Miniature inverted-repeat transposable elements mobilize diverse antibiotic resistance genes in Enterobacteriaceae

Tn3-derived inverted-repeat miniature elements (TIMEs) that mobilize antibiotic resistance genes | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Tn 3 -derived inverted-repeat miniature elements (TIMEs) that mobilize antibiotic resistance genes View ORCID Profile Ryota Gomi , View ORCID Profile Hirokazu Yano doi: https://doi.org/10.1101/2025.11.05.686661 Ryota Gomi a Research Center for Water Environment Technology, School of Engineering, The University of Tokyo , Bunkyo-ku, Tokyo, Japan b Department of Environmental Engineering, Graduate School of Engineering, Kyoto University , Katsura, Nishikyo-ku, Kyoto, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ryota Gomi For correspondence: gomi{at}env.t.u-tokyo.ac.jp gomi.ryota.34v{at}kyoto-u.jp Hirokazu Yano c Antimicrobial Resistance Research Center, National Institute of Infectious Diseases, Japan Institute for Health Security , Higashimurayama, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hirokazu Yano Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Miniature inverted-repeat transposable elements (MITEs) are nonautonomous mobile genetic elements (MGEs) that can be mobilized by transposases provided by the relevant autonomous MGEs. MITEs originating from Tn 3 -family transposons were previously termed Tn 3 -derived inverted-repeat miniature elements (TIMEs). Composite transposon-like structures bounded by two copies of TIME, called TIME-COMPs, were shown to mobilize the intervening sequences. However, their association with antibiotic resistance genes (ARGs) has not yet been systematically studied. This study thus aimed to identify new TIME-COMP-like structures containing ARGs in the genomic sequences of the clinically important bacterial family Enterobacteriaceae in public databases. TIME-COMP-like structures were first searched for in the plasmid database PLSDB, focusing on small plasmids, using a self-against-self blastn approach to identify repeated elements. Then, newly and previously identified MITEs (including TIMEs) were searched for in the NCBI core nucleotide database to identify TIME-COMP-like structures located on other replicons. Bioinformatic analysis identified multiple previously unreported TIME-COMPs containing ARGs, which are bounded by directly or inversely oriented TIMEs, namely, IS 101 , MITESen1, and a novel 244-bp TIME termed TIME244. TIME244 contains a putative resolution site related to that of Tn 21 . These TIMEs were predominantly detected in plasmids and very rarely in chromosomes. The ARGs embedded in newly identified TIME-COMPs were bla KPC-2 , floR , qnrS1 , and tet (A). Notably, the bla KPC-2 carbapenemase gene was found in TIME-COMPs bounded by TIME244 and a TIME-COMP bounded by IS 101 . These findings highlight a potential role for TIMEs in the spread of diverse ARGs. IMPACT STATEMENT Bacterial miniature inverted-repeat transposable elements (MITEs) are a group of short (50 bp–500 bp) nonautonomous transposable elements that are thought to have originated from insertion sequences or transposons. Although MITEs can theoretically mobilize antibiotic resistance genes (ARGs) in the presence of transposases, only a few studies have reported their association with ARGs, probably due to difficulties in identifying MITEs in genomic sequences. This study provides evidence, based on bioinformatic analysis of public Enterobacteriaceae genomes, that a subset of MITEs, called Tn 3 -derived inverted-repeat miniature elements (TIMEs), mobilizes ARGs by forming composite transposon-like structures. A novel 244-bp TIME, designated TIME244, was present in more than 100 Enterobacteriaceae plasmids in the current RefSeq database, suggesting its further transmission in bacterial populations through horizontal gene transfer. This study reveals that TIMEs were often overlooked when analyzing the genetic contexts of ARGs in previous studies. These findings highlight the importance of TIMEs in bacterial gene acquisition and underscore the need for new tools that can detect TIMEs in bacterial genomes for ARG surveillance. DATA SUMMARY Accession numbers of sequence data analyzed in this study are provided within the article or in supplementary data files. INTRODUCTION Antibiotic-resistant bacteria, particularly those with multidrug resistance (MDR), are a global health concern. Bacteria can accumulate antibiotic resistance genes (ARGs) with the help of mobile genetic elements (MGEs), which can often lead to MDR. MGEs can be categorized into two types: those mobilizing ARGs from one cell to another cell (i.e., intercellular mobilization) and those mobilizing ARGs from one replicon to another replicon or to different locations within the same replicon (i.e., intracellular mobilization). 