Identification Of Plasmid Replication Origin Of Plasmid P26 From Bacillus Thuringiensis Subsp. Finitimus Strain YBT-020

Bacillus thuringiensis subsp. finitimus strain YBT-020 harbores thee indigenous plasmids. In our previous study, a novel replicon had been isolated from plasmid p26, whose replication origin contains two ORFs:ORF15 and ORF16. Sequence alignment indicated that orf15 encoded a putative protein with unknown function. It contained a HTH-domain which was conserved in replication protein, and it is believable that the protein might be classified as a novel replication protein. Orf16 encoded a protein with high sequence identity to the PcrA helicase which contains seven conserved domains, and so the protein was named as PcrA26. This work has done to prove the ORF15 protein as a novel replication protein, and to further define the function of ORF16 and their role in replication of plasmid p26.1. We predicted that the ORI site of plasmid p26 is located at the inside of orf15. And the ORF15 protein was over-expressed in E. coli and purified. Binding experiment between the ORF15 protein and the DNA fragment containing ORI site in vitro indicated that ORF15 protein could bind to the DNA fragment and initiate replication. So the ORF15 protein was named as Rep26.2. We conducted the yeast two-hybrid experiment to confirm the interaction between Rep26 and ORF16 and their polymerization. The results showed that Rep26 protein could form an oligomer by self-polymerize. The protein was suggested as a hybrid protein involving in both the initiation of replication and the segregation of plasmid p26. PcrA26 could not self-polymerize to oligomer, which indicated that it functioned as monomer. Our results also confirmed that there was no interaction between PcrA26 and Rep26, which suggested that PcrA26 was independent of replication protein when performing its helicase function.Our study characterizes the replication origin of p26 plasmid. Rep26 is considered to be a novel hybrid protein, involving in both the initiation of replication and the segregation of plasmid p26. PcrA26 acts as a monomer during theθ-replication, and being recruited independent of Rep protein. We inferred that the PcrA26 protein interact with the DNA fragment of replication origin directly. Based on the evidences above, we suggest the the plasmid of p26 exist a novel type of replication mechanism.