximine (MSX), an inhibitor of GS (Ranzio et al., 1969; Meister, 1974), and azaserine (AZS), an inhibitor of 2-oxoglutarate amido transferase (GO-GAT) (Wallsgrove et al., 1977; Elrifi and Turpin, 1986), were purchased from the Sigma Chemical Company. Stock solutions were stored at 4 [degrees] C and routinely tested by measuring their effectiveness in inhibiting ammonium uptake by cultured 3N813A cells. In all experiments, MSX was used at a final concentration of 200 [[micro]molar] and AZS at 1 mM.
Extraction and measurement of free amino acid pools
Samples of algae, or of animal homogenates centrifuged at 1000 X g for 5 min to remove algae, were added to absolute ethanol to give 80% ethanol (v:v) and extracted for 24 h at 4 [degrees] C. Protein contents of homogenates were determined using the Bradford method (Bradford, 1976). In some cases, algal samples were filtered at low vacuum through Whatman GF/C filters, which were then extracted twice in 80% ethanol. Extracts were dried in vacuo and resuspended in an appropriate volume of 12.5 [[micro]molar] [Alpha]-amino-butyric acid (AABA), which acted as an internal standard. Amino acid contents of aliquots of extracts were determined by o-phthaldialdehyde pretreatment and reverse-phase HPLC as previously described (McAuley, 1992). MSX but not AZS was detectable using this system; the MSX peak occurred 0.8 min after that of serine.
Glutamine and methylamine uptake
Glutamine uptake was determined from cultured 3N813A cells resuspended in 10 mM MES buffer pH 7 at a density of 5 x [10.sup.7] cells [m.sup.-1]. After 30 min of preincubation, the assay was started by adding 2-20 [[micro]molar] glutamine containing 3.7 [Mu]Bq [[U.sup.-14]C]-glutamine (Amersham International; specific activity 9.25 GBq m[mol.sup.-1]) with or without additional 20 [[micro]molar] AZS. At intervals of 1, 2, and 3 min after addition of radioactivity, 200-[[micro]liter] samples were filtered onto Whatman GF/C filters at low vacuum and washed with 20 ml distilled water. Filters were dried in scintillation vials, 10 ml of scintillation fluid was added (McAuley, 1988), and samples were counted on an LKB 1214 Rackbeta scintillation counter. [V.sub.max] and [K.sub.M] were determined from Lineweaver Burke double reciprocal plots of uptake rates against substrate concentration.
Uptake of [14C]-methylamine was determined by the same method as glutamine uptake, except that [14C]-methylamine (Amersham International; specific activity 2.22 GBq m[mol.sup.-1]) was added to give a final concentration of 5 [[micro]molar]. At intervals, 100-[[micro]liter] samples were filtered onto Whatman GF/C filters and radioactivity was determined as described above.
About 100 hydra, previously starved for 3 days, were homogenized in extraction buffer as described by Rees (1986). The homogenate was centrifuged at 13000 x g for 5 min at 4 [degrees] C, and the supernatant was assayed for GOGAT activity by determining the rate of oxidation of NAD(P)H at 340 nm by the method of Bhandari and Nicholas (1981). Blanks were run without addition of [Alpha]-ketoglutarate.
Effect of ammonium on symbionts in intact hydra
Incubation of EE hydra for 7 days in M solution supplemented with 50 [[micro]molar] ammonium chloride had no effect on the size of the glutamate and glutamine pools of their algal symbionts (Table I). However, significantly more algae (one-way ANOVA, P [less than] 0.05, n = 6 independent experiments) were present in digestive cells of hydra maintained in ammonium-supplemented medium (24.94 [+ or -] 1.71 SD) than in controls (22.58 [+ or -] 1.48).
Effect of MSX and AZS on ammonium uptake
As previously observed (Rees, 1986; McAuley, 1990), MSX not only inhibited ammonium assimilation but also caused release of ammonium from both intact hydra and cultured 3N813A algae (Table II). AZS inhibited ammonium assimilation but did not cause release in 3N813A algae; ammonium release by green hydra treated with AZS was seven times lower than that observed with MSX treatment.
