Expression of virulence and antibiotic resistance in anEscherichia coli transconjugant carrying a large plasmid pCAT120 ofShigella dysenteriae type I and its spontaneous fragmentations

The role of a 120-kb plasmid in relation to virulence and drug resistance factor inShigella 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 ofS. dysenteriae toE. coli K-12 was not successful. Introduction of anE. coli fertility factor plasmid F, did not help to mobilize the plasmid. Low transfer frequencies of antibiotic markers toE. coli were achieved by treatment of the donorS. 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 ofS. 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 avirulentE. coli K12 by trans-formation. 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. Presented in part at the57th Annual Meeting of Society of Biological Chemists (India), New Delhi, October, 1988 (Abstr. No. 269 & 272) andIndo-UK Workshop on Diarrhoeal Diseases, Calcutta, January 1989 (Abstr. Page No. 215-217).