1 Intercellular mobilization mechanisms such as conjugation can directly contribute to horizontal transfer of ARGs and thus the emergence and spread of MDR. Intracellular mobilization can also contribute to the increase of MDR in multiple ways; for example, by mobilizing ARGs on a plasmid to different plasmids, thereby increasing the potential of the ARGs to be transmitted to additional bacterial recipients via conjugation. 2 MGEs mediating intracellular mobilization of ARGs include well-studied elements such as insertion sequences (ISs) and transposons. These are autonomous (i.e., capable of self-transposition), in contrast to nonautonomous elements, which can also mediate intracellular mobilization but need the transposase activity provided by autonomous elements in the same cell. 1 Among the known nonautonomous elements are structures called miniature inverted-repeat transposable elements (MITEs). 3 MITEs are thought to have originated from ISs or transposons. They retain the terminal inverted repeats (IRs) but lack the internal regions, including the transposase gene(s). They are typically 50 bp–500 bp and create direct repeats (DRs) upon insertion. 4 A subset of MITEs originating from Tn 3 -family transposons and carrying Tn 3 -related IRs are called Tn 3 -derived inverted-repeat miniature elements (TIMEs), and they include elements such as IS 101 , integron mobilization unit (IMU), MITESen1, and TIME1–TIME4. 5 – 8 Composite transposon-like structures bounded by two copies of TIME, known as TIME-COMPs, were shown to mobilize the intervening sequences when the transposase activity of autonomous Tn 3 -family elements was provided in trans . 5 , 7 However, to date, known TIME-COMPs carrying an ARG(s) are limited to IMU-bounded TIME-COMPs carrying a carbapenemase gene bla GES-5 and an IS 101 -bounded TIME-COMP carrying a quinolone resistance gene qnrD1 , even in the clinically important bacterial family Enterobacteriaceae ( Figure S1 ). 7 , 9 , 10 MITEs other than TIMEs should also be capable of mobilizing an ARG when the ARG is embedded within a TIME-COMP-like structure; however, to our knowledge, no such non-TIME MITE-bounded structures have been reported in Enterobacteriaceae. Tn 3 -family transposons have been identified in nearly all bacterial phyla and are classified into several subgroups (including Tn 3 and Tn 21 ) based on the phylogeny of their transposases. 11 These elements mobilize through a “paste-and-copy” mechanism involving two distinct steps: cointegrate formation and cointegrate resolution. The first step is mediated by the transposase (the tnpA gene product), IRs, and the host replication machinery, generating a fusion replicon in which the donor and target replicons are bridged by directly repeated copies of the transposon. In the second step, the cointegrate is resolved into two separate molecules, each carrying a transposon insertion. This resolution is typically mediated by the transposon-encoded resolvase (the tnpR gene product) and its cognate target site ( res ), or alternatively through homologous recombination between the duplicated transposon sequences. TIME and TIME-COMP elements are hypothesized to employ a similar “paste-and-copy” mechanism; however, whether these elements contain functional resolution sites remains unclear in most cases, with the exception of IS 101 , which possesses a functional res site. 12 Transposition of an IMU-bounded TIME-COMP was previously demonstrated by providing a transposase of IS Sod9 belonging to the Tn 21 subgroup. 7 IS 101 cointegrate formation and resolution were demonstrated in the presence of Tn 1000 (also called gamma-delta) belonging to the Tn 3 subgroup. 12 Known TIMEs and TIME-COMPs create 5-bp (or sometimes 6-bp) DRs upon transposition like Tn 3 -family transposons. 