Origin of transfer: Difference between revisions

An '''origin of transfer''' (''oriT'') is a short sequence ranging from 40-500 base pairs in length<ref name=":0">{{cite journal | vauthors = de la Cruz F, Frost LS, Meyer RJ, Zechner EL | title = Conjugative DNA metabolism in Gram-negative bacteria | journal = FEMS Microbiology Reviews | volume = 34 | issue = 1 | pages = 18–40 | date = January 2010 | pmid = 19919603 | doi = 10.1111/j.1574-6976.2009.00195.x | s2cid = 24003194 | doi-access = free }}</ref><ref>{{Citation|last=Frost|first=L. S.|title=Conjugation, Bacterial|date=2009-01-01|url=https://www.sciencedirect.com/science/article/pii/B9780123739445000079|workencyclopedia=Encyclopedia of Microbiology (Third Edition)|pages=517–531|editor-last=Schaechter|editor-first=Moselio|place=Oxford|publisher=Academic Press|language=en|doi=10.1016/b978-012373944-5.00007-9|isbn=978-0-12-373944-5|access-date=2021-12-03}}</ref> that is necessary for the transfer of DNA from a [[Gram-negative bacteria|gram-negative]] bacterial donor to recipient during [[bacterial conjugation]].<ref name=":5">{{cite journal | vauthors = Kiss J, Szabó M, Hegyi A, Douard G, Praud K, Nagy I, Olasz F, Cloeckaert A, Doublet B | display-authors = 6 | title = Identification and Characterization of <i>''oriT</i>'' and Two Mobilization Genes Required for Conjugative Transfer of <i>''Salmonella</i>'' Genomic Island 1 | journal = Frontiers in Microbiology | volume = 10 | pages = 457 | date = 2019 | pmid = 30894848 | pmc = 6414798 | doi = 10.3389/fmicb.2019.00457 | doi-access = free }}</ref><ref name=":6">{{cite journal | vauthors = Howard MT, Nelson WC, Matson SW | title = Stepwise assembly of a relaxosome at the F plasmid origin of transfer | language = English | journal = The Journal of Biological Chemistry | volume = 270 | issue = 47 | pages = 28381–28386 | date = November 1995 | pmid = 7499340 | doi = 10.1074/jbc.270.47.28381 | doi-access = free }}</ref><ref name=":2">{{cite journal | vauthors = Lanka E, Wilkins BM | title = DNA processing reactions in bacterial conjugation | journal = Annual Review of Biochemistry | volume = 64 | issue = 1 | pages = 141–169 | date = June 1995 | pmid = 7574478 | doi = 10.1146/annurev.bi.64.070195.001041 }}</ref>. The transfer of DNA is a critical component for [[antimicrobial resistance]] within bacterial cells<ref>{{cite journal | vauthors = Gyles C, Boerlin P | title = Horizontally transferred genetic elements and their role in pathogenesis of bacterial disease | journal = Veterinary Pathology | volume = 51 | issue = 2 | pages = 328–340 | date = March 2014 | pmid = 24318976 | doi = 10.1177/0300985813511131 | s2cid = 206510894 | doi-access = free }}</ref> and the ''oriT'' structure and mechanism within plasmid DNA is complimentarycomplementary forto its function in bacterial conjugation. The first ''oriT'' to be identified and cloned was on the [[RK2 plasmid|RK2]] (IncP) conjugative plasmid, which was done by Guiney and Helinski in 1979.<ref>{{cite journal | vauthors = Guiney DG, Helinski DR | title = The DNA-protein relaxation complex of the plasmid RK2: location of the site-specific nick in the region of the proposed origin of transfer | journal = Molecular & General Genetics | volume = 176 | issue = 2 | pages = 183–189 | date = October 1979 | pmid = 393953 | doi = 10.1007/BF00273212 | s2cid = 23889133 }}</ref>.

# [[Nic site|''nic'' site]]: where the unwound plasmid DNA is cut; usually site-specific.<ref name=":6" /><ref name=":7">{{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 }}</ref><ref>{{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 }}</ref>.

# 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.<ref name=":5" /><ref name=":7" /><ref>{{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 }}</ref>.

# AT-rich region: important for DNA strand opening and is located adjacent to the inverted repeat sequences.<ref name=":0" /><ref name=":5" /><ref name=":2" /><ref name=":7" /><ref>{{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 = 1-21–2 | pages = 219–225 | date = June 1987 | pmid = 3039307 | doi = 10.1007/BF00330445 | s2cid = 11985769 }}</ref><ref name=":8">{{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 }}</ref>.

