Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT

The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined, A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively, Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence, The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT, Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold, Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence, Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation, A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.