5 , 7 , 11 Thus, past transposition of a TIME-COMP can be inferred from the presence of 5-bp or 6-bp DRs flanking the outermost IRs of the TIME-COMP. MITEs (including TIMEs) tend to be overlooked in standard annotation pipelines due to their lack of transposase gene(s). Furthermore, as of January 2026, the number of MITE sequences registered in the ISFinder database remained at 65, with only three originating from Enterobacteriaceae. 13 Thus, the contribution of MITEs to the intracellular mobility of ARGs is likely underestimated. Hence, this study aimed to identify previously overlooked TIME-COMP-like structures carrying ARGs in Enterobacteriaceae, by data-mining. METHODS Plasmid sequences Plasmid sequences (n = 72,556) were downloaded from PLSDB (v. 2024_05_31_v2), which is a curated and non-redundant plasmid database sourced from the NCBI database. 14 Plasmid sequences meeting the following criteria were extracted: (i) at least one ARG was detected by Abricate (v1.0.1, https://github.com/tseemann/abricate ) with the ncbi database; 15 (ii) at least one plasmid replicon was detected by Abricate (v1.0.1) with the “--db plasmidfinder” option 16 (this was to exclude sequences that are not likely to be true plasmids and to be conservative; for example, NZ_CP107371 is composed almost entirely of ARGs and ISs/transposons, suggesting a non-plasmid circular element or a misassembled circular contig); (iii) the plasmid host is Enterobacteriaceae; and (iv) the length is 20,000 bp or shorter. Here, we focused on small plasmids because their small size allows manual curation of the results of bioinformatic analysis (described in detail below), and previously reported TIME-COMPs with ARGs in Enterobacteriaceae were located on small plasmids (<10 kbp). 7 , 9 , 10 Plasmid sequences meeting the above criteria (n = 1,007) were subjected to self-against-self blastn analysis as described below to detect repeated sequences that could be MITEs. Detection of TIME-COMP-like structures carrying ARGs The BLAST+ (v2.16.0) program was used for similarity searches. The makeblastdb and blastn functions were used with defaults parameters. Plasmid sequences with self-against-self blastn hits meeting the following criteria were retained: (i) the alignment length is between 50 bp and 500 bp; (ii) the percentage of identical matches is 98 % or higher; (iii) a sequence longer than 500 bp is present between the aligned part of the query sequence and that of the subject sequence. These criteria were applied to detect repeat sequences (50 bp–500 bp) surrounding a stretch of sequence (> 500 bp) in a plasmid (see Figure S2 for the rationale for these criteria). To confirm that the 50-bp to 500-bp repeat sequences are actually MITEs, a self-against-self blastn search was performed for each repeat sequence to identify IRs, which are the signature of MITEs. The blastn search was performed by using a word size of six to increase the sensitivity, and the result was visualized using CLC Main Workbench 24 (Qiagen, Hilden, Germany) ( Figure S3 ). The putative MITE sequences were checked for identity to any known elements in the ISfinder database. 13 Only TIME-COMP-like structures flanked by DRs, which are typically created upon insertion of the entire structure, were considered for further analysis. This was to exclude cases where two MITEs were independently inserted and coincidentally formed a TIME-COMP-like structure. The lengths of DRs looked for were determined with reference to those of the related ISs or transposons. Plasmids with TIME-COMP-like structures carrying ARGs were annotated using ISfinder and the blastn web tool ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The MITE(s) found in the above approach, previously described TIMEs associated with ARGs in Enterobacteriaceae (i.e., IMU and IS 101 ), 7 , 9 , 10 and Enterobacteriaceae MITEs listed in the ISfinder database (i.e., MITEKpn1 and MITESen1, excluding MITEEc1, which is an enterobacterial repetitive intergenic consensus [ERIC] sequence and present in multiple copies on the chromosome of Escherichia coli ), 13 , 17 , 18 were searched for in the NCBI core nucleotide database (core_nt) using the blastn web tool (last accessed July 2025). Replicons with two or more blastn hits with ≥95 % identity and ≥95 % coverage were manually screened to detect additional TIME-COMP-like structures carrying an ARG in Enterobacteriaceae. This enabled identification of TIME-COMP-like structures in small plasmids not indexed in PLSDB, in longer plasmids, and also in chromosomes. Putative