INVITRO AND INVIVO ANALYSIS OF TRANSCRIPTION WITHIN THE REPLICATION REGION OF PLASMID PIP501

Derivatives of the conjugative streptococcal plasmid pIP501 replicate stably in Bacillus subtilis. The region essential for replication of pIP501 has been narrowed down to a 2.2 kb DNA segment, the sequence of which has been determined. This region comprises two genes, copR and repR, proposed to be involved in copy control and replication. By in vitro and in vivo transcriptional analysis we characterized three active promoters, p(I), p(II), and p(III) within this region. A putative fourth promoter (p(IV)) was neither active in vitro nor in vivo. We showed that copR is transcribed from promoter p(I) while the repR gene is transcribed from promoter p(II) located just downstream of copR. The p(II) transcript encompasses a 329 nucleotide (nt) long leader sequence. A counter transcript that was complementary to a major part of this leader was found to originate from a third promoter p(III). The secondary structure of the counter transcript revealed several stem-loop regions. A regulatory function for this antisense RNA in the control of repR expression is proposed. Comparative analysis of the replication regions of pAM-beta-1 and pSM19035 suggested a similar organization of transcriptional units, suggesting that an antisense RNA is produced by these plasmids also.