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 Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wa ...

bacterial donor to recipient during bacterial conjugation. The transfer of DNA is a critical component for

antimicrobial resistance Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that protect them from the effects of antimicrobials. All classes of microbes can evolve resistance. Fungi evolve antifungal resistance. Viruses evolve antiviral resistance. P ...

within bacterial cells and the ''oriT'' structure and mechanism 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: # ''nic'' site: where the unwound plasmid DNA is cut; usually site-specific. # An

inverted repeat An inverted repeat (or IR) is a single stranded sequence of nucleotides followed downstream by its complementarity (molecular biology), reverse complement. The intervening sequence of nucleotides between the initial sequence and the reverse compleme ...

sequence: signals the end of replication of donor DNA and is responsible for transfer frequency, plasmid mobilization, and secondary DNA structure formation. # AT-rich region: important for DNA strand opening and is located adjacent to the inverted repeat sequences. 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 A relaxase is a single-strand DNA transesterase enzyme produced by some prokaryotes and viruses. Relaxases are responsible for site- and strand-specific nicks in unwound double-stranded DNA . Known relaxases belong to the rolling circle replicati ...

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. Plasmid oriT

''oriT''s are primarily '' cis-''acting, which allows for a more efficient DNA transfer. BacterialConjugation

Mechanism and function in bacterial conjugation

At the start of bacterial conjugation, a donor cell will elaborate a

pilus A pilus (Latin for 'hair'; plural: ''pili'') is a hair-like appendage found on the surface of many bacteria and archaea. The terms ''pilus'' and '' fimbria'' (Latin for 'fringe'; plural: ''fimbriae'') can be used interchangeably, although some r ...

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 The bacterial type IV secretion system, also known as the type IV secretion system or the T4SS, is a secretion protein complex found in gram negative bacteria, gram positive bacteria, and archaea. It is able to transport proteins and DNA across t ...

, a protein complex that forms the transfer channel between the donor and recipient, starting the formation of the relaxation complex known as the

relaxosome The relaxosome is the Multiprotein complex, complex of proteins that facilitates plasmid transfer during bacterial conjugation. The proteins are encoded by the tra operon on a fertility plasmid in the region near the origin of transfer, Origin of ...

at the ''oriT''. A plasmid's ''oriT'' sequence serves as both a recognition point and a substrate for the enzymes in the relaxosome, therefore the first step of bacterial conjugation occurs at the ''nicn'' site of the ''oriT'' region of the plasmid.

Relaxase A relaxase is a single-strand DNA transesterase enzyme produced by some prokaryotes and viruses. Relaxases are responsible for site- and strand-specific nicks in unwound double-stranded DNA . Known relaxases belong to the rolling circle replicati ...

enzymes, otherwise known as DNA strand transferases part of the relaxosome complex, catalyze a strand- and site-specific

phosphodiester bond In chemistry, a phosphodiester bond occurs when exactly two of the hydroxyl groups () in phosphoric acid react with hydroxyl groups on other molecules to form two ester bonds. The "bond" involves this linkage . Discussion of phosphodiesters is d ...

cleavage at the ''nicn'' site and are specific to each plasmid. This reaction is a trans-esterification, which produces a nicked double-stranded DNA with the 5' end bound to a

tyrosine -Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. The word "tyrosine" is from the G ...

residue in the relaxase. The relaxase then moves toward the 3' end of the strand to unwind the DNA in the plasmid. The other strand of the plasmid, the strand that was not nicked by the relaxase, is a template for further synthesis by

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create ...

. Once the relaxase reaches the upstream section of the ''oriT'' again where there is an

, the process is terminated by reuniting the ends of the plasmid and releasing a single-stranded plasmid in the recipient.

Applications

Genetic engineering

Conjugation allows for the transfer of target genes to many recipients, including

yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constit ...

, 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 marker A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, a ...

s on the plasmids generate bacteria that have resistance to antibiotics like

ampicillin Ampicillin is an antibiotic used to prevent and treat a number of bacterial infections, such as respiratory tract infections, urinary tract infections, meningitis, salmonellosis, and endocarditis. It may also be used to prevent group B stre ...

and

kanamycin Kanamycin A, often referred to simply as kanamycin, is an antibiotic used to treat severe bacterial infections and tuberculosis. It is not a first line treatment. It is used by mouth, injection into a vein, or injection into a muscle. Kanamyci ...

Antimicrobial resistance

The interaction between the DNA o''riT'' and relaxase enables

via

horizontal gene transfer Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between unicellular and/or multicellular organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). H ...

(Figure 1). Various ''oriT'' regions in plasmid DNA contain inverted repeats onto which relaxase proteins are able bind. Major contributors of

drug resistance Drug resistance is the reduction in effectiveness of a medication such as an antimicrobial or an antineoplastic in treating a disease or condition. The term is used in the context of resistance that pathogens or cancers have "acquired", that is ...

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

genes, they are very similar to conjugative plasmids that are responsible for the prevalence of drug resistance among bacterial cells. A 2017 study on MGIs revealed that they are able to integrate themselves into the genome of the receiving bacterial cells by themselves via ''int'', a gene 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.

References