ancestral plasmids without insertion of TIME-COMP-like structures were identified by performing the online blastn analysis against the NCBI core_nt database, using a sequence, which was prepared by joining the sequences flanking the TIME-COMP-like structure minus one copy of the DR, as a query sequence (last accessed January 2026). Detection of MITEs in RefSeq Enterobacteriaceae genomes To investigate the prevalence of MITEs that were found to be associated with ARGs in the above analysis, we downloaded RefSeq Enterobacteriaceae genomes with an assembly level of “complete genome” using the NCBI Datasets command-line tools (v18.9.0) in October 2025 (n = 11,572). 19 MITE sequences were searched for in the retrieved genomes using makeblastdb and blastn in BLAST+ (v2.16.0) with default parameters. We used the same criteria as the online blastn search to define the presence of MITE sequences (i.e., ≥95 % identity and ≥95 % coverage). RESULTS AND DISCUSSION Identification of TIME-COMP-like structures carrying ARGs First, we conducted self-against-self blastn analysis to identify TIME-COMP-like structures in a subset of small Enterobacteriaceae plasmids registered in the curated plasmid database, PLSDB. This relatively small dataset (n = 1,007) made manual curation of the blastn results feasible, which includes confirmation of the IRs in putative MITEs and DRs flanking the entire TIME-COMP-like structures. These procedures revealed TIME-COMP-like structures carrying ARGs in six plasmids ( Table S1 ). All these structures are bounded by TIMEs and thus are TIME-COMPs. One of these plasmids is pKFu015_4, which was previously reported to carry the IS 101 -bounded TIME-COMP containing qnrD1 ( Figure S1 ). 10 The remaining five plasmids contain the same TIME-COMP structure with bla KPC-2 and a truncated bla TEM gene, which is bounded by two copies of a 244-bp TIME (hereafter named TIME244). pCHE-A and pCHE-A1, which were previously reported to contain IMU-bounded TIME-COMPs ( Figure S1 ), were detected by the self-against-self blastn searches but were filtered out in manual curation, because the TIME-COMP structures in these plasmids are not flanked by DRs. 7 , 9 We also detected six other plasmids carrying TIME-COMP structures with ARGs but not flanked by DRs, all bounded by MITESen1. These TIME-COMP structures may have the potential to transpose as a unit; however, these cases were filtered out to restrict the dataset to plasmids showing direct evidence of transposition. To identify additional TIME-COMP-like structures in replicons other than small plasmids indexed in PLSDB, we performed online blastn searches using TIME244 and previously reported MITEs, including those classified as TIMEs (IS 101 , IMU, and MITESen1) and one non-TIME MITE (MITEKpn1), as query sequences (see Table S2 for the sequence of each MITE). This online blastn analysis identified 141 additional replicons carrying TIME-COMPs with ARGs. These included 136 plasmids and one chromosome carrying TIME244-bounded TIME-COMPs (most of which contain tet (A)), one plasmid carrying an IS 101 -bounded TIME-COMP, and three plasmids carrying MITESen1-bounded TIME-COMPs ( Table S1 ). The genetic contexts of TIME-COMPs identified using the above approaches were thoroughly inspected as described below. TIME244-bounded TIME-COMPs As mentioned above, we detected TIME244-bounded TIME-COMPs in a total of 142 replicons (i.e., five identified by the self-against-self blastn analysis, and 137 identified by the online blastn analysis). Three main types of TIME244-bounded TIME-COMPs were identified. One of them is a TIME244-bounded TIME-COMP carrying tet (A) detected in 133 replicons ( Table S1 ). This TIME-COMP is bounded by inversely oriented TIME244 copies and contains part of Tn 1721 , an 11-kb Tn 3 -family element carrying tetracycline resistance determinant genes originally identified on an E. coli plasmid ( Figure 1a ). 