Mapping studies and genetic analysis of transfer genes of the multi-resistant IncHI2 plasmid, R478

A bank of transfer-defective Tn7 insertion mutants of the multi-resistant IncHI2 megaplasmid, R478, was generated. Complementation analysis of these mutations identified a large 144-kb transfer-associated region of R478. A 6.8-kb segment from the transfer region was sequenced. The precise locations of Tn7 insertion within four distinct R478::Tn7 transfer-defective mutants were mapped and each insertion was found to disrupt a specific open reading frame. These transfer-associated determinants of R478 were designated htdB (H transfer determinant), htdD, htdT and htdC. Both htdB and htdC encoded amino acid sequences that showed a low homology with pilus biosynthetic proteins encoded by the F plasmid.