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DNA Structure at the Plasmid Origin-Of-Transfer Indicates Its www.nature.com/scientificreports OPEN DNA structure at the plasmid origin-of-transfer indicates its potential transfer range Received: 12 May 2016 Jan Zrimec1,2,3 & Aleš Lapanje1,4,5 Accepted: 10 January 2018 Horizontal gene transfer via plasmid conjugation enables antimicrobial resistance (AMR) to spread Published: xx xx xxxx among bacteria and is a major health concern. The range of potential transfer hosts of a particular conjugative plasmid is characterised by its mobility (MOB) group, which is currently determined based on the amino acid sequence of the plasmid-encoded relaxase. To facilitate prediction of plasmid MOB groups, we have developed a bioinformatic procedure based on analysis of the origin-of-transfer (oriT), a merely 230 bp long non-coding plasmid DNA region that is the enzymatic substrate for the relaxase. By computationally interpreting conformational and physicochemical properties of the oriT region, which facilitate relaxase-oriT recognition and initiation of nicking, MOB groups can be resolved with over 99% accuracy. We have shown that oriT structural properties are highly conserved and can be used to discriminate among MOB groups more efciently than the oriT nucleotide sequence. The procedure for prediction of MOB groups and potential transfer range of plasmids was implemented using published data and is available at http://dnatools.eu/MOB/plasmid.html. Antimicrobial resistance (AMR) is a pressing global issue, as it diminishes the activity of 29 antibiotics and conse- quently leads to over 25,000 deaths each year in Europe alone1,2. Te development of AMR in microbial commu- nities is facilitated by horizontal gene transfer (HGT) of conjugative elements (including plasmids and integrative elements)3 carrying antibiotic resistance genes along with virulence genes4,5. It is therefore important to deter- mine the routes of plasmid transfer among bacteria6,7, based on determining their host range8. It is currently known that each of the 6 established mobility superclasses of conjugative elements have limited transfer host range8. Conjugation systems of each of these MOB groups are classifed according to the conserva- tion of the amino acid sequences of relaxase, the central enzyme that enables relaxation and transfer of elements from donor to recipient cells9,10. Besides relaxases, the relative conservative nature of MOB groups can be detected among other protein components of conjugation systems, which are comprised of (i) auxiliary proteins that take part in formation of the relaxation complex (relaxosome) in the origin of transfer (oriT) DNA region11, (ii) cou- pling protein (type IV)12,13, which connects the relaxosome with (iii) the mating complex (type IV secretion system, T4SS) that forms the transfer channel between donor and recipient cells14. Tese protein components were shown to coevolve to a large extent within their respective MOB groups12,13,15. In addition to the conservative nature of proteins involved in DNA transfer, it has also been observed that a relaxase from a certain MOB group enables the most efcient transfer only of plasmids belonging to that same group16. Terefore, one can expect that the substrate for relaxases, the bare noncoding sites in oriT, should also possess some MOB-specifc properties that enable their cognate relaxases to initiate the conjugation process most efciently (Fig. 1, Table 1). Te specifc conservation of oriT properties within MOB groups can also be expected, since DNA binding proteins recognize a particular site on DNA by a physicochemical interaction with the DNA. Prior to binding, proteins slide on DNA in controlled 1D difusion processes in search of their active binding sites17,18. Terefore, some of the essential features of DNA recognition that optimize the protein-DNA indirect readout process are the conformational and physicochemical DNA structural properties at the specifc binding sites and around them19,20. In the case of initiation of conjugation, the oriT region is a recognition site and it is also an enzymatic 1Institute of Metagenomics and Microbial Technologies, 1000, Ljubljana, Slovenia. 2Faculty of Health Sciences, University of Primorska, 6320, Izola, Slovenia. 3Department of Biology and Biological Engineering, Chalmers University of Technology, 412 96, Göteborg, Sweden. 4Department of Nanotechnology, Saratov State University, 410012, Saratov, Russian Federation. 5Department of Environmental Sciences, Institute Jožef Štefan, 1000, Ljubljana, Slovenia. Correspondence and requests for materials should be addressed to J.Z. (email: janzrimec@ gmail.com) or A.L. