Construction of pYGK, an Actinobacillus actinomycetemcomitans-Escherichia coli shuttle vector

A shuttle vector that is capable of replicating in Actinobacillus actinomycetemcomitans (Aa) and Escherichia coli (Ec) was constructed by modifying the Actinobacillus pleuropneumoniae (Ap) plasmid pYG53. A DNA fragment containing the KmR gene was inserted into pYG53 to generate pYGK, which confers resistance to kanamycin in both Aa and Ec. By electroporation, Ec DH5alpha and 17 strains of Aa were transformed with pYGK with efficiencies ranging from 0.5 to 3 X 10(6) colonies per microgram of DNA. Plasmid pYGK exists at approx. 3-4 copies per cell in Ec. This plasmid will facilitate the genetic manipulation of Aa strains and the molecular analysis of virulence factors expressed by this organism.