INTRODUCTION

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Coral bleaching is the disruption of the symbiotic association between coral hosts and their photosynthetic microalgal endosymbionts, referred to as zooxanthellae (12). Coral bleaching events of unprecedented frequency and global extent have been reported during the last two decades (13). It has been suggested that coral bleaching is triggered by environmental factors which impose stress on the coral. The most frequently reported stress condition is increased seawater temperature (5, 16). Thus, it is possible that global warming could result in alteration or destruction of coral reef systems. Consequently, it is essential to understand the mechanism(s) of coral bleaching.

Bleaching of the coral Oculina patagonica from the Mediterranean Sea is the result of a bacterial infection (17, 18). The causative agent, Vibrio shiloi, was obtained in pure culture and was shown to cause bleaching in controlled aquarium experiments. Furthermore, it was shown that bacterium-induced bleaching by V. shiloi could be inhibited by antibiotics. The bacterial infection and resulting coral bleaching were temperature dependent, occurring only at elevated seawater temperatures (25 to 30°C).

Using the V. shiloi-O. patagonica model system to study coral bleaching, Toren et al. demonstrated that the first step in the infectious process was adhesion of V. shiloi to a -galactoside-containing receptor on the coral surface (32). After V. shiloi adheres to O. patagonica, it penetrates into the exodermal layer of the coral (2). However, the mechanism by which the bacterium kills the algae is unknown. Recently, we reported that V. shiloi secretes extracellular materials that inhibit photosynthesis and bleach and lyse zooxanthellae isolated from corals (4). The material responsible for inhibition of photosynthesis was heat stable and was produced only when the bacteria were grown at elevated seawater temperatures.

In the present paper we describe production, purification, and characterization of a proline-rich dodecapeptide from V. shiloi that rapidly inhibits photosynthesis of zooxanthellae in the presence of NH₃.

	MATERIALS AND METHODS

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Bacterial strain and growth conditions. V. shiloi AK1 (= ATCC BAA-91), isolated from bleached coral as previously described (6), was used in this study. The strain was maintained on MB agar (1.8% marine broth [Difco] plus 0.9% NaCl solidified with 1.8% agar). After streaking, the plates were incubated at 30°C for 2 days and then allowed to stand for 1 week. For experiments described here, the bacteria were grown in MBT medium (1.8% marine broth, 0.75% tryptone, 0.9% NaCl), CA medium (0.75% Casamino Acids, 2% NaCl), and CAG medium (0.5% Casamino Acids, 0.5% glycerol, 2% NaCl) at 29°C with shaking.

Preparation of zooxanthellae from coral. Intact colonies of the coral O. patagonica were collected from a depth of 1 to 3 m along the Mediterranean coast of Israel. Within 2 h of collection, each colony was split into several pieces, and the pieces were placed into 2-liter aerated aquaria containing filtered (pore size, 0.45 µm) seawater that were maintained at 25°C. The aquaria were illuminated with a fluorescent lamp by using cycles consisting of 12 h of light and 12 h of darkness. To obtain zooxanthellae, a healthy coral fragment (surface area, ~1 cm²) was removed from an aquarium and rinsed gently with filter-sterilized seawater, and then the tissue was disrupted with a dental water pick by using ca. 50 ml of sterile seawater. The suspension was centrifuged for 30 min at 2,000 × g. The pellet, resuspended in 1 ml of seawater, was then centrifuged in an Eppendorf centrifuge (model 5402) for 4 min at 10,000 rpm. The pellet, resuspended in 1 ml of seawater, was then centrifuged for 21 min at 1,200 rpm. The final pellet was resuspended in seawater to a concentration of ca. 5 × 10⁶ algae per ml (based on hemacytometer counts). Fresh zooxanthella preparations were used in all experiments.

Purification of toxin P. Cultures of V. shiloi, grown in MBT medium for 72 h at 29°C, were centrifuged at 12,000 × g for 10 min at 4°C. The supernatant fluid was then passed through a 0.2-µm-pore-size Millipore membrane filter. Ammonium sulfate was added with stirring at 0°C to the cell-free supernatant fluid to a final concentration of 80% saturation. After the preparation stood overnight at 4°C, the precipitate was collected by centrifugation and dissolved in 1/10th the initial volume of water. The concentrated crude toxin P was then extracted three times with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and evaporated to dryness in vacuo at 30°C.

Measurement of photosynthetic quantum yield of zooxanthellae. A portable underwater mini pulse-amplitude-modulation fluorometer (Walz) was used to measure the quantum yield of zooxanthellae. This instrument allows direct noninvasive measurement of the effective quantum yield of photosystem II under ambient light conditions (15, 23-25). Good correlations between measurements of quantum yield and photosynthetic rates (determined by O₂ evolution and CO₂ uptake) have been reported for plants (10) and cyanobacterial symbionts of lichens (31).

