Origin of transfer
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An origin of transfer (oriT) is a short sequence ranging from 40-500 base pairs in length[1][2] that is necessary for the transfer of DNA from a gram-negative bacterial donor to recipient during bacterial conjugation.[3][4][5] The transfer of DNA is a critical component for antimicrobial resistance within bacterial cells[6] and the oriT structure and mechanism within plasmid DNA is complementary to its function in bacterial conjugation. The first oriT to be identified and cloned was on the RK2 (IncP) conjugative plasmid, which was done by Guiney and Helinski in 1979.[7]
oriT regions are central to the process of transferring DNA from the donor to recipient and contain several important regions that facilitate this:
- nic site: where the unwound plasmid DNA is cut; usually site-specific.[4][8][9]
- An inverted repeat sequence: signals the end of replication of donor DNA and is responsible for transfer frequency, plasmid mobilization, and secondary DNA structure formation.[3][8][10]
- AT-rich region: important for DNA strand opening and is located adjacent to the inverted repeat sequences.[1][3][5][8][11][12]
The oriT is a noncoding region of the bacterial DNA.[13] Due to its important role in initiating bacterial conjugation, the oriT is both an enzymatic substrate and recognition site for the relaxase proteins.[1][13][14] Relaxosomes have oriT-specific auxiliary factors that help it to identify and bind to the oriT.[1] Upstream of the oriT nic site is a termination sequence.[5]
oriTs are primarily cis-acting, which allows for a more efficient DNA transfer.[5][12][15]
Mechanism and function in bacterial conjugation
At the start of bacterial conjugation, a donor cell will elaborate a pilus and signal to a nearby recipient cell to get in close contact. This identification of a suitable recipient cell will begin the mating pair formation process.[1][16] This process of bringing the two cells together recruits the type IV secretion system, a protein complex that forms the transfer channel between the donor and recipient, starting the formation of the relaxation complex known as the relaxosome at the oriT.[13]
A plasmid's oriT sequence serves as both a recognition point and a substrate for the enzymes in the relaxosome,[13] therefore the first step of bacterial conjugation occurs at the nicn site of the oriT region of the plasmid.[4][14] Relaxase enzymes, otherwise known as DNA strand transferases part of the relaxosome complex, catalyze a strand- and site-specific phosphodiester bond cleavage at the nicn site and are specific to each plasmid.[17] This reaction is a trans-esterification, which produces a nicked double-stranded DNA with the 5' end bound to a tyrosine residue in the relaxase.[4][5][14][17] The relaxase then moves toward the 3' end of the strand to unwind the DNA in the plasmid.[17]
The other strand of the plasmid, the strand that was not nicked by the relaxase, is a template for further synthesis by DNA polymerase.[17]
Once the relaxase reaches the upstream section of the oriT again where there is an inverted repeat, the process is terminated by reuniting the ends of the plasmid and releasing a single-stranded plasmid in the recipient.[5][15][18]