20 , 21 This TIME-COMP was inserted in three unique locations with DRs of AGCAA (n = 114), TATAA (n = 18), and TACTT (n = 1) ( Figure 1a and Table S1 ). This indicates that three independent transposition events of this TIME-COMP had occurred in the past. In 114 replicons with DRs of AGCAA, two types of the TIME-COMP were identified: one with an inverted internal structure (n = 52) and the other without (n = 62) (see below for detailed discussion). Furthermore, there were several indels within the TIME-COMP structure, two of which involved alterations of ARG contents ( Figure S4 , see Table S1 for detailed description). Briefly, in a plasmid, a resistance region containing bla TEM-1B and bla CTX-M-206 is inserted within the left TIME244 copy. In the other plasmid, the Tn 1721 -derived region is partially deleted and replaced by a truncated Tn 3 -like transposon and a truncated Tn 4401 -like transposon containing bla KPC-2 (i.e., a variant of non-Tn 4401 elements [NTE KPC ], which contains only a portion of Tn 4401 , the most common bla KPC -containing mobile element belonging to the Tn 3 family 22 ). In these cases, the DR sequences flanking the TIME-COMPs are the common ones (AGCAA); thus the indels were likely introduced after the insertion of the original TIME-COMP carrying tet (A). The outermost IRs within the altered TIME-COMP structures are intact, implying the feasibility of further transposition if the relevant transposase activity is provided in trans ( Figure S4 ). Download figure Open in new tab Figure 1. (a) TIME244-bounded TIME-COMP carrying tet (A) and DRs flanking the TIME-COMP. The expanded diagram of the TIME-COMP is shown on the top. Major replicon types and the numbers of replicons are shown on the right side. *There are variants in TIME-COMPs flanked by AGCAA (see Table S1 for details). (b) TIME244-bounded TIME-COMP carrying bla KPC-2 and a truncated bla TEM gene, and plasmids carrying the TIME-COMP. The expanded diagram of the TIME-COMP is shown above the plasmids. For the TIME-COMP structure in each plasmid, only TIMEs and ARGs are depicted for clarity. The TIME-COMP was inserted into a pRJKP36-5-like plasmid and a p4-N2251932-like plasmid, leading to pKPC45a and pKQPS142b (and four other IncQ plasmids showing >99% identity and >99% coverage against pKQPS142b, as in Table S1 ), respectively. In pKPN535a, three copies of TIME244 are present, and a sequence identical to pSYCC1_6 is present between two of the TIME244 copies. This indicates a cointegrate formation by “paste-and-copy” transposition of TIME244, leading to fusion of a pKQPS142b-like plasmid and pSYCC1_6. The online blastn analysis identified eight putative ancestral plasmids (including pRJKP36-5) for pKPC45 and seven putative ancestral plasmids (including p4-N2251932) for pKQPS142b, showing >90% identity and >98% coverage against the uninterrupted version of each plasmid. (c) TIME244-bounded TIME-COMP carrying qnrS1 , and plasmids carrying the TIME-COMP. The TIME-COMP was likely inserted in a pCP102-like plasmid, leading to pK814-QNRS. pK814-QNRS then acquired a ∼35-kb region with high similarity to the partial sequence of Klebsiella phage KP12 clone KP12_2 (OM835952) (96% identity and 93% coverage), leading to pK1326-qnrS. The online blastn analysis identified three putative ancestral plasmids (including pCP102) for pK814-QNRS, showing >90% identity and >80% coverage against the uninterrupted version of pK814-QNRS. Red arrows indicate ARGs, gray pointed boxes indicate ISs, blue boxes indicate TIMEs, and black arrows indicate other genes. The IRs within each TIME are shown by a black (left, IRL) or white (right, IRR) triangle (see TableS2 for the basis of the orientations of TIMEs). DRs are shown as differently shaped flags, and their sequences are shown in boxes. The accession numbers of the plasmids are as follows: pRJKP36-5, CP100081 ; pKPC45a, MH595534 ; p4-N2251932, CP165866 ; pKQPS142b, CP023480 ; pKPN535a, MH595533 ; pSYCC1_6, CP113183 ; pCP102, CP104015 ; pK814-QNRS, CP183858 ; and pK1326-qnrS, CP183864 . Another TIME-COMP carries bla KPC-2 and a truncated bla TEM gene, which is bounded by directly oriented TIME244 copies ( Figure 1b ). This genetic context was previously described as a variant of NTE KPC , designated NTE KPC -IId. 23 The authors noted 243-bp repeats at its boundaries, but did not identify them as MITEs or TIMEs. This TIME-COMP was inserted in the same site in six highly related IncQ1 plasmids, with the same DRs (TGTTTA), and in a single Col-like plasmid with DRs of TATAT ( Figure 1b and Table S1 ). In one of the IncQ1 plasmids (pKPN535a), three copies of TIME244 are present, which could be explained by the fusion of two plasmids. We also detected another TIME-COMP, which contains qnrS1 and is bounded by inversely oriented TIME244 copies, in two untypeable plasmids ( Figure 1c ). 