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Determination of toxin P activity. In the initial growth experiments, toxin P activity was determined by removing a sample from a culture, centrifuging it to remove the cells, extracting the supernatant fluid with ethyl acetate three times, evaporating the combined ethyl acetate extracts to dryness in vacuo, and dissolving the residue in 50% ethanol. Dilutions of the solution (in sterile seawater) were then used to measure inhibition of photosynthesis of zooxanthellae in the presence of 12.5 mM NH₄Cl (unless stated otherwise), as described above. Inhibition due to NH₄Cl alone and ethyl acetate-extracted media (controls) was subtracted before toxin P activity was calculated. One unit of toxin P activity was defined as a 10% decrease in the quantum yield after 10 min of incubation. The same procedure was used for assaying purified toxin P except that the ethyl acetate step was omitted.

Micro sequence analysis. Automated Edman degradation of the purified peptide was performed with pulse liquid automatic sequentor (Applied Biosystems model 473A) by Technion Protein Laboratory, Haifa, Israel.

Solid-phase peptide synthesis of toxin (PYPVYAPPPVVP). Val-to-Val couplings are often sluggish, and Pro at the carboxyl terminus of a peptide has been associated with high levels of diketopiperazine formation during solid-phase peptide synthesis (28). Given the fact that the peptide toxin had a Val-Val-Pro sequence at the carboxyl terminus, a synthetic strategy that would minimize diketopiperazine formation during the Val-Val coupling step was developed. Accordingly, synthesis was carried out on a 2-chlorotrityl chloride resin because the bulk of the trityl handle minimized diketopiperazine formation during tripeptide formation. The tripeptide 9-fluorenylmethoxy-carbonyl (Fmoc)-Val-Val-Pro-Resin was synthesized manually, and after evaluation of its quality, chain assembly was completed by using an Applied Biosystems synthesizer.

	RESULTS

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Growth and toxin P production. V. shiloi grew exponentially with approximately the same doubling times (40 min) in MBT, CA, and CAG media, reaching final cell turbidities (A₆₀₀) of 1.7, 1.8, and 2.8, respectively (Fig. 1A). Extracellular photosynthetic inhibitor (toxin P) activity appeared after growth ceased and increased during the stationary phase (Fig. 1B). Although MBT medium supported the lowest cell yield, it yielded the highest toxin P activity (2.6 U/ml). Addition of glycerol to Casamino Acids medium (CAG medium) increased the cell yield but both delayed and inhibited toxin P production. The small amount of toxin P activity that was produced appeared only after the glycerol had been depleted from the medium. Furthermore, in glycerol-salts medium, V. shiloi grew to a final turbity (A₆₀₀) of 1.85 but produced no toxin P activity. Thus, glycerol, which is an excellent carbon source for growth of V. shiloi, inhibits toxin P production. Ethyl acetate extraction of cells from exponential- and stationary-phase cultures in MBT, CA, and CAG media resulted in only traces of toxin P activity, indicating that the cells did not accumulate the toxin (data not shown).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Kinetics of growth and toxin P production in different media. An overnight culture of V. shiloi was inoculated into MBT (), CA (), and CAG () media and incubated with shaking at 29°C. At intervals samples were removed for determinations of growth turbidity (A) and toxin P activity (B) as described in Materials and Methods.

Purification of toxin P. Toxin P was purified to homogeneity by five purification steps (Table 1). Precipitation with an 80% (NH₄)₂SO₄ solution, followed by ethyl acetate extraction, concentrated and partially purified the activity. When a cation-exchange (RQ) column was used, all of the activity that was recovered eluted as a single peak in the flowthrough volume. Toxin P activity eluted as a single peak with an apparent molecular weight of approximately 14,000 as determined by gel filtration (Fig. 2A). In the final purification step, in which a hydrophobic column was used, the activity eluted as a symmetrical peak at 35% ACN (Fig. 2B). The overall yield was 26%, and the toxin was purified 35-fold from the ammonium sulfate fraction to the final product. HPLC and thin-layer chromatography demonstrated that the product was more than 95% pure.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Chromatographic purification of toxin P. (A) Partially purified toxin P was applied to a Superdex Peptide HR 10/30 (gel filtration) column and eluted with 50% ethanol. The arrows indicate the elution volumes of standard molecular weight markers. (B) The active fractions from the gel filtration column (14 to 16 ml) were concentrated, applied to an RP18 hydrophobic column, and eluted with increasing ACN concentrations.

