Origin of transfer

{{short description|Sequence of DNA}}

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

1. nic site: where the unwound plasmid DNA is cut; usually site-specific.{{cite journal | vauthors = Francia MV, Varsaki A, Garcillán-Barcia MP, Latorre A, Drainas C, de la Cruz F | title = A classification scheme for mobilization regions of bacterial plasmids | journal = FEMS Microbiology Reviews | volume = 28 | issue = 1 | pages = 79–100 | date = February 2004 | pmid = 14975531 | doi = 10.1016/j.femsre.2003.09.001 | doi-access = free }}{{cite journal | vauthors = Zhang S, Meyer R | title = The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer | journal = Molecular Microbiology | volume = 25 | issue = 3 | pages = 509–516 | date = August 1997 | pmid = 9302013 | doi = 10.1046/j.1365-2958.1997.4861849.x | s2cid = 26826243 }}
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.{{cite journal | vauthors = Scherzinger E, Lurz R, Otto S, Dobrinski B | title = In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins | journal = Nucleic Acids Research | volume = 20 | issue = 1 | pages = 41–48 | date = January 1992 | pmid = 1738602 | pmc = 310323 | doi = 10.1093/nar/20.1.41 }}
3. AT-rich region: important for DNA strand opening and is located adjacent to the inverted repeat sequences.{{cite journal | vauthors = Coupland GM, Brown AM, Willetts NS | title = The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing | journal = Molecular & General Genetics | volume = 208 | issue = 1–2 | pages = 219–225 | date = June 1987 | pmid = 3039307 | doi = 10.1007/BF00330445 | s2cid = 11985769 }}{{cite journal | vauthors = Fu YH, Tsai MM, Luo YN, Deonier RC | title = Deletion analysis of the F plasmid oriT locus | journal = Journal of Bacteriology | volume = 173 | issue = 3 | pages = 1012–1020 | date = February 1991 | pmid = 1991706 | pmc = 207219 | doi = 10.1128/jb.173.3.1012-1020.1991 }}

The oriT is a noncoding region of the bacterial DNA.{{cite journal | vauthors = Zrimec J, Lapanje A | title = DNA structure at the plasmid origin-of-transfer indicates its potential transfer range | journal = Scientific Reports | volume = 8 | issue = 1 | pages = 1820 | date = January 2018 | pmid = 29379098 | pmc = 5789077 | doi = 10.1038/s41598-018-20157-y | bibcode = 2018NatSR...8.1820Z }} Due to its important role in initiating bacterial conjugation, the oriT is both an enzymatic substrate and recognition site for the relaxase proteins.{{cite journal | vauthors = Byrd DR, Matson SW | title = Nicking by transesterification: the reaction catalysed by a relaxase | journal = Molecular Microbiology | volume = 25 | issue = 6 | pages = 1011–1022 | date = September 1997 | pmid = 9350859 | doi = 10.1046/j.1365-2958.1997.5241885.x | s2cid = 35753372 | doi-access = free }} 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.

File:Plasmid oriT.png.]]

oriTs are primarily cis-acting, which allows for a more efficient DNA transfer.{{cite journal | vauthors = Lee CA, Grossman AD | title = Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis | journal = Journal of Bacteriology | volume = 189 | issue = 20 | pages = 7254–7261 | date = October 2007 | pmid = 17693500 | pmc = 2168444 | doi = 10.1128/JB.00932-07 }}

File:BacterialConjugation.jpg. (1) relaxase and helicase bind to the plasmid (F-factor) at the origin of transfer (OriT). Helicase unwinds the plasmid DNA and relaxase attaches to the transfer DNA strand. (3) Relaxase carries the transfer DNA strand through the pilus connecting the two bacterial cells. (4) The remaining strand is rewound with a complementary strand of DNA. (5) Relaxase joins the two ends of the transfer DNA into a circular plasmid. (6) Relaxase detaches from the plasmid. (7) New plasmid DNA is rewound with a complementary strand of DNA.]]

