Copy number control of the streptococcal plasmio plP501 occurs at three levels

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Abstract
Transcriptional analysis of the replication region of plasmid pUPSOU has revealed three active promoters. The repR gene which is essential for pDP501 replication was transcribed from promoter pM. A small antisense RNA (136 rot, RMADDi) generated from promoter pm was complementary to the leader region of the repR mRNA. Introduction of either point mutations or deletions into promoter pm or RWAlli resulted in a 5–20fold increased plasmid copy number suggesting a negative regulatory function for RNAill The copR gene, the complete DMA and amino acid sequence of which is reported, was dispensable for plP501 replication. However, deletion of the copR promoter pl 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 pIII/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 pII. 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 pDP501 interact to regulate its copy number.