IN-VIVO DEFINITION OF THE FUNCTIONAL ORIGIN OF LEADING-STRAND REPLICATION ON THE LACTOCOCCAL PLASMID PFX2

The lactococcal plasmid pFX2 belongs to a family of plasmids, whose pr ototype is the streptococcal plasmid pMV158, that replicates by the ro lling circle mechanism. Determination of the nucleotide sequence of th e repX gene of pFX2 allowed us to make some minor corrections in the p ublished sequence, and to show that the repX gene is identical to the rep gene of plasmid pWV01. We have established pFX2 in Escherichia col i and in Streptococcus pneumoniae. In the latter host, we have defined in vivo the nick site introduced by the RepX protein. Plasmid pFX2 an d the pMV158 derivative pLS1 exhibit a moderate degree of incompatibil ity in S. pneumoniae. Cloning of the double strand origin (dso) of pFX 2 into a high-copy-number plasmid that is compatible with the pMV158 r eplicon led to an increase in incompatibility toward pLS1. Plasmids pF X2 and pLS1 exhibit homologies in their Rep proteins and in their dso sequences, but not in their negative control elements. Thus, the obser ved incompatibility indicates that cross-recognition of Rep proteins a nd dso takes place.