24 , 25 In these plasmids, the TIME-COMP structure is inserted in the same position with DRs of ATCCA; therefore, a total of one unique transposition event was detected. These two plasmids and their putative ancestral plasmid were classified as phages by geNomad (v1.11.2). 26 Thus, these are putative phage-plasmids, which transfer horizontally between cells as viruses and vertically within cellular lineages as plasmids. 27 An IS 101 -bounded TIME-COMP IS 101 , a 209-bp element, is among the earliest identified TIMEs and was originally discovered on a recombinant E. coli plasmid, pSC101. 6 We identified a previously unreported IS 101 -bounded TIME-COMP in one Enterobacter kobei IncX3 plasmid, pEkFL23_IncX3 ( Figure 2 ). This TIME-COMP is bounded by inversely oriented copies of IS 101 and carries bla KPC-2 embedded within a truncated Tn 4401b structure. 28 This genetic context was previously reported as Tn 4401k , but the presence of IS 101 was not addressed in the previous study. 29 The sequence between the two IS 101 copies in this TIME-COMP corresponds to the partial sequence of plasmids such as pKPC_FCF/3SP, and the whole TIME-COMP structure was flanked by 5-bp DRs (ATTCT). These indicate that IS 101 -mediated transposition of part of a pKPC_FCF/3SP-like plasmid into another plasmid generated pEkFL23_IncX3. Download figure Open in new tab Figure 2. IS 101 -bounded TIME-COMP carrying bla KPC-2 and a plasmid carrying the TIME-COMP. The TIME-COMP was inserted into a p3-N4292977-like plasmid, leading to pEkFL23_IncX3. The online blastn analysis identified 19 putative ancestral plasmids (including p3-N4292977) for pEkFL23_IncX3, showing >99% identity and >90% coverage against the uninterrupted version of pEkFL23_IncX3. Genes and elements are shown as in Figure 1 . The accession numbers of the plasmids are as follows: pKPC_FCF/3SP, CP004367 ; p3-N4292977, CP165819 ; and pEkFL23_IncX3, OQ434669 . MITESen1-bounded TIME-COMPs MITESen1 is a 256-bp TIME first identified on an IncX1 plasmid from Salmonella enterica . 8 We identified two different MITESen1-bounded TIME-COMPs in the same location with the same flanking 5-bp DRs (TAATA) in two IncX1-IncX2 hybrid plasmids ( Figure 3a ). 30 , 31 One TIME-COMP contains qnrS1 , and the other contains floR . MITESen1 copies are directly oriented in these TIME-COMPs. Interestingly, there are IncX1-X2 hybrid plasmids carrying a single MITESen1 copy inserted in the same location. We speculate that the two plasmids with TIME-COMPs were generated by integration of a hypothetical circular molecule containing MITESen1 and an ARG into a plasmid already carrying a single copy of MITESen1. The hypothetical circular molecules might be generated by recombination between MITESen1 in a TIME-COMP or by intramolecular replicative transposition of MITESen1 (also see Figure S5 for another possible pathway for generation of the floR -containing circular element). Integration of the circular molecule might occur by homologous recombination between MITESen1 copies. Download figure Open in new tab Figure 3. (a) MITESen1-bounded TIME-COMPs carrying qnrS1 and floR , and plasmids carrying these TIME-COMPs. pLAO96 and pSC-KP585-2 might have been generated by integration of a hypothetical circular molecule into a pCUWW06A-like plasmid. pLAO96 (45,300 bp) and pSC-KP585-2 (44,092 bp) share ∼35.6 kb of highly similar sequence (>99% identity), excluding the TIME-COMP regions (1,894 bp and 4,800 bp) and regions bounded by IS 26 (7,824 bp and 3,609 bp). (b) MITESen1-bounded TIME-COMP carrying qnrD1 and a plasmid carrying the interrupted TIME-COMP. A putative intermediate plasmid carrying the intact TIME-COMP is also shown. “in trans ” indicates intramolecular replicative transposition (in trans ) of IS 26 . The online blastn analysis identified one putative ancestral plasmid (p2025CK-00294_2) for pECC-003-1, showing >99% identity and 86% coverage against the uninterrupted version of the putative intermediate. Genes and elements are shown as in the previous figures. The accession numbers of the plasmids are as follows: pCUWW06A, CP182140 ; pLAO96, OP242301 ; pSC-KP585-2, CP123874 ; p2025CK-00294_2, CP194151 ; and pECC-003-1, CP143772 . A plasmid with the repE(pEh60-7) replicon (pECC-003-1) was found to carry a remnant of a MITESen1-bounded TIME-COMP carrying qnrD1 ( Figure 3b ). 32 One copy of MITESen1 is interrupted by IS 26 , while the truncated remnant of MITESen1 is present next to another copy of IS 26 that is oriented in the opposite direction. This arrangement is consistent with intramolecular replicative transposition (in trans ) of an IS 26 element into MITESen1 in the intact TIME-COMP structure. 33 The presence of 8-bp DRs (ATATCAAC or its reverse complement) next to each IS 26 element on the MITESen1-sides supports this, though the intact version of this TIME-COMP structure was not present in the NCBI core_nt database. Putative resolution sites in TIMEs All TIMEs forming the TIME-COMP structures identified in the present study (i.e., TIME244, IS 101 , and MITESen1) carry IRs related to Tn 3 -family transposons ( Figure 4a ). IRs of TIME244 and the previously reported IMU show more similarities to IRs of Tn 21 than to those of Tn 3 , IS 101 , MITESen1, and Pseudomonas -derived TIME2 that is not associated with ARGs. 5 Thus, TIME244 and IMU seem to have originated from the Tn 21 subgroup of the Tn 3 family. 11 To deduce whether TIME244 contains a res site, we conducted all-against-all SSEARCH using sequences of TIMEs, Tn 21 res , Tn 1721 res , and Tn 3 res . 34 A significant match (Smith-Waterman score [S-W] >100) was detected for pairs of TIME244-Tn 21 res (S-W = 148), TIME244-Tn 1721 res (S-W = 152), Tn 21 res -Tn 1721 res (S-W = 188), and IS 101 -Tn 3 res (S-W = 139). Figure 4b shows alignment of the TIME244 internal sequence and the experimentally determined res sites of Tn 21 and Tn 1721 . 35 The res sites of the Tn 3 family consist of three subsites (site I, site II, and site III) where TnpR dimers bind. Strand exchange takes place at the center of site I (C/O in Figure 4b ). 11 The TIME244 internal sequence shows similarity to both Tn 21 res and Tn 1721 res at all three subsites. Therefore, we speculate that TIME244 contains a functional res site of the Tn 21 subgroup. On the other hand, the internal sequences of IMU and MITESen1 did not show similarity to well-characterized res sites from either autonomous Tn 3 -family elements or other TIMEs (S-W <70), although this does not exclude the possibility that these TIMEs carry resolution sites that serve as targets of site-specific recombinase(s). Download figure Open in new tab Figure 4. (a) Multiple alignment of IRs of TIMEs, Tn 3 , and Tn 21 . (b) Multiple alignment of res sites of Tn 21 , TIME244, and Tn 1721 . Definition of subsites follows a previous study. 35 See Table S2 for the sequences of TIME244, IMU, IS 101 , and MITESen1. The remaining sequences are derived from the following accession numbers: Tn 21 , AF071413; Tn 3 , HM749966; TIME2, KJ920398; and Tn 1721 , X61367. (c) Two frequent forms of TIME244-bounded TIME-COMPs carrying tet (A) inserted in the same location. The genetic contexts in two IncN plasmids (LC771585 and AP026485) are shown as examples. 40 (d) Deduced transposition pathways of TIME-COMPs bounded by inversely oriented TIME244 copies. Cointegrate formation (left) is mediated by TnpA and IRs, followed by replication. Cointegrate resolution (right) is mediated by RecA-dependent homologous recombination (HR) between duplicated TIME-COMPs or by TnpR/ res -dependent site-specific recombination (SSR). SSR can occur between either pair of the directly oriented TIME244 copies. Nicking sites in the donor molecule are indicated by red arrows. In principle, the functionality of res affects the stability of the TIME-COMP structures. When res sites are directly oriented, the segment between two res sites can be deleted in the presence of TnpR. According to studies on autonomous Tn 3 -family elements, when res sites are inversely oriented, the segment between the two res sites can be inverted by TnpR in vivo , 36 , 37 likely because knotted substrates carrying inversely oriented res sites, which are efficient substrates for TnpR, 38 are produced in vivo . Among 142 replicons containing TIME244-bounded TIME-COMPs, only seven replicons contain directly oriented TIME244 copies, while 135 replicons contain inversely oriented TIME244 copies ( Table S1 ). This orientation bias is consistent with the idea that TIME-COMPs carrying directly repeated TIME244 copies are structurally unstable due to the intrinsic resolution activity in the presence of Tn 21 -related TnpR. The most prevalent TIME244-bounded TIME-COMP was that carrying tet (A). This TIME-COMP carries TIME244 copies in inverse orientation. The TIME-COMPs carrying tet (A