IDENTIFICATION OF A NEW GENE IN THE STREPTOCOCCAL PLASMID PLS1 - THE RNAI GENE

The streptococcal plasmid pMV158 has been reported to harbor five gene s: three involved in initiation of rolling circle replication and its control (copG, repB, and maII), one involved in conjugative mobilizati on (mobM), and the fifth one specifying constitutive resistance to tet racycline (tet). The mobM gene was removed in the construction of the pMV158-derivative plasmid pLS1, which was used in this study. By in vi tro transcription assays, primer extension experiments, and constructi on of mutations, here we demonstrate the presence of another gene (the sixth of pMV158), termed mal, which is transcribed in opposite orient ation with respect to the plasmid mRNAs, to lender RNA I. The 5'-end o f RNA I has an 8-nt sequence which is complementary to a region of the lagging-strand origin (ssoA) comprising a 6-nt consensus sequence inv olved in lagging strand synthesis. This suggested that RNA I could inf luence, positively or negatively, initiation of lagging strand synthes is from the pLS1-ssoA. However, plasmids defective in RNA I synthesis exhibited a phenotype similar to the wild type in terms of efficiency of replication from the ssoA and copy number. When the mal gene was cl oned into a compatible plasmid, the resulting recombinants did not exh ibit incompatibility toward plasmids with the pLS1 replicon. Thus, RNA I does not seem to be a true copy number control element. We postulat e that transcription from the mal promoter may facilitate extrusion of the hairpin of the plasmid double-strand origin, which is the target of the initiator of replication protein, (C) 1998 Academic Press.