User:Ddelrio08/Origin of transfer

An origin of transfer (oriT) is a short sequence ranging from 40-500 base pairs in length that is necessary for the transfer of DNA from a gram-negative bacterial donor to recipient during bacterial conjugation. The transfer of DNA is a critical component for antimicrobial resistance within bacterial cells and the oriT structure and location within plasmid DNA is complimentary for 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.

Structure
oriT regions are central to the process of transferring DNA from the donor to recipient and contain several important regions that facilitate this:

The oriT is a noncoding region of the bacterial DNA. Due to its important role in initiating bacterial conjugation, the oriT is both an enzymatic substrate and recognition site for the relaxase proteins. Relaxosomes have oriT-specific auxiliary factors that help it to identify and bind to the oriT. Upstream of the oriT nic site is a termination sequence.
 * 1) nic site: where the unwound plasmid DNA is cut; usually site-specific.
 * 2) 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) AT-rich region: important for DNA strand opening and is located adjacent to the inverted repeat sequences.

oriTs are primarily cis-acting, which allows for a more efficient DNA transfer.

oriT within Plasmid DNA
The oriT region in plasmid DNA is the site where strands or fragments of DNA can be transferred from cell to cell during the conjugation of plasmids (see bacterial conjugation below) and the cloning of DNA. The initiation of transfer and replication activities begins at the nick site specific to a region on the plasmid DNA. Studies published in 1984 determined the location of these single-stranded nick sites on F plasmid DNA. Researchers used gel electrophoresis to restriction map the regions of plasmid DNA and employed restriction endonucleases to further refine the DNA fragments. They were able to procure a functioning oriT region at 373 base pairs and found that nic sites were dispersed over a region of 20 base pairs at the end of the oriT region.

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. 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.

A relaxase protein will nick the DNA at the oriT and begin conjugation. The nicked DNA strand, known as the T-strand, is then transferred to the recipient cell in a 5’ to 3’ direction beginning at the oriT. Synthesis of the complementary DNA and recircularization of the T-strand back at the oriT results in both the donor and recipient cells being capable of plasmid transfer.

Antimicrobial Resistance
The interaction between the DNA oriT and relaxase enables antimicrobial resistance via horizontal gene transfer (Figure 1). Various oriT regions in plasmid DNA contain inverted repeats onto which relaxase proteins are able bind. Major contributors of drug resistance are mobile genomic islands (MGIs), or segments in DNA that are found in similar strains of bacteria and are factors in diversification of bacteria. MGIs provide resistance to their host cells, and through bacterial conjugation, spread this advantage to other cells. With bacterial cell MGIs having their own oriT sequences and being in close proximity to relaxosome genes, they are very similar to conjugative plasmids that are responsible for the prevalence of drug resistance among bacterial cells.

Applications
Conjugation allows for the transfer of target genes to many recipients, including yeast, mammalian cells , and diatoms.

Diatoms could be useful plasmid hosts as they have the potential to autotrophically produce biofuels and other chemicals. There are some methods for genetic transfer for diatoms, but they are slow compared to bacterial conjugation. By designing plasmids for the diatoms P. tricornutum and T. pseudonana based on sequences for yeast and developing a method for conjugation from E. coli to the diatoms, researchers hope to advance genetic manipulation in diatoms.

One of the main problems in using bacterial conjugation in genetic engineering is that certain selectable markers on the plasmids generate bacteria that have resistance to antibiotics like ampicillin and kanamycin.

A 2017 study on mobile genomic islands revealed that MGIs are able to integrate themselves into the genome of the receiving bacterial cells by themselves via int, a gene that that codes for the integrase enzyme. After the OriT of the MGI are processed by the relaxosomes encoded by integrative and conjugative elements (ICE), the MGI are able to enter the genome of the receiver cells and allow for the multiformity of bacteria that leads to antimicrobial resistance