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:1820 | DOI:10.1038/s41598-018-20157-y 1 www.nature.com/scientificreports/ Figure 1. Schematic representation of available experimental data on oriT regions from four MOB groups. oriT data from MOB F (blue), P (green), Q (red) and V (cyan) supports that the conservation of structural properties within each MOB group is greater than between groups. Known binding sites for auxiliary proteins and relaxases are marked (colored squares) and are frequently characterized by inverted repeats (<IR>). Relaxase binding sites are nearest to the nic site (between 0 and 1 bp). General characteristics of MOB groups are: (F) system of multiple auxiliary proteins including the DNA-bending protein IHF44,55, (P) up to 5 proteins including relaxase involved in relaxation (RP4)47,54,61 (Q) a shorter oriT region of only 38 bp that covers besides relaxase 2 auxiliary proteins without clear binding sites (RSF1010)48,49,62, (V) no known auxiliary proteins50,51,63. Experimental oriT structural features Reference Predicted properties Deformability S Direct and inverted repeats of DNA sequence around nic that defne extensive 44,64 Def secondary structures (e.g. hairpins) and act as protein-DNA recognition regions Duplex stability SStab DNA melting bubbles and destabilizations that facilitate relaxase nicking, aided by 21,33,65,66 Termally induced duplex lower duplex stability around nic and by DNA thermal dynamics destabilizations STIDD Bending propensity S Intrinsically curved or fexible regions that facilitate binding and changes in oriT 44,55,67 Bend structure around nic (e.g. IHF protein binding in MOB F) Persistence length SPer 68 Diferences in DNA spacing and orientation between binding sites and nic Helical repeats SHel Table 1. oriT structural properties that enable relaxasome formation and nicking of DNA to initiate transfer of conjugative elements. Shown are experimentally determined oriT structural features and predicted structural properties that were used to interpret them. substrate, since the relaxase recognizes specifc DNA as well as makes a nick in the DNA to initiate conjuga- tion21,22. However, contrary to the conserved amino or nucleic acid sequences of relaxases and auxiliary proteins, the oriT is a noncoding region and low conservation of nucleotide sequence is expected9,11. Terefore, in order to pinpoint the specifc properties in oriT that are conserved within plasmids of a par- ticular MOB group, the conventional approach based on clustering of similar DNA sequences is unlikely to be successful. A more advanced approach is required to classify MOB groups based on the analysis of oriT structural properties. Te aims of the present study were to (i) analyze the DNA structural properties of oriT regions from diferent MOB groups (Fig. 1, Table 1), (ii) determine if DNA structural properties are conserved within MOB groups and can be used to discriminate among them and (iii) implement the classifcation procedure as a webtool available to the wider research community. Methods oriT datasets. To construct and analyze statistical and predictive models a training and a testing dataset were used. Te training dataset comprised nucleotide sequences of oriT regions of 64 elements that were obtained from the Genbank database. In these sequences the oriT regions were identifed and aligned according to published experimental information on nic sites (Supp. Table S1). Despite the scarce amount of published data, which lim- ited the amount of MOB groups used and the size of the training dataset, the dataset was balanced, with approx- imately 16 elements from each MOB and contained oriTs from all known MOB subgroups10. For the testing dataset we obtained 136 oriT regions from plasmids, for which the only previously available information was that of their MOB groups, determined on the basis of amino acid sequences of relaxases10. Te locations of nic sites in these plasmids were determined by fnding the minimal Euclidean distance between structural properties of training oriTs and the testing dataset. Te positions of resulting oriT regions were verifed using experimental data and relaxase locations10 (Supp. Table S2). By combining the training and testing datasets, the expanded data- set of 200 elements was of an appropriate size to support a statistical and machine learning analysis (Supp. Fig. S1: SCIENTIFIC REPORTS | (2018) 8:1820 | DOI:10.1038/s41598-018-20157-y 2 www.nature.com/scientificreports/ Figure 2. Overview of the performed computational analysis. DNA sequences of oriT regions and their MOB groups were used to compare (A) the conventional approach based on analysis of primary sequences with (B) our new approach based on DNA structure prediction. oriT regions were ligned up to the nic functional site. see learning curves). Te testing dataset was thus used for cross validations as well as training of predictive mod- els. In both datasets
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