A new mobilizable cosmid vector for use in Vibrio cholerae and other gram-negative bacteria

Abstract

A new mobilizable cosmid vector, pCOS5, was engineered for use in Vibrio cholerae (Vc). Plasmid pCOS5 is small in size (7286 bp), contains the oriT from plasmid RK2, and has several unique restriction sites. The complete nucleotide sequence of pCOS5 was deduced from the DNA fragments used in its construction. Biparental matings using Escherichia coli (Ec) SM10 and triparental matings using Ec DH5 alpha[pRK2013] were used to measure the conjugation frequency of pCOS5 and pAJM1, a clone containing a 40-kb insert of chromosomal DNA from Vc ligated into pCOS5. Transfer of pCOS5 or pAJM1 to Vc occurred at a frequency of between 10(-2)-10(-3) transconjugants per recipient cell. The promiscuous nature of RP4/RK2 transfer functions makes pCOS5 a potentially useful vector for mobilizing large fragments of cloned DNA between different Gram- bacteria that support replication of ColE1 plasmids or as a mobilizable suicide vector in Gram- bacteria where replication of ColE1 plasmids is not supported.