Phosphorylation and inactivation of streptomycin by plant pathogenic Pseudomonas lachrymans.

Sir: Inactivation of aminoglycoside antibiotics in clinical isolates has been studied by many research workers". Streptomycin(SM)-inactivating enzymes were reported to exist in SM-resistant strains of Staphylococcus auren.s2-11, Escherichia coli l',, and Pseudomonas aeruginosal 1-11), in which are operative 2 mechanisms, phosphorylation and adenylylation, i.e., SM 3"-phosphotransferase; APH(3"), SM 6-phosphotransferase; APH(6), SM 3"-adenylyltransferase; AAD (3"), SM 6-adenylyltransferase; AAD(6). Psendomonas lachr,'mans is known to be the cucumber angular leaf spot bacterium and SM-resistant strains have been recently isolated"'. However, the mechanisms of SM-resistance in plant pathogenic P. lachrymans have not been studied. Thirteen SM-resistant P. lachrl•mans were selected and biochemical mechanisms of SM-resistance in these strains were investigated. Peptone water was used for liquid culture, which consisted of 10 g peptone, 5 g NaCI, and 1,000 ml of deionized water. Bacteria were inoculated in peptone water and shaken at 27' C. After 20 hours of incubation, cells were harvested. The S-30 fraction, the supernatant of 30,000g centrifugation, was prepared as described in a previous paper''. An incubation reaction was carried out at 27~C for I hour and then stopped by heating in boiling water for 3 minutes. Antibiotic activity remaining in the reaction mixture was determined by bioassay using Bacillus subtilis ATCC6633 as test organism. As shown in Table 1, five P. lachrrmans strains inactivated SM but the remaining 8 strains did not inactivate the drug under these conditions. The enzymatic transfer of the ^V-32p or '1C-AMP from isotope-labeled ATP into SM was carried out as described previously,'. From the result, we concluded that 5 strains inactivated SM by phosphorylation. ,'-''_'P-SM was prepared by the inactivation reaction using the extract of one of the 5 strains N-7554. The reaction mixture contained: 100 /if of S-30 fraction (5 mg of protein/ml), 20 /tl of I /lCi of ^'-32P-ATP (515 mCi/mmol) , 20 /rl of i mtit of SM, 2011 of 0.02 M MgCl2, and 40 /if of 0.2 M iris-HCI buffer (pH 7.0). After 60 minutes of incubation at 27-C, the reaction was stopped by heating in boiling water and centrifuged. The supernatant was pipetted onto a phosphocellulose paper and washed with deionized water, extracted with 10 nil of 0.5 N HCl and lyophilized. ,'Plabeled phosphoryl SM thus obtained, the authentic samples of SM-3"-phosphate prepared from P. aeruginosa TI-13 and SM-6-phosphate from P. aeruginosa GN573 were developed with the following solvent system on a thin-layer of silica gel (Tokyo Kasei). The radioactive spot of 32P-phosphoryl SM coincided exactly with the SAKAGUCHi reaction'''-positive spot of SM3"-phosphate (Rf 0.36), while Rf value of SM-6phosphate was 0.23, on thin-layer chromatography using CH3OH H2O 15'1 NaCl (9: 1 : 5 in volume). On high-voltage paper electrophoresis under 3,500 volts for 20 minutes using formic acid acetic acid water (25: 75: 900 in volume), the 32P-phosphoryl SM and SM-3"phosphate moved toward the cathode 9.4 cm. From these results, it was concluded that the chemical structure of SM inactivated by P. lachrymans was SM-3"-phosphate. The biochemical mechanisms of SM resistance in the remaining 8 strains which could not inactivate the drug will be described elsewhere.