DNA structure at the plasmid origin-of-transfer indicates its potential transfer range

Abstract

Horizontal gene transfer via plasmid conjugation enables antimicrobial resistance (AMR) to spread 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 efficiently 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 consequently leads to over 25,000 deaths each year in Europe alone 1,2 . The development of AMR in microbial communities is facilitated by horizontal gene transfer (HGT) of conjugative elements (including plasmids and integrative elements) 3 carrying antibiotic resistance genes along with virulence genes 4,5 . It is therefore important to determine the routes of plasmid transfer among bacteria 6,7 , based on determining their host range 8 . It is currently known that each of the 6 established mobility superclasses of conjugative elements have limited transfer host range 8 . Conjugation systems of each of these MOB groups are classified according to the conservation of the amino acid sequences of relaxase, the central enzyme that enables relaxation and transfer of elements from donor to recipient cells 9,10 . 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 region 11 , (ii) coupling 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 cells 14 . These protein components were shown to coevolve to a large extent within their respective MOB groups 12,13,15 . 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 efficient transfer only of plasmids belonging to that same group 16 . Therefore, one can expect that the substrate for relaxases, the bare noncoding sites in oriT, should also possess some...

Multiple plasmid origin-of-transfer substrates enable the spread of natural antimicrobial resistance to human pathogens

“…Horizontal gene transfer of antibiotic resistance (AMR) genes occurs via the processes of transformation and conjugation. The former mediates especially narrow range, intra-genus transfers 1,2 , whereas the latter is implicated in a wider range of transfer hosts 3,4 and potentially enables AMR to overcome the toughest phylogenetic and ecological transmission barriers [5][6][7][8][9][10] .…”

“…Consequently, the interaction between conjugative relaxase enzymes and their DNA origin-of-transfer (oriT) substrates facilitates the majority of all AMR transfers in nature 11,12 and is especially important for ones related to human infection complications 13 . However, the current knowledge on conjugative transfer mechanisms and systems 4,[14][15][16] is unable to describe the unprecedented amount of observed horizontal transfer 7,8,12,17 that seems to transcend all transfer barriers between resistance reservoirs and human hosts [5][6][7]9,10,12,13,18 .…”

“…However, besides the possibility of yet unidentified enzymes and mobility groups 15,[20][21][22][23][24] , multiple new processes have recently been uncovered that might confer additional mobility to plasmids and involve the origin-of-transfer (oriT) DNA substrate. These include (i) broadened relaxase binding specificities to multiple different oriT sequence variants [25][26][27][28][29] , which, according to the evolutionary theory of such DNA regions 4,30,31 , indicates the possibility of plasmids carrying multiple functional secondary oriTs, and (ii) trans-mobilization of plasmids carrying oriTs triggered by relaxases from co-resident plasmids acting in trans on the non-cognate oriTs [32][33][34][35] . The latter mechanism demonstrates that oriT regions are the only elements of the conjugation machinery required in cis 21 and suggests that many plasmids classified as non-mobile due to the absence of putative relaxases may in fact be mobilizable 33 .…”

“…A major problem with uncovering oriT regions is that, apart from being experimentally laborious, it is computationally challenging due to multiple molecular mechanisms and a variety of DNA 3 sequence elements present and coevolving in the DNA substrate 4 , even among plasmids belonging to a single species such as Staphylococcus aureus 32 . OriT typing requires algorithms beyond simple sequence-based alignment 36,37 , which can recognise and process more complex molecular motifs such as inverted repeats and hairpins [38][39][40] as well as underlying DNA physicochemical and conformational features.…”

“…OriT typing requires algorithms beyond simple sequence-based alignment 36,37 , which can recognise and process more complex molecular motifs such as inverted repeats and hairpins [38][39][40] as well as underlying DNA physicochemical and conformational features. These underpin key protein-DNA readout and activity mechanisms 4,[41][42][43] as well as define conserved niches of structural variants that enable good resolution between MOB groups and subgroups 4 . The use of DNA structural representations has indeed led to improvements in algorithms for identification of other regulatory regions, such as promoters and replication origins [44][45][46][47][48] .…”