Origin of transfer

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An origin of transfer (oriT) is a short sequence ranging from 40-500 base pairs in length [1] [2] that is necessary for the transfer of DNA from a gram-negative bacterial donor to recipient during bacterial conjugation. [3] [4] [5] The transfer of DNA is a critical component for antimicrobial resistance within bacterial cells [6] 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. [7]

Contents

Structure

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. [4] [8] [9]
  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] [8] [10]
  3. AT-rich region: important for DNA strand opening and is located adjacent to the inverted repeat sequences. [1] [3] [5] [8] [11] [12]

The oriT is a noncoding region of the bacterial DNA. [13] Due to its important role in initiating bacterial conjugation, the oriT is both an enzymatic substrate and recognition site for the relaxase proteins. [1] [13] [14] Relaxosomes have oriT-specific auxiliary factors that help it to identify and bind to the oriT. [1] Upstream of the oriT nic site is a termination sequence. [5]

Figure 1 ^ Region of oriT sequence on plasmid DNA. Plasmid oriT.png
Figure 1 ▲ Region of oriT sequence on plasmid DNA.

oriTs are primarily cis-acting, which allows for a more efficient DNA transfer. [5] [12] [15]

Figure 2 ^ Two bacterial cells undergoing bacterial conjugation. (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. BacterialConjugation.jpg
Figure 2 ▲ Two bacterial cells undergoing bacterial conjugation. (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.

Mechanism and 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. [1] [16] 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. [13]

A plasmid's oriT sequence serves as both a recognition point and a substrate for the enzymes in the relaxosome, [13] therefore the first step of bacterial conjugation occurs at the nicn site of the oriT region of the plasmid. [4] [14] 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. [17] 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. [4] [5] [14] [17] The relaxase then moves toward the 3' end of the strand to unwind the DNA in the plasmid. [17]

The other strand of the plasmid, the strand that was not nicked by the relaxase, is a template for further synthesis by DNA polymerase. [17]

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. [5] [15] [18]

Applications

Genetic engineering

Conjugation allows for the transfer of target genes to many recipients, including yeast, [19] mammalian cells, [20] [21] and diatoms. [22]

Diatoms could be useful plasmid hosts as they have the potential to autotrophically produce biofuels and other chemicals. [22] 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. [22]

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. [23]

Antimicrobial resistance

The interaction between the DNA oriT and relaxase enables antimicrobial resistance via horizontal gene transfer (Figure 1). [13] Various oriT regions in plasmid DNA contain inverted repeats onto which relaxase proteins are able bind. [3] 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. [3] [24] MGIs provide resistance to their host cells, and through bacterial conjugation, spread this advantage to other cells. [3] 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. [3] 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. [24]

See also

Related Research Articles

<span class="mw-page-title-main">Bacterial conjugation</span> Method of bacterial gene transfer

Bacterial conjugation is the transfer of genetic material between 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 mode of reproduction in bacteria.

<span class="mw-page-title-main">Pilus</span> A proteinaceous hair-like appendage on the surface of bacteria

A pilus is a hair-like appendage found on the surface of many bacteria and archaea. The terms pilus and fimbria can be used interchangeably, although some researchers reserve the term pilus for the appendage required for bacterial conjugation. All conjugative pili are primarily composed of pilin – fibrous proteins, which are oligomeric.

<span class="mw-page-title-main">Plasmid</span> Small DNA molecule within a cell

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the internet.

<span class="mw-page-title-main">Horizontal gene transfer</span> Type of nonhereditary genetic change

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 evolution of many organisms. HGT is influencing scientific understanding of higher order evolution while more significantly shifting perspectives on bacterial evolution.

<span class="mw-page-title-main">Hfr cell</span>


A high-frequency recombination cell is a bacterium with a conjugative plasmid integrated into its chromosomal DNA. The integration of the plasmid into the cell's chromosome is through homologous recombination. A conjugative plasmid capable of chromosome integration is also called an episome. When conjugation occurs, Hfr cells are very efficient in delivering chromosomal genes of the cell into recipient F− cells, which lack the episome.

<span class="mw-page-title-main">Transformation (genetics)</span> Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

<i>Agrobacterium tumefaciens</i> Bacterium, genetic engineering tool

Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA, from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors.

