COPY NUMBER CONTROL OF THE STREPTOCOCCAL PLASMID PIP501 OCCURS AT 3 LEVELS

Transcriptional analysis of the replication region of plasmid pIP501 has revealed three active promoters. The repR gene which is essential for pIP501 replication was transcribed from promoter p((II). A small antisense RNA (136 nt, RNAIII) generated from promoter p(III) was complementary to the leader region of the repR mRNA. Introduction of either point mutations or deletions into promoter p(III) or RNAIII resulted in a 5-20fold increased plasmid copy number suggesting a negative regulatory function for RNAIII. The copR gene, the complete DNA and amino acid sequence of which is reported, was dispensable for pIP501 replication. However, deletion of the copR promoter p(I) and/or the copR coding sequence led to a 10-20fold increase in plasmid copy number. This effect was also observed when a -1 frameshift mutation was introduced into the CopR coding region. Mutations in copR and p(III)/RNAIII were not additive. It is, therefore, proposed that both components act at the same level of copy number control most likely in a sequential way. A second level of copy number control was found to involve an inverted repeat structure upstream of and overlapping with promoter p(II). Destruction of this repeat sequence by deletion caused an increase in copy number 2-3fold higher than that observed for either RNAIII or copR mutations. A working model is proposed how different components of pIP501 interact to regulate its copy number.