Effect of MSX and AZS on internal pools of glutamate and glutamine in 3N813A
3N813A algae were incubated with either MSX or AZS in the absence or presence of a nitrogen source (either glutamine or ammonium), and amino acid pools were compared to those of untreated controls (Table III). MSX and AZS had distinct effects on internal glutamine and glutamate pools irrespective of the nitrogen supply, and these effects were consistent with inhibition of GS or GOGAT respectively. MSX significantly decreased glutamine and AZS significantly decreased glutamate in all treatments. In algae without a nitrogen source or supplied with glutamine, MSX also significantly decreased glutamate pools; in algae without a nitrogen source or supplied with ammonium, AZS significantly increased glutamine pools.
Table I Effect of incubation of intact hydra in ammonium on glutamate and glutamine pools of algal symbionts
Ammonium Controls treated
Glu 574.1 [+ or -] 38.0 578.9 [+ or -] 56.7 Gln 150.3 [+ or -] 18.5 146.7 [+ or -] 29.3 Gln:Glu 0.26 [+ or -] 0.03 0.26 [+ or -] 0.06
EE Hydra were starved for 7 days in M solution [+ or -]+50 [[micro]molar] ammonium chloride; incubation solutions were changed every day. After 7 days, hydra were washed five times in large volumes of M solution and homogenized, and amino acid pools in SDS-washed freshly isolated symbionts were determined as described in Materials and Methods. Values are the means [+ or -] SD of amino acid content (amol [cell.sup.-1]) of glutamate and glutamine pools determined from three independent experiments.
[TABULAR DATA FOR TABLE II OMITTED]
Supply of nitrogen also affected glutamine and glutamate pools. Glutamine increased in algae supplied with ammonium and glutamine, and glutamate increased in algae supplied with glutamine (one-way ANOVA, P [less than] 0.05). Tukey post-hoc tests showed that the size of the glutamine pools was similar in all MSX and all AZS treatments (P [greater than] 0.05). The size of the glutamate pools was also similar in all AZS treatments (P [greater than] 0.05), but glutamate levels were higher in algae treated with MSX in the presence of glutamine than in the presence of ammonium or the absence of a nitrogen source.
Effect of MSX and AZS on internal pools of glutamate and glutamine in freshly isolated symbionts and in the intact symbiosis
The effect of maltose release on the changes the two inhibitors produce in glutamate and glutamine pools was tested on freshly isolated symbionts treated at pH 5 and at pH 7. Maltose release is pH dependent (Cernichiari et al., 1969) and occurs at the former but not the latter pH.
When freshly isolated symbionts were treated with MSX and AZS at pH 7, the effects were similar to those seen when 3N813A algae were treated without a nitrogen source: MSX caused a significant decrease in glutamine and glutamate; AZS caused a significant decrease in glutamate and an increase in glutamine (Table IV). At pH [TABULAR DATA FOR TABLE III OMITTED] 5, AZS treatment also caused a significant decrease in glutamate and an increase in glutamine, but whereas MSX treatment decreased glutamate, glutamine levels tended to increase, although this trend was not significant. Uptake of MSX at pH 5 was considerable (over 3000 amol [cell.sup.-1]), and the large MSX peak, adjacent to that of glutamine, may have interfered with detection of glutamine. When an amount of MSX equivalent to that found in the algae was added to a sample containing 10 pmol glutamine, the apparent amount of glutamine detected by HPLC increased 30%. The smaller MSX peak at pH 7 did not interfere with glutamine detection.
[TABULAR DATA FOR TABLE IV OMITTED]
Treatment of freshly isolated symbionts at pH 5 caused a significant reduction in both glutamate and glutamine pools compared to those at pH 7 (one-way ANOVA, P [less than] 0.05).
Incubation of intact green hydra in MSX caused no significant change in the glutamate or glutamine pools of either the symbiotic algae or the host (Table V). Only 16.2 [+ or -] 30.2 SD amol [cell.sup.-1] MSX was detected in algal pools after 24-h treatment of intact hydra, compared to high levels in freshly isolated symbionts after only 90 min of treatment (3126.0 [+ or -] 227.6 amol [cell.sup.-1] in freshly isolated symbionts treated at pH 5; 495.3 [+ or -] 175.7 amol [cell.sup.-1] in freshly isolated symbionts treated at pH 7). Treatment of intact hydra with AZS increased glutamine pools in both hydra and algae, although the increase was significant only in hydra. AZS treatment did not cause a reduction in either host or algal glutamate pools, in contrast to the consistent effect of AZS on glutamate pools measured in both freshly isolated symbionts and 3N813A irrespective of pH or nitrogen supply.