<span class="mw-page-title-main">Ti plasmid</span>

A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of Agrobacterium, including A. tumefaciens, A. rhizogenes, A. rubi and A. vitis.

The F-plasmid allows genes to be transferred from one bacterium carrying the factor to another bacterium lacking the factor by conjugation. The F factor was the first plasmid to be discovered. Unlike other plasmids, F factor is constitutive for transfer proteins due to a mutation in the gene finO. The F plasmid belongs to F-like plasmids, a class of conjugative plasmids that control sexual functions of bacteria with a fertility inhibition (Fin) system.

Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host can only contain one fosmid molecule. Fosmids can hold DNA inserts of up to 40 kb in size; often the source of the insert is random genomic DNA. A fosmid library is prepared by extracting the genomic DNA from the target organism and cloning it into the fosmid vector. The ligation mix is then packaged into phage particles and the DNA is transfected into the bacterial host. Bacterial clones propagate the fosmid library. The low copy number offers higher stability than vectors with relatively higher copy numbers, including cosmids. Fosmids may be useful for constructing stable libraries from complex genomes. Fosmids have high structural stability and have been found to maintain human DNA effectively even after 100 generations of bacterial growth. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence.

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 replication (RCR) initiator superfamily of enzymes and fall into two broad classes: replicative (Rep) and mobilization (Mob). The nicks produced by Rep relaxases initiate plasmid or virus RCR. Mob relaxases nick at origin of transfer (oriT) to initiate the process of DNA mobilization and transfer known as bacterial conjugation. Relaxases are so named because the single-stranded DNA nick that they catalyze lead to relaxation of helical tension.

<span class="mw-page-title-main">Prokaryotic DNA replication</span> DNA Replication in prokaryotes

Prokaryotic DNA Replication is the process by which a prokaryote duplicates its DNA into another copy that is passed on to daughter cells. Although it is often studied in the model organism E. coli, other bacteria show many similarities. Replication is bi-directional and originates at a single origin of replication (OriC). It consists of three steps: Initiation, elongation, and termination.

The relaxosome is the 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, oriT. The most important of these proteins is relaxase, which is responsible for beginning the conjugation process by cutting at the nic site via transesterification. This nicking results in a DNA-Protein complex with the relaxosome bound to a single strand of the plasmid DNA and an exposed 3' hydroxyl group. Relaxase also unwinds the plasmid being conjugated with its helicase properties. The relaxosome interacts with integration host factors within the oriT.

The traA gene codes for relaxase, which is an enzyme that initiates plasmid DNA transfer during bacterial conjugation. Relaxase forms a relaxosome complex with auxiliary proteins to initiate conjugation. Relaxosome binds to the origin of transfer (oriT) sequence and cleaves the DNA strand that will be transferred.

Transfer genes or tra genes, are some genes necessary for non-sexual transfer of genetic material in both gram-positive and gram-negative bacteria. The tra locus includes the pilin gene and regulatory genes, which together form pili on the cell surface, polymeric proteins that can attach themselves to the surface of F-bacteria and initiate the conjugation. The existence of the tra region of a plasmid genome was first discovered in 1979 by David H. Figurski and Donald R. Helinski In the course of their work, Figurski and Helinski also discovered a second key fact about the tra region – that it can act in trans to the mobilization marker which it affects.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

<span class="mw-page-title-main">TraJ-II RNA motif</span>

The traJ-II RNA motif is a conserved RNA structure discovered in bacteria by using bioinformatics. traJ-II RNAs appear to be in the 5' untranslated regions of protein-coding genes called traJ, which functions in the process of bacterial conjugation. A previously identified motif known as TraJ 5' UTR is also found upstream of traJ genes functions as the target of FinP antisense RNAs, so it is possible that traJ-II RNAs play a similar role as targets of an antisense RNA. However, some sequence features within the traJ-II RNA motif suggest that the biological RNA might be transcribed from the reverse-complement strand. Thus is it unclear whether traJ-II function as cis-regulatory elements. traJ-II RNAs are found in a variety of Pseudomonadota.

