EXPRESSION OF VIRULENCE AND ANTIBIOTIC-RESISTANCE IN AN ESCHERICHIA-COLI TRANSCONJUGANT CARRYING A LARGE PLASMID PCAT120 OF SHIGELLA-DYSENTERIAE TYPE-I AND ITS SPONTANEOUS FRAGMENTATIONS

The role of a 120-kb plasmid in relation to virulence and drug resistance factor in Shigella dysenteriae was studied. For characterization of plasmids, the mating system is a useful and efficient means of transferring both large and small plasmids to a new host. The conjugative transfer of a 120-kb (pCAT120) ampicillin-resistant plasmid of S. dysenteriae to E. coli K-12 was not successful. Introduction of an E. coli fertility factor plasmid F, did not help to mobilize the plasmid. Low transfer frequencies of antibiotic markers to E. coli were achieved by treatment of the donor S. dysenteriae with N-methyl-N'-nitro-N-nitrosoguanidine. The transconjugants showed resistance to ampicillin, chloramphenicol, tetracycline and cadmium. A transconjugant carrying the 120-kb plasmid of S. dysenteriae produced keratoconjunctivitis in guinea pigs. Repeated subculture of Clm(r) transconjugant (pCAT120) on tryptic soya agar plates became Clm(s) and showed four distinct DNA bands ranging from 3 to 10 kb in size on agarose gel electrophoresis. Utilization of organic acids, metal resistance (Cd), dye-binding properties (Crb+, Ebr+) and drug resistance (Amp, Tet) were identified on 10, 7, 4 and 3-kb plasmid DNA fragment of pCAT120 respectively. Crb+ 4-kb DNA fragment of pCAT120 was isolated, purified and transferred to an avirulent E. coli K12 by transformation. However, transformant (pET4) showed poor growth on solid media and its growth in liquid culture was only possible after supplementation of the unknown low-molar-mass thermolabile factor(s) secreted by the recipient strain. A 130-kDa outer membrane protein was synthesized by the transformant (pET4) carrying a 4-kb Congo red binding plasmid DNA fragment of pCAT120. A highly reduced rate of synthesis of a few low-molar-mass outer membrane proteins was also observed among the transformant (pET4) in relation to the recipient strain. Transconjugant carrying four plasmid DNA fragments of pCAT120 and Crb+ transformant (pET4) failed to produce keratoconjunctivitis in guinea pigs.