Transfer of the chromosomal bla gene from Enterobacter cloacae to Escherichia coli by RP4::mini-Mu

Abstract

The resistance gene for beta-lactamase-stable cephalosporins from Enterobacter cloacae was transferred to Escherichia coli by the aid of RP4::mini-Mu. The R-prime plasmids generated carried 60 to 80 kilobases (kb) of E. cloacae DNA and coded for the chromosomal E. cloacae beta-lactamase. The gene was fully expressed in the recipient. Restriction endonuclease EcoRI fragments of the R-prime plasmid pBP100 were cloned into the vector pBP328, yielding the plasmid pBP102 with a size of 14 kb. A restriction map of this plasmid was constructed. By digesting pBP102 into seven PstI fragments, ligating the fragments, and looking for the smallest plasmid generated, pBP103 was isolated. It consisted of three PstI fragments, two of them (together 4.2 kb) necessary for resistance. During the experiment (performed in a recA+ background) the largest PstI fragment had undergone a substitution of a 0.3-kb segment of pBP102 by a 0.7-kb segment in pBP103 (as deduced by heteroduplex analysis). The bla gene of resistant E. cloacae strains was dominant over the gene of susceptible organisms.