<span class="mw-page-title-main">Plasmid-mediated resistance</span> Antibiotic resistance caused by a plasmid

Plasmid-mediated resistance is the transfer of antibiotic resistance genes which are carried on plasmids. Plasmids possess mechanisms that ensure their independent replication as well as those that regulate their replication number and guarantee stable inheritance during cell division. By the conjugation process, they can stimulate lateral transfer between bacteria from various genera and kingdoms. Numerous plasmids contain addiction-inducing systems that are typically based on toxin-antitoxin factors and capable of killing daughter cells that don't inherit the plasmid during cell division. Plasmids often carry multiple antibiotic resistance genes, contributing to the spread of multidrug-resistance (MDR). Antibiotic resistance mediated by MDR plasmids severely limits the treatment options for the infections caused by Gram-negative bacteria, especially family Enterobacteriaceae. The global spread of MDR plasmids has been enhanced by selective pressure from antimicrobial medications used in medical facilities and when raising animals for food.

Bacterial recombination is a type of genetic recombination in bacteria characterized by DNA transfer from one organism called donor to another organism as recipient. This process occurs in three main ways:

Integrative and conjugative elements (ICEs) are mobile genetic elements present in both gram-positive and gram-negative bacteria. In a donor cell, ICEs are located primarily on the chromosome, but have the ability to excise themselves from the genome and transfer to recipient cells via bacterial conjugation.