{{Further|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.{{cite journal | vauthors = Arutyunov D, Frost LS | title = F conjugation: back to the beginning | journal = Plasmid | volume = 70 | issue = 1 | pages = 18–32 | date = July 2013 | pmid = 23632276 | doi = 10.1016/j.plasmid.2013.03.010 | series = Special Issue based on the International Society for Plasmid Biology Meeting: Santander 2012 }} 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 cleavage at the nicn site and are specific to each plasmid.{{cite journal | vauthors = Guasch A, Lucas M, Moncalián G, Cabezas M, Pérez-Luque R, Gomis-Rüth FX, de la Cruz F, Coll M | display-authors = 6 | title = Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC | journal = Nature Structural Biology | volume = 10 | issue = 12 | pages = 1002–1010 | date = December 2003 | pmid = 14625590 | doi = 10.1038/nsb1017 | s2cid = 27050728 }} 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. 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.

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.{{cite journal | vauthors = Frost LS, Ippen-Ihler K, Skurray RA | title = Analysis of the sequence and gene products of the transfer region of the F sex factor | journal = Microbiological Reviews | volume = 58 | issue = 2 | pages = 162–210 | date = June 1994 | pmid = 7915817 | pmc = 372961 | doi = 10.1128/mr.58.2.162-210.1994 }}

{{Main|Diatom}}

Conjugation allows for the transfer of target genes to many recipients, including yeast,{{cite journal | vauthors = Heinemann JA, Sprague GF | title = Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast | journal = Nature | volume = 340 | issue = 6230 | pages = 205–209 | date = July 1989 | pmid = 2666856 | doi = 10.1038/340205a0 | bibcode = 1989Natur.340..205H | s2cid = 4351266 }} mammalian cells,{{cite journal | vauthors = Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V | title = Genetic transformation of HeLa cells by Agrobacterium | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 98 | issue = 4 | pages = 1871–1876 | date = February 2001 | pmid = 11172043 | pmc = 29349 | doi = 10.1073/pnas.98.4.1871 | bibcode = 2001PNAS...98.1871K | doi-access = free }}{{cite journal | vauthors = Waters VL | title = Conjugation between bacterial and mammalian cells | journal = Nature Genetics | volume = 29 | issue = 4 | pages = 375–376 | date = December 2001 | pmid = 11726922 | doi = 10.1038/ng779 | s2cid = 27160 }} and diatoms.{{cite journal | vauthors = Karas BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips AP, Noddings CM, Brunson JK, Valas RE, Deerinck TJ, Jablanovic J, Gillard JT, Beeri K, Ellisman MH, Glass JI, Hutchison CA, Smith HO, Venter JC, Allen AE, Dupont CL, Weyman PD | display-authors = 6 | title = Designer diatom episomes delivered by bacterial conjugation | journal = Nature Communications | volume = 6 | issue = 1 | pages = 6925 | date = April 2015 | pmid = 25897682 | pmc = 4411287 | doi = 10.1038/ncomms7925 | bibcode = 2015NatCo...6.6925K }}

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.{{cite journal | vauthors = Lopatkin AJ, Meredith HR, Srimani JK, Pfeiffer C, Durrett R, You L | title = Persistence and reversal of plasmid-mediated antibiotic resistance | journal = Nature Communications | volume = 8 | issue = 1 | pages = 1689 | date = November 2017 | pmid = 29162798 | pmc = 5698434 | doi = 10.1038/s41467-017-01532-1 | bibcode = 2017NatCo...8.1689L }}

{{Further|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.{{cite journal | vauthors = Carraro N, Rivard N, Burrus V, Ceccarelli D | title = Mobilizable genomic islands, different strategies for the dissemination of multidrug resistance and other adaptive traits | journal = Mobile Genetic Elements | volume = 7 | issue = 2 | pages = 1–6 | date = 2017-03-04 | pmid = 28439449 | pmc = 5397120 | doi = 10.1080/2159256X.2017.1304193 }} 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 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.

• Bacterial conjugation
• Origin of replication
• Recombinant DNA
• Horizontal gene transfer

{{Reflist|30em}}

Category:DNA