A CONJUGATIVE PLASMID FROM EPIPHYTIC ENTEROBACTER CLOACAE THAT CONFERS MULTI-DRUG RESISTANCE M SATOl, K WATANABEl and Y SATO2 lNational Institute of Sericultural and Entomological Science, Tsukuba, lbaraki 305-8634, Japan; 2National Institute of Agro-Environmental Sciences, Tsukuba, lbaraki 305-8604, Japan Backgrand and objectives Several plant pathogenic bacteria including Erwinia amylovora, Pseudomonas syringae pathovars and Xanthomonas campestris pv. vesicatoria harbor indigenous plasmids that confer the resistance to streptomycin (Smr) [1, 2, 3]. The Smr determinants from these strains were shown to be homologous to the strA-strB genes of the broad-host-range enterobacterial plasmid RSF1010 [1, 3, 4]. These findings suggest that gene transfer has played an important role in the dissemination of Smr genes in plant pathogenic bacteria, which might acquire the genes by conjugating with other epiphytic bacteria. In the present study, we detected a conjugative plasmid conferred the resistance to Sm and other several antibiotics in epiphytic Enterobacter colacae. Materials and methods Identification of bacteria was performed by an API 20E kit and other bacteriological properties. A wild strain designated Mull and its variant Mull (1010), which means that the plasmid RSF1010 as a marker was introduced into the strain Mull, were mainly used. Plasmid isolation was performed by alkali method. Southern blot analysis was performed using a prove pRSF2 which is 2.1 kb Hincll fragment containing strA-strB from RSF1010. Mating was performed by an ordinarily filter method, and transconjugants were isolated on the selected medium supplemented with antibiotics. Results and conclusion Several strains of E. cloacae were isolated from mulberry leaves. These strains exhibited almost the same bacteriological properties as those of clinical strains of E. cloacae, and were not pathogenic to mulberry. The strain Mull exhibited the resistance to several drugs, such as Sm (50 mg/ml), kanamycin (20 mg/ml), ampicillin (60 mg/ml), tetracyecline (40 mg/ml) and chloramphenicol (60 mg/ml). On the other hand, a spontaneous mutant designated "Mul(1010) pM-" from Mull (1010) did not exhibit any resistance to these antibiotics except for Sm (originated from RSF1010). Plasmid profile of these strains was analyzed. The former harbored a large plasmid designated pMUL1, while the latter did not it, having only RSF1010. Then, Mull and Mull (1010) were mated with Erwinia spp. and Pseudomonas spp. Several strains of epiphytic or pathogenic Erwinia spp. including E. ananas and E. herbicoia acquired the plasmid pMUL1 at a very high frequency, and also did the resistance to above-mentioned antibiotics except for ampicillin (native phenotype of Erwinia spp.), simultaneously. The plasmid pMUL1 from Mull and transconjugants hybridized with pRSF2 prove. Thus, pMUL1 is conjugative and codes multi-drug resistance genes including Smr genes to be homologous to the strA-strB genes of RSF1010. These findings suggest that epiphytic E. cloacae may play a role of in the dissemination of drug-resistance genes including Smr genes in plant pathogenic bacteria. References 1. Sundin GW, Bender CL 1993. Appi. Environ. Microbiol. 59, 1018-1024. 2. Minsavage GV, Canteros, BJ, Stall RE, 1990. Phytopathology 80:719-723. 3. Chiou CS. Jones AL, 1993. J. Bacteriol. 175:732-740. 4. Nakajima M, Yamashita S, Takikawa Y, Tsuyumu S, Hibi T, Goto M, 1995. Ann. Phytopathol. Soc. Jpn. 61:489-492.