Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae

Abstract

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29 degrees C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH(4)Cl, pure natural or synthetic toxin P (10 microM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH(4)Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH(4)Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH(3) into the cell. It is known that uptake of NH(3) into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.

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.

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.

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.

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.

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.

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 (○).