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, unlike gram-positive bacteria, do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelope consists ...

bacterial donor to recipient during

bacterial conjugation Bacterial conjugation is the transfer of genetic material between Bacteria, bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual cycle, parasexual mode ...

. The transfer of DNA is a critical component for

antimicrobial resistance Antimicrobial resistance (AMR or AR) occurs when microbes evolve mechanisms that protect them from antimicrobials, which are drugs used to treat infections. This resistance affects all classes of microbes, including bacteria (antibiotic resista ...

within bacterial cells 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.

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 complem ...

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 Non-coding DNA (ncDNA) sequences are components of an organism's DNA that do not encode protein sequences. Some non-coding DNA is transcribed into functional non-coding RNA molecules (e.g. transfer RNA, microRNA, piRNA, ribosomal RNA, and regula ...

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

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 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 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 conditionally essential amino acid with a polar side group. The word "tyrosine" is ...

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 t ...

. 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 (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are est ...

, mammalian cells, and

diatom A diatom (Neo-Latin ''diatoma'') is any member of a large group comprising several Genus, genera of algae, specifically microalgae, found in the oceans, waterways and soils of the world. Living diatoms make up a significant portion of Earth's B ...

s. Diatoms could be useful plasmid hosts as they have the potential to

autotroph An autotroph is an organism that can convert Abiotic component, abiotic sources of energy into energy stored in organic compounds, which can be used by Heterotroph, other organisms. Autotrophs produce complex organic compounds (such as carbohy ...

ically produce

biofuel Biofuel is a fuel that is produced over a short time span from Biomass (energy), biomass, rather than by the very slow natural processes involved in the formation of fossil fuels such as oil. Biofuel can be produced from plants or from agricu ...

s 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 ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...

'' to the diatoms, researchers hope to advance genetic manipulation in diatoms. One of the main problems in using bacterial conjugation in

genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of Genetic engineering techniques, technologies used to change the genet ...

is that certain

selectable marker A selectable marker is a gene introduced into cell (biology), cells, especially bacteria or cells in cell culture, culture, which confers one or more traits suitable for artificial selection. They are a type of reporter gene used in laboratory micr ...

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

ampicillin Ampicillin is an antibiotic belonging to the aminopenicillin class of the penicillin family. The drug is used to prevent and treat several bacterial infections, such as respiratory tract infections, urinary tract infections, meningitis, s ...

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. Kanamy ...

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 organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). HGT is an important factor in the e ...

(Figure 1). Various ''oriT'' regions in plasmid DNA contain

s 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 island A genomic island (GI) is part of a genome that has evidence of horizontal origins. The term is usually used in microbiology, especially with regard to bacteria. A GI can code for many functions, can be involved in symbiosis or pathogenesis, and ...

s (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. 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 Retroviral integrase (IN) is an enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme ...

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