The ''oriT'' is a [[Non-coding DNA|noncoding region]] of the bacterial DNA.<ref name=":1">{{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 }}</ref>. Due to its important role in initiating bacterial conjugation, the ''oriT'' is both an enzymatic substrate and recognition site for the [[relaxase]] proteins.<ref name=":0" /><ref name=":1" /><ref name=":9">{{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 }}</ref>. [[Relaxosome|Relaxosomes]]s have ''oriT-''specific auxiliary factors that help it to identify and bind to the ''oriT''.<ref name=":0" />. Upstream of the ''oriT nic'' site is a termination sequence.<ref name=":2" />.

''oriT''s are primarily ''[[Cis-regulatory element|cis]]-''acting, which allows for a more efficient DNA transfer.<ref name=":2" /><ref name=":8" /><ref name="Identification of the origin of tra">{{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 }}</ref>.

== Mechanism and Functionfunction in Bacterialbacterial Conjugationconjugation ==

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.<ref name=":0" /><ref>{{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 }}</ref>. 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''.<ref name=":1" />.

A plasmid's ''oriT'' sequence serves as both a recognition point and a substrate for the enzymes in the relaxosome,<ref name=":1" />, therefore the first step of bacterial conjugation occurs at the ''nicn'' site of the ''oriT'' region of the plasmid.<ref name=":6" /><ref name=":9" />. [[Relaxase]] enzymes, otherwise known as DNA strand transferases part of the [[Relaxosome|relaxosome complex]], catalyze a strand- and site-specific [[phosphodiester bond]] cleavage at the ''nicn'' site and are specific to each plasmid.<ref name=":3">{{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 }}</ref>. This reaction is a [[Transesterification|trans-esterification]], which produces a nicked double-stranded DNA with the 5' end bound to a [[tyrosine]] residue in the relaxase.<ref name=":6" /><ref name=":2" /><ref name=":9" /><ref name=":3" />. The relaxase then moves toward the 3' end of the strand to unwind the DNA in the plasmid.<ref name=":3" />.

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.<ref name=":2" /><ref>{{cite journal | vauthors name= 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 = 10.1128tra"/JB.00932-07 }}</ref><ref>{{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 }}</ref>.

Conjugation allows for the transfer of target genes to many recipients, including [[yeast]],<ref>{{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 }}</ref> [[Eukaryote|mammalian cells]],<ref>{{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 }}</ref><ref>{{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 }}</ref> and [[diatom]]s.<ref name=":4">{{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 }}</ref>

Diatoms could be useful plasmid hosts as they have the potential to [[Autotroph|autotrophicallyautotroph]]ically produce [[Biofuel|biofuelsbiofuel]]s and other chemicals.<ref name=":4" /> There are some methods for [[Transformation (genetics)|genetic transfer]] for diatoms, but they are slow compared to bacterial conjugation. By designing plasmids for the diatoms ''[[Phaeodactylum tricornutum|P. tricornutum]]'' and ''[[Thalassiosira pseudonana|T. pseudonana]]'' based on sequences for yeast and developing a method for conjugation from ''[[Escherichia coli|E. coli]]'' to the diatoms, researchers hope to advance genetic manipulation in diatoms.<ref name=":4" />

One of the main problems in using bacterial conjugation in [[genetic engineering]] is that certain [[selectable marker]]s on the plasmids generate bacteria that have resistance to antibiotics like [[ampicillin]] and [[Kanamycin A|kanamycin]].<ref>{{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 }}</ref>

=== Antimicrobial Resistanceresistance ===

The interaction between the DNA o''riT'' and relaxase enables [[antimicrobial resistance]] via [[horizontal gene transfer]] (Figure 1).<ref name=":1" />. Various ''oriT'' regions in plasmid DNA contain [[Invertedinverted repeat|inverted repeats]]s onto which relaxase proteins are able bind.<ref name=":5" />. Major contributors of [[drug resistance]] are mobile [[Genomicgenomic island|genomic islands]]s (MGIs), or segments in DNA that are found in similar strains of bacteria and are factors in diversification of bacteria.<ref name=":5" /><ref name="Mobilizable genomic islands, differ">{{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 }}</ref>. MGIs provide resistance to their host cells, and through bacterial conjugation, spread this advantage to other cells.<ref name=":5" />. 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.<ref name=":5" />. 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 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.<ref>{{cite journal | vauthors name= 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 = 10.1080differ"/2159256X.2017.1304193 }}</ref>.