References

  1. 1 2 3 4 5 de la Cruz F, Frost LS, Meyer RJ, Zechner EL (January 2010). "Conjugative DNA metabolism in Gram-negative bacteria". FEMS Microbiology Reviews. 34 (1): 18–40. doi: 10.1111/j.1574-6976.2009.00195.x . PMID   19919603. S2CID   24003194.
  2. ↑ Frost, L. S. (2009-01-01), "Conjugation, Bacterial", in Schaechter, Moselio (ed.), Encyclopedia of Microbiology (Third Edition), Oxford: Academic Press, pp. 517–531, doi:10.1016/b978-012373944-5.00007-9, ISBN   978-0-12-373944-5 , retrieved 2021-12-03
  3. 1 2 3 4 5 6 7 Kiss J, Szabó M, Hegyi A, Douard G, Praud K, Nagy I, et al. (2019). "Identification and Characterization of oriT and Two Mobilization Genes Required for Conjugative Transfer of Salmonella Genomic Island 1". Frontiers in Microbiology. 10: 457. doi: 10.3389/fmicb.2019.00457 . PMC   6414798 . PMID   30894848.
  4. 1 2 3 4 Howard MT, Nelson WC, Matson SW (November 1995). "Stepwise assembly of a relaxosome at the F plasmid origin of transfer". The Journal of Biological Chemistry. 270 (47): 28381–28386. doi: 10.1074/jbc.270.47.28381 . PMID   7499340.
  5. 1 2 3 4 5 6 Lanka E, Wilkins BM (June 1995). "DNA processing reactions in bacterial conjugation". Annual Review of Biochemistry. 64 (1): 141–169. doi:10.1146/annurev.bi.64.070195.001041. PMID   7574478.
  6. ↑ Gyles C, Boerlin P (March 2014). "Horizontally transferred genetic elements and their role in pathogenesis of bacterial disease". Veterinary Pathology. 51 (2): 328–340. doi: 10.1177/0300985813511131 . PMID   24318976. S2CID   206510894.
  7. ↑ Guiney DG, Helinski DR (October 1979). "The DNA-protein relaxation complex of the plasmid RK2: location of the site-specific nick in the region of the proposed origin of transfer". Molecular & General Genetics. 176 (2): 183–189. doi:10.1007/BF00273212. PMID   393953. S2CID   23889133.
  8. 1 2 3 Francia MV, Varsaki A, Garcillán-Barcia MP, Latorre A, Drainas C, de la Cruz F (February 2004). "A classification scheme for mobilization regions of bacterial plasmids". FEMS Microbiology Reviews. 28 (1): 79–100. doi: 10.1016/j.femsre.2003.09.001 . PMID   14975531.
  9. ↑ Zhang S, Meyer R (August 1997). "The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer". Molecular Microbiology. 25 (3): 509–516. doi:10.1046/j.1365-2958.1997.4861849.x. PMID   9302013. S2CID   26826243.
  10. ↑ Scherzinger E, Lurz R, Otto S, Dobrinski B (January 1992). "In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins". Nucleic Acids Research. 20 (1): 41–48. doi:10.1093/nar/20.1.41. PMC   310323 . PMID   1738602.
  11. ↑ Coupland GM, Brown AM, Willetts NS (June 1987). "The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing". Molecular & General Genetics. 208 (1–2): 219–225. doi:10.1007/BF00330445. PMID   3039307. S2CID   11985769.
  12. 1 2 Fu YH, Tsai MM, Luo YN, Deonier RC (February 1991). "Deletion analysis of the F plasmid oriT locus". Journal of Bacteriology. 173 (3): 1012–1020. doi:10.1128/jb.173.3.1012-1020.1991. PMC   207219 . PMID   1991706.
  13. 1 2 3 4 5 Zrimec J, Lapanje A (January 2018). "DNA structure at the plasmid origin-of-transfer indicates its potential transfer range". Scientific Reports. 8 (1): 1820. Bibcode:2018NatSR...8.1820Z. doi:10.1038/s41598-018-20157-y. PMC   5789077 . PMID   29379098.
  14. 1 2 3 Byrd DR, Matson SW (September 1997). "Nicking by transesterification: the reaction catalysed by a relaxase". Molecular Microbiology. 25 (6): 1011–1022. doi: 10.1046/j.1365-2958.1997.5241885.x . PMID   9350859. S2CID   35753372.
  15. 1 2 Lee CA, Grossman AD (October 2007). "Identification of the origin of transfer (oriT) and DNA relaxase required for conjugation of the integrative and conjugative element ICEBs1 of Bacillus subtilis". Journal of Bacteriology. 189 (20): 7254–7261. doi:10.1128/JB.00932-07. PMC   2168444 . PMID   17693500.
  16. ↑ Arutyunov D, Frost LS (July 2013). "F conjugation: back to the beginning". Plasmid. Special Issue based on the International Society for Plasmid Biology Meeting: Santander 2012. 70 (1): 18–32. doi:10.1016/j.plasmid.2013.03.010. PMID   23632276.
  17. 1 2 3 4 Guasch A, Lucas M, Moncalián G, Cabezas M, Pérez-Luque R, Gomis-Rüth FX, et al. (December 2003). "Recognition and processing of the origin of transfer DNA by conjugative relaxase TrwC". Nature Structural Biology. 10 (12): 1002–1010. doi:10.1038/nsb1017. PMID   14625590. S2CID   27050728.
  18. ↑ Frost LS, Ippen-Ihler K, Skurray RA (June 1994). "Analysis of the sequence and gene products of the transfer region of the F sex factor". Microbiological Reviews. 58 (2): 162–210. doi:10.1128/mr.58.2.162-210.1994. PMC   372961 . PMID   7915817.
  19. ↑ Heinemann JA, Sprague GF (July 1989). "Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast". Nature. 340 (6230): 205–209. Bibcode:1989Natur.340..205H. doi:10.1038/340205a0. PMID   2666856. S2CID   4351266.
  20. ↑ Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V (February 2001). "Genetic transformation of HeLa cells by Agrobacterium". Proceedings of the National Academy of Sciences of the United States of America. 98 (4): 1871–1876. Bibcode:2001PNAS...98.1871K. doi: 10.1073/pnas.98.4.1871 . PMC   29349 . PMID   11172043.
  21. ↑ Waters VL (December 2001). "Conjugation between bacterial and mammalian cells". Nature Genetics. 29 (4): 375–376. doi:10.1038/ng779. PMID   11726922. S2CID   27160.
  22. 1 2 3 Karas BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips AP, Noddings CM, et al. (April 2015). "Designer diatom episomes delivered by bacterial conjugation". Nature Communications. 6 (1): 6925. Bibcode:2015NatCo...6.6925K. doi:10.1038/ncomms7925. PMC   4411287 . PMID   25897682.
  23. ↑ Lopatkin AJ, Meredith HR, Srimani JK, Pfeiffer C, Durrett R, You L (November 2017). "Persistence and reversal of plasmid-mediated antibiotic resistance". Nature Communications. 8 (1): 1689. Bibcode:2017NatCo...8.1689L. doi:10.1038/s41467-017-01532-1. PMC   5698434 . PMID   29162798.
  24. 1 2 Carraro N, Rivard N, Burrus V, Ceccarelli D (2017-03-04). "Mobilizable genomic islands, different strategies for the dissemination of multidrug resistance and other adaptive traits". Mobile Genetic Elements. 7 (2): 1–6. doi:10.1080/2159256X.2017.1304193. PMC   5397120 . PMID   28439449.
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