Physical and genetic analyses of streptococcal plasmid pAM beta 1 and cloning of its replication region

Abstract

Plasmid pAM.beta.1, originally isolated from Streptococcus faecalis DS5, mediates resistance to the MLS (macrolide, lincosamide and streptogramin B.alpha.) group of antibiotics. A restriction endonuclease map of the 26.5 kilobase (kb) pAM.beta.1 molecule was constructed by using the enzymes AvaI, HpaII, EcoRI, PvuII, KpnI, BstEII, HpaI, HhaI and HindIII. A comparison of this map to those of 4 independently isolated deletion derivatives of pAM.beta.1 located the MLS resistance determinant within a 2 kb DNA segment and at least 1 conjugative function within an 8 kb region. The 5.0 kb EcoRI-B fragment from pAM.beta.1 was ligated onto the 4.0 kb Escherichia coli plasmid vector, pACKC1, and used to transform E. coli HB101. The 9.0 kb chimeric plasmid was then used to transform S. sanguis Challis with concurrent expression of the E. coli kanamycin resistance determinant. The 5.0 kb EcoRI-B fragment from pAM.beta.1 was subsequently used as a vector to clone a streptomycin resistance determinant from a strain of S. mutans containing no detectable plasmid DNA. Subcloning experiments, using a HindIII partial digest of pAM.beta.1 DNA, narrowed the replication region of this plasmid to a 2.95 kb fragment.