Mapping of Streptococcus faecalis plasmids pAD1 and pAD2 and studies relating to transposition of Tn917

Abstract

Plasmids pAD1 (37.8 megadaltons) and pAD2 (17.1 megadaltons) of Streptococcus faecalis strain DS16 have been mapped with restriction enzymes. The location of a hemolysin-bacteriocin determinant on the conjugative pAD1 plasmid was derived from analyses of transposon insertions. Electron microscope and hybridization analyses located Tn917(Em) and the streptomycin (Sm) and kanamycin (Km) resistance determinants on the nonconjugative pAD2 plasmid. It was shown previously that the erythromycin (Em) resistance associated with Tn917 is inducible and that transposition from pAD2 to pAD1 is also stimulated by exposure of cells to low concentrations of Em. Here we show that inducing concentrations of Em also increase the conjugative transfer potential of pAD1; this is possibly related to a mild and short-lived inhibitory stress placed on the cells before full induction of resistance. Selection of Em-resistant transconjugants arising from matings between DS16 and a plasmid-free recipient gave rise to transconjugants which primarily harbor stable pAD1::pAD2 cointegrates. A 30-min exposure of donors to Em (0.5 microgram/ml) before mating resulted in a severalfold increase in the number of such transconjugants. However, a small fraction (e.g., 3 of 40) of these Emr Smr Kmr transconjugants harbored pAD1::Tn917 and pAD2 molecules. Since we believe pAD2 is incapable of being mobilized by pAD1 without being covalently linked, it is likely that transfer in these cases involved cointegrates representing structural intermediates in the transposition of Tn917 from pAD2 to pAD1. It follows that such intermediates probably had two copies of Tn917 and readily resolved after transfer. (These cointegrates are different from the stable cointegrates which were shown to have only a single copy of Tn917; the latter are assumed not to be related to transposition.) Two variants with altered Tn917 transposition properties were derived. One of them transposed at an elevated frequency, whereas the other showed no detectabel transposition. In neither case was transposition influenced by Em exposure; however, both remained inducible for Em resistance.