Chemical properties of toxin P. Pure toxin P was subjected to Edman degradation, which gave the following 12-residue sequence: PYPVYAPPPVVP. This sequence fits precisely the molecular mass of 1,295.54 Da determined by mass spectroscopy. Thus, toxin P is a linear, proline-rich dodecapeptide. The calculated pI is 5.99. Toxin P had an absorbance maximum at 275 nm at pH 7.0, and a molecular extinction coefficient of 2,800, which is typical of a peptide with two tyrosine residues.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Gel filtration of toxin P in 50% ethanol () and 50 mM Tris-HCl buffer (pH 8.0) (). Purified toxin P was eluted on a Superdex Peptide HR 10/30 column, and fractions were analyzed for photosynthesis-inhibiting activity as described in Materials and Methods. The arrows indicate the positions of elution of standard molecular weight markers.

Inhibition of algal photosynthesis by toxin P. In the presence of NH₄Cl, natural toxin P rapidly inhibited photosynthesis of zooxanthellae (Fig. 4). One minute after addition of the toxin and NH₄Cl to the algae, the photosynthetic quantum yield decreased 27%, from 0.55 to 0.40. Inhibition became progressively stronger for the next 3 min and then leveled off at 64%. Under the same conditions, NH₄Cl by itself inhibited the quantum yield by 18% after 5 min, and then the quantum yield remained constant. Similar results were obtained with the synthetic toxin P. In the absence of NH₄Cl, toxin P had no effect on algal photosynthesis.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Kinetics of inhibition of algal photosynthesis by toxin P. The photosynthetic quantum yield of fresh zooxanthellae was measured in seawater containing 20 mM Tris-HCl buffer (pH 8.0) and 10 µM toxin P (), 12.5 mM NH4Cl (), or 10 µM toxin P plus 12.5 mM NH4Cl (). A control containing zooxanthellae in seawater and buffer gave the same data as toxin P alone.

View larger version (10K): [in this window] [in a new window]   FIG. 5.   Inhibition of algal photosynthesis as a function of toxin P concentration. The experiment was performed as described in Materials and Methods by using 12.5 mM NH4Cl and different concentrations of the chemically synthesized peptide. The quantum yield was recorded after 10 min of incubation.

Binding of toxin P to zooxanthellae. To test if the effect of toxin P and NH₄Cl on zooxanthella photosynthesis was reversible, algae were washed extensively after different treatments. The quantum yields of the washed algae were then measured before and after addition of NH₄Cl (Table 2). The algae in the seawater control maintained a high photosynthetic quantum yield (0.60) after the washing treatment. Addition of 10 mM NH₄Cl to the washed control resulted in a 13% decrease in the quantum yield. Algae that were initially treated with NH₄Cl completely recovered their quantum yield after the washing procedure and showed a 15% decrease after a second addition of NH₄Cl. Interesting data were obtained with the algae treated initially with toxin P and toxin P plus NH₄Cl. As expected (from the data presented in Fig. 4), toxin P alone had no effect on quantum yield, whereas toxin P plus NH₄Cl resulted in 67% inhibition. Algae treated with toxin P and then washed extensively in seawater retained all of their photosynthetic activity, but when they were treated with NH₄Cl, they showed a 48% decrease in quantum yield. Thus, toxin P remained bound to the algal cells and subsequently exhibited its synergistic effect with NH₄Cl. Algae treated initially with both toxin P and NH₄Cl recovered photosynthesis efficiency only partially after washing. Addition of NH₄Cl to the washed cells led to strong inhibition of the quantum yield (83%), again indicating that toxin P remained bound to the zooxanthellae. The experiments described above were preformed with both natural toxin P and synthetic toxin P, which yielded identical data.

Toxin P-induced pH changes. When cells take up NH₃ more rapidly than NH₄⁺, the pH of the bulk liquid decreases as a result of dissociation of NH₄⁺ to NH₃ and H⁺. To test the hypothesis that toxin P catalyzes movement of NH₃ into zooxanthellae, pH changes were monitored in algal suspensions containing NH₄Cl and NH₄Cl plus natural toxin P (Fig. 6). The pH of algae in unbuffered seawater remained constant at 7.8. Addition of 15 mM NH₄Cl caused the pH to drop to 7.6 in 5 min, and then the pH remained constant. Toxin P by itself had no effect on the pH (data not shown). However, addition of toxin P plus 15 mM NH₄Cl resulted in a large, rapid decrease in the pH, which reached 7.45 after 2 min and 7.2 after 7 min. Thus, toxin P facilitated transport of NH₃ into the algae. Titration of 15 mM NH₄Cl in seawater from pH 7.8 to 7.2 required 1.7 mM HCl. Thus, ca. 1.7 mM NH₃ (the equivalent amount of NH₄⁺ converting to NH₃ plus H⁺) must have entered the algae. Accordingly, each toxin molecule (concentration in the experiment, 10 µM) must have been responsible for transport of ca. 170 molecules of NH₃.

View larger version (18K): [in this window] [in a new window]   FIG. 6.   Toxin P-induced pH changes. To 1 ml of zooxanthellae (5 × 106 cells/ml) in unbuffered seawater 15 mM NH4Cl () or 10 µM toxin P plus 15 mM NH4Cl () was added. At intervals the pH of the suspension was measured. Algae in seawater served as a control ().

	DISCUSSION

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The data presented here demonstrate that toxin P is a linear, proline-rich dodecapeptide. It is likely that toxin P is produced from a larger peptide by proteolysis. The amino acid sequence of toxin P shows strong similarity (10 of the 12 amino acids are identical) to the amino acid sequence of an internal peptide in the vrg-6 gene product of Bordetella pertussis (GenBank accession number M77374). It has been suggested that the vrg gene products facilitate intracellular survival and the persistence of the bacterium in the host (3). In this regard it is interesting that V. shiloi is also an intracellular pathogen (2).

The high hydrophobicity predicted from the amino acid sequence of toxin P may help explain the observed binding of the toxin to zooxanthellae. Bound toxin P by itself did not affect algal photosynthesis. However, addition of NH₄Cl to algae containing the bound toxin resulted in rapid inhibition of photosynthesis and a concurrent decrease in the pH of the bulk liquid. Inhibition of photosynthesis by ammonia is well established (1, 7, 35). Ammonia acts as an uncoupler of photosynthesis by passing across membranes, thereby destroying the pH gradient across the thylakoid membrane (27, 34). Many membrane-penetrating peptides, including melittin (33), gaegurin (29), cecropin (22), and buforin (21), contain prolyl residues. Although the exact role of prolyl in the function of these peptides is unknown, proline causes kinks in helical polypeptides and increases the flexibility of peptide chains in its immediate environment. Some of these peptides cause lysis of cell membranes, while others act as ion channel formers (30). In addition, some membrane-active peptides orient parallel to the bilayer, whereas others orient perpendicular to the plane of the membrane (26). Very recently, a hydrophilic proline-rich domain from the C terminus of L-type calcium channels was shown to remain membrane associated (11). Cyclic peptides rich in proline have been found to bind alkali metals (9, 19). Since toxin P is a dodecapeptide, it cannot traverse the membrane bilayer. However, it could form aggregates of the head-to-head or head-to-tail type, as found for gramicidin A. Furthermore, toxin P aggregates in aqueous solution (Fig. 3), supporting the hypothesis that it can enter the algal membrane and act as an ammonia channel. However, the possibility that ammonia activates toxin P has not been eliminated.

Proline-rich antibacterial peptides are also produced by mammalian neutrophils (20). These peptides are mainly active against gram-negative bacteria. Investigation of the secondary structure of one of these peptides (PR-39), which contains 39 amino acids (19 Pro residues), suggests that it exists in a polyproline II conformation in water. After interacting with the membrane, PR-39 rapidly enters human microvascular endothelial cells and binds to a number of cytoplasmic proteins (6). Now that pure chemically synthesized toxin P is available, it should be possible to examine its structure and mode of action in more detail. It will also be interesting to examine synthetic peptides with defined amino acid replacements in order to determine their ability to inhibit photosynthesis.

In considering the natural role of toxin P in pathogenesis (coral bleaching), we should mention that high levels of toxin P were found in coral tissues shortly after infection with V. shiloi (8). The toxin is produced only at the elevated seawater temperatures (25 to 30°C) necessary for bacterial bleaching of corals (16). Presumably, once V. shiloi penetrates into the coral tissue, it produces toxin P and ammonia (from metabolism of coral cytoplasmic protein). The resulting inhibition of photosynthesis in the intracellular zooxanthellae would damage the algae and contribute to coral bleaching (loss of the algae). It should be noted that the equilibrium of NH₄⁺ dissociation to NH₃ is shifted towards increased NH₃ formation with increasing temperature. For example, three times more NH₃ is produced at 25°C than at 10°C at a constant pH and a constant total NH₃-NH₄⁺ concentration. It should be noted that O. patagonica in the eastern Mediterranean Sea experiences a shift in temperature from 16°C (winter) to 29 to 30°C (summer, when bleaching occurs) (16). This may contribute to magnification of the toxin P-enhanced toxicity of ammonia for zooxanthellae in infected coral during the summer.