Molecular cloning of an Escherichia coli plasmid determinant that encodes for the production of heat stable enterotoxin

Abstract

A conjugative plasmid, ESF0041, was isolated from an enterotoxigenic strain of E. coli from calves. ESF0041 was found to be 65 x 106 daltons in mass and a member of the F incompatibility complex. Acquisition of ESF0041 by E. coli K 12 was invariably associated with the capacity to produce heat stable (ST) enterotoxin. ESF0041 and pSC101 deoxyribonucleic acids were cleaved with EcoRI, and the fragments were ligated with polynucleotide ligase. Transformation of E. coli K 12 with the ligation mixture led to the isolation of an ST+ clone. Further analysis of the plasmid deoxyribonucleic acid from this clone showed that the structural gene(s) associated with ST biosynthesis had been isolated as a 5.7 x 106 dalton ESF0041 fragment in pSC101. In turn, the 5.7 x 106 dalton fragment was ligated to a multicopy ColE1 derivative, RSF2124, so that toxin synthesis was amplified about threefold.