ExcA proteins of IncI1 plasmid R64 and IncIγ plasmid R621a recognize different segments of their cognate TraY proteins in entry exclusion

Entry exclusion is a process whereby plasmid transfer between donor and recipient cells harboring identical or closely related conjugative plasmids is inhibited. Exclusion proteins in the recipient cells are responsible for entry exclusion. Although IncI1 Plasmid R64 and IncIγ plasmid R621a exhibit similar genome structure in replication, transfer, and leading regions, they belong to different incompatibility and exclusion groups. The amino acid sequences of TraY and ExcA proteins are significantly different between R64 and R621a. In the present study, TraY proteins of R64 and R621a were exchanged. Transfer of R64 derivative carrying R621a TraY was inhibited by recipient R621a ExcA but not R64 ExcA and transfer of R621a derivative carrying R64 TraY was inhibited by recipient R64 ExcA but not R621a ExcA. This indicates that R64 and R621a TraY proteins in the donor cells are the targets of cognate ExcA proteins in the recipient proteins. Since two segments, an internal and a C-terminal segment, were found to vary between R64 and R621a TraY proteins, various chimera TraY proteins were constructed. Conjugation experiments suggested that the R64 internal variable segment recognizes R64 ExcA protein and the R621a C-terminal variable segment recognizes R621a ExcA protein.

► R64 and R621a ExcA were not required in the donor cells for entry exclusion.
► TraY proteins of plasmids R64 and R621a were exchanged.
► R64 and R621a TraY proteins were found to be the targets of their ExcA proteins.
► R64 ExcA recognized the internal variable segment of R64 TraY.
► R621a ExcA recognized the C-terminal variable segment of R621a TraY.