Effect of MSX and AZS on glutamine and methylamine uptake by 3N813A algae
[14C]-glutamine uptake by 3N813A algae was competitively inhibited by a low concentration of AZS [ILLUSTRATION FOR FIGURE 1 OMITTED]. [K.sub.M] was 17.3 [[micro]molar] in the absence of AZS and 28.6 [[micro]molar] in the presence of AZS. In both cases, [V.sub.max] was 250 amol glutamine [cell.sup.-1] [h.sup.-1].
There was linear uptake of the ammonium analog [14C]-methylamine in the presence of both MSX and AZS, although uptake rates were reduced by 25.1% and 33.4% respectively, in comparison to controls [ILLUSTRATION FOR FIGURE 2 OMITTED]. In contrast, ammonium chloride inhibited methylamine uptake [ILLUSTRATION FOR FIGURE V OMITTED] by 98% for the first 4 min, although the uptake increased gradually thereafter. Calculations based on ammonium uptake rates in Table I indicated that the algae would have greatly depleted ammonium concentration at the time that uptake of methylamine began to increase.
Uptake of glutamine by freshly isolated symbionts
Short-term experiments showed that incubation of freshly isolated symbionts in 100 [[micro]molar] glutamine caused an increase in internal glutamine and glutamate pools at both pH 5 and pH 7 [ILLUSTRATION FOR FIGURE 3 OMITTED].
Neither NADH- or NADPH-specific GOGAT activity was detectable in hydra tissue.
Several lines of evidence suggest that assimilation of ammonium in the green hydra-Chlorella symbiosis is due to GS in the animal host rather than in the algal symbiont. Incubation of green hydra in medium supplemented with ammonium chloride caused a small, significant increase in numbers of algae per host digestive cell, but there was no effect on glutamine and glutamate pools or on the Gln: Glu ratio of the algae (Table I), the latter being an indicator of nitrogen sufficiency in microalgae (Flynn et al., 1989). In marine symbioses, in which it is believed that symbionts are able to assimilate ammonium from seawater, ammonium supplementation not only causes large increases in the population density of symbionts (Muscatine et al., 1989; Hoegh-Guldberg and Smith, 1989; Dubinsky et al., 1990; Fitt and Cook, 1990; Stambler et al., 1991; Muller-Parker et al., 1994), but, in a coral and a hydroid, also increases the size of internal glutamine pools of symbionts and hence their Gln:Glu ratio (McAuley, 1994; McAuley and Cook, 1994).
Further, although MSX inhibits ammonium uptake by intact hydra (Rees, 1986; McAuley, 1990; Table II, this paper), almost no MSX was detected in symbiotic algae isolated from hydra that had been incubated in MSX for 24 h, and there was no change in algal internal pools of glutamate and glutamine. In contrast, freshly isolated symbionts treated with MSX rapidly accumulated large amounts of the inhibitor, and MSX treatment caused predictable changes in glutamate and glutamine pools of freshly isolated symbionts and cultured 3N813A algae in a variety of conditions (Tables III and IV).
In addition to inhibiting GS, MSX has been shown to be a noncompetitive inhibitor of active ammonium transport in Anabaena flos-aquae (Turpin et al., 1984). In 3N813A, both MSX and AZS caused a degree of inhibition of uptake of methylamine [ILLUSTRATION FOR FIGURE 2 OMITTED], a structural analog of ammonium that is taken up by the same transport system (Hackette et al., 1970; Pelley and Bannister, 1979; Smith, 1982; Wright and Syrett, 1983). However, if both MSX and AZS caused noncompetitive inhibition of entry of ammonium into symbionts, release of ammonium from the intact symbiosis would presumably be the same in both treatments. Instead, release was seven times higher in hydra treated with MSX than in hydra treated with AZS (Table II). As shown in 3N813A cells, MSX blocks assimilation of ammonium into glutamine via GS (Ranzio et al., 1969; Meister, 1974), leading to low glutamine pools and release of ammonium. AZS blocks subsequent metabolism of glutamine to glutamate via GOGAT (Wallsgrove et al., 1977; Elrifi and Turpin, 1986), leading to accumulation of glutamine, low glutamate pools, and no ammonium release. That hydra release more ammonium when treated with MSX than with AZS is consistent with inhibition of GS activity pathways rather than with noncompetitive inhibition of ammonium transport into the algae. Because very little MSX was detected in symbionts after treatment of intact hydra, the ammonium release observed during MSX treatment of the intact symbiosis must have been due to inhibition of host GS.
No GOGAT was detected in crude, alga-free extracts of homogenized green hydra. However, treatment of intact hydra with AZS, an inhibitor of GOGAT, prevented uptake of ammonium from the medium by the intact symbiosis and caused a significant increase in host glutamine pools (Tables II and V). This may be explained if glutamine resulting from activity of host GS was taken up by the algae and convened into glutamate via GOGAT. Uptake of glutamine by algae would also explain why MSX treatment did not cause an increase in host glutamine pools.
AZS treatment may have affected this coupling in two ways. First, AZS may have specifically inhibited algal GOGAT, although AZS treatment of the intact symbiosis did not cause the reduction in glutamate and increase in glutamine consistently observed in 3N813A or in freshly isolated symbionts. Second, AZS, which competitively inhibited uptake of glutamine in 3N813A algae [ILLUSTRATION FOR FIGURE 1 OMITTED], prevented entry of glutamine into either the perialgal vacuole or the symbiotic algae themselves. In either case, the glutamine transport system must be specific for glutamine and AZS but not MSX. Given that symbionts accumulated MSX in vitro but not in hospice, it is possible that transport systems in the host perialgal vacuolar membrane differentially control entry of substances into the perialgal vacuole.
Supply of nitrogen to algal symbionts as glutamine or other amino acids rather than as ammonium may be important because a supply of carbon is required for assimilation of ammonium into amino acids (Turpin, 1991), and symbionts release a high proportion of fixed carbon to their host in the form of maltose (Mews, 1980). In cultured 3N813A, maltose synthesis and release inhibits ammonium uptake because it diverts photosynthetically fixed carbon from assimilation of ammonium into amino acids (McAuley, 1992). Maltose release is stimulated by low pH, and ammonium uptake by freshly isolated symbionts and cultured maltose-releasing algae falls as pH is reduced and maltose release increases (Rees, 1989). Below a critical pH value, the algae actually begin to release ammonium, not only because fixed carbon is diverted from the TCA cycle to maltose release, but also because amino acids may be deaminated to provide carbon skeletons for maltose synthesis (Rees, 1990).
Given that high rates of maltose release and ammonium assimilation are incompatible, amino acids may provide an alternative source of nitrogen for algae in symbiosis with green hydra. Freshly isolated symbionts have active amino acid transport systems (McAuley, 1986b), and algae in the intact symbiosis can assimilate a variety of amino acids supplied by host feeding (McAuley, 1987b, 1988, 1991). Further, the rate of glutamine uptake by freshly isolated symbionts peaks between pH 4 and 5, the same range over which release of maltose is maximum (McAuley, 1991). Maltose-releasing symbionts using glutamine as a nitrogen source would require carbon skeletons in the form of 2-oxoglutarate to produce two molecules of glutamate from each molecule of glutamine. But even if one molecule of glutamate was deaminated, the carbon recycled to 2-oxoglutarate, and the ammonium released to be reassimilated by the host, the algae would still gain a new molecule of glutamate for each molecule of glutamine taken up. Short-term experiments showed that uptake of glutamine by freshly isolated symbionts at both pH 5 (with maltose release) and pH 7 (without maltose release) caused an increase in the size of internal glutamate pools [ILLUSTRATION FOR FIGURE 3 OMITTED]. Although the increase in glutamate pools was similar, it should be noted that the rate of glutamine uptake by freshly isolated symbionts is more than 5 times higher at pH 5 than at pH 7 (McAuley, 1991).
This scheme is qualitatively but not quantitatively different from that in marine algal-invertebrate symbioses, in which symbiotic zooxanthellae appear to assimilate ammonium via coupled GS/GOGAT (for review, see Spencer-Davies, 1992). In green hydra, although both GS and GOGAT are involved in ammonium assimilation, they may be located in different compartments, and the necessity for glutamine to traverse the perialgal vacuole would produce a point at which nitrogen supply to the algae could be regulated.
This work was supported by grants from the Royal Society and the Natural Environmental Research Council. My thanks to Mr. Harry Hodge for his help in HPLC analysis of amino acids, and to an anonymous referee for helpful comments on a draft of this paper.
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|Publication:||The Biological Bulletin|
|Date:||Apr 1, 1995|
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