Inhibition of Ornithin Carbamyl Transferase from Bean Plants by the Toxin of Pseudomonas phaseolicola

Abstract

Pseudomonas phaseolicola (Burkh.) Dowson, a pathogen of bean (Phaseolus vulgaris L.) plants, causes chlorotic haloes in infected host leaves (7). In vitro the pathogen produces an extra-cellular toxin which when injected into bean leaves also produces chlorotic haloes (2). Whether the symptoms are caused by infection or by injection of toxin, ornithine accumulates in the chlo-rotic tissues (9). Here we present evidence which indicates that ornithine accumulation is caused by the inhibition of ornithine carbamyl transferase of the host by the toxin. Toxin Preparation. The halo blight toxin was partially purified from the culture filtrate of a virulent strain of P. phaseolicola grown in the YEP medium (9). Five hundred milliliters of the filtrate were concentrated to 20 ml under reduced pressure at 50 C, and the concentrate was desalted with 10 volumes of methanol. The extract was concentrated and the procedure repeated two more times. The final extract was evaporated to dryness and made up to 10 ml with distilled water. Four milliliters of this sample were applied to a 2-X 45-cm column of Sephadex G-10. The column was eluted with distilled water at a flow rate of 17.6 nl/hr (fraction volume, 2.2 ml). An identical batch of the medium, which was not inoculated, was subjected to the same procedure including incubation under the growth conditions, as a control and is designated nongrown. The culture filtrates are designated grown. Aliquots (25 ,ul) from every third fraction collected from the Sephadex column were assayed for halo-inducing activity on young trifoliate leaves of 4-week-old Red Kidney bean plants in a growth chamber (2). The same fractions were diluted 10-fold with distilled water and 80 Iul of the diluted fraction were used to test its inhibitory activity against OCT2 of bean leaves. To determine ve/vo (retention volume/void volume) of the active material, fractions with halo-inducing activity were concentrated to 2 ml and put on an analytical column (90 x 1.2 cm) of Sephadex G-10. The elution rate was 10.8 ml/hr (fraction volume, 1.8 ml). Enzyme Preparation. Acetone powder was prepared from tri-foliate leaves of 3 to 4 week-old Red Kidney bean plants as described before (4). Five grams of the acetone powder were stirred for 1 hr in 90 ml of 0.1 M potassium phosphate buffer, pH 7.0, containing 5 X 10-3 M 2-mercaptoethanol. The slurry was cen