Department of Biology, McMaster University, Hamilton, Ontario, L8S 4K1 Canada
The 1700 kb plasmid of Sinorhizobium melilpti possesses a repABC operon that controls replication and partitioning of the plasmid. We are engaging in genetic and biochemical analyses to determine the nature of cis and trans acting elements that control replication, segregation, copy number control, and incompatibility functions in the plasmid.
We have isolated a minimal, functioning replication region of 5.4 kb and the resulting mini-pExo derivative has been used in genetic studies to deduce the functions of encoded gene products and c/s-acting regions. The 5.4 kb fragment encodes three genes (repA, repB and repC) and confers replication, efficient segregation, and incompatibility against the parental pExo (pSymB) plasmid when cloned into an otherwise non-replicating vector. Interestingly, a 4.6 kb fragment that contains the rep A promoter and approximately 500 bp downstream of repC is not efficiently segregated during cell division. This indicates that stability determinates lie outside these boundaries.
On the basis of genetic analyses, we conclude that the repABC genes are transcribed from a single promoter upstream of rep A. This promoter exists within 100 bp upstream of the predicted translational start of RepA. An insertion in rep A eliminates replication likely due to polar effects on the expression of RepC. A 2.3 kb subclone containing only the repC open reading frame (plus upstream and downstream non-coding DNA) confers replication indicating that the expression of RepC is sufficient for replication. However, replication is only manifest when this fragment is cloned downstream of a vector based lac promoter suggesting that a promoter does not exist immediately upstream of repC.
The repABC gene fragment exerts incompatibility against its parental plasmid. A 242 bp sequence isolated from the intergenic region between repB and repC is sufficient to confer this incompatibility. Interestingly, a fine scale genetic dissection of this region indicates that the incompatibility locus is at least 130 bp long and that the entire region is needed for incompatibility. A frameshift mutation in repA partially relieves the incompatibility effect suggesting that RepA is a trans-acting incompatibility factor.
Both the repB-repC intergenic region directly upstream of repC and a nucleotide region downstream of the repC gene are essential for replication. Subcloning experiments wherein each region was deleted independently abolished replication.
We are continuing to delineate the minimal genetic requirements for replication, segregation and incompatibility.
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