Abstract
A region of the IncHI2 plasmid R478, encoding the phenotypes of tellurite resistance (Ter), phage inhibition (Phi), and colicin resistance (PacB), was cloned and sequenced. Analysis indicated seven open reading frames (ORFs), whose genes were designated terZ, -A, -B, -C, -D, -E, and -F. Five of these predicted ORFs (A to E) had extensive amino acid homology with the previously reported ORFs of the IncHI2 Ter operon from plasmid pMER610. There were domains of highly conserved amino acid residues within the group TerA, -D, -E, and -F and within TerD, -E, and -Z, but no consensus could be found among all five putative polypeptides. There were also regions of good identity and similarity between individual pairs of ORFs which was not reflected in the multiple alignments. The three phenotypes were expressed in Escherichia coli DH5 alpha by an 8.4-kb EcoRI insert subcloned from a cosmid of R478. The latter insert was clonable only as a double insertion with a 4.5-kb fragment, and forced deletion of the smaller fragment was lethal to cells. This lethality was not dependent on the cloned orientation of either fragment, suggesting that there is a trans-acting element in the 4.5-kb fragment. Tn1000 mutagenesis of one of the double-insert clones, pDT2575, showed that the phenotypes, including multiple colicin resistance, were genetically linked. Transpositions into terD, terC, and terZ reduced or abolished all phenotypes, while inserts into terE and terF had no effect on the phenotypes. Insertions in terA reduced phage inhibition levels only. The presence of the terZ and terF ORFs in pMER610 was confirmed, and derivatives of this plasmid mediated Phi, PacB, and Ter.