pIGWZ12 – A cryptic plasmid with a modular structure

• The structure of cryptic plasmid pIGWZ12 isolated from clinical E. coli was studied.
• Identified REP module belongs to IncF family.
• MOB module from pIGWZ12 belongs to MOBQ family.
• Iterons from origin of replication form a multifunctional region indispensable to plasmid replication.
• The role of MOB elements (Mob proteins, origin of transfer) was determined.

We studied the detailed structure of the cryptic plasmid pIGWZ12, which was isolated from an Escherichia coli strain. pIGWZ12 is composed of two structural modules of distinct evolutionary origin. The REP module, which contains all the features necessary for replication and stable maintenance in the bacterial cell, was assigned by genotyping to the IncF family. The MOB module, which is responsible for plasmid mobilization, shows significant homology to MOBQ modules from broad-host-range plasmids belonging to the RSF1010/R1162 family. We showed that iterons located in the origin of replication are the target for specific binding by the replication initiator protein RepApIGWZ12. Furthermore, we proved that the promoter for the repA gene overlaps with the iterons, and that the latter are the sole determinant of incompatibility. We performed a mutagenesis analysis of the MOBpIGWZ12 module and characterized the roles played by all identified genes (mobA and mobC), as well as the role played by oriT in mobilization. Finally, we showed that it was possible to remove the MOB module from pIGWZ12 without any loss in plasmid replication and stability. Furthermore, the MOBpIGWZ12 module was fully functional after subcloning into another plasmid. Therefore, pIGWZ12 is yet another example of modular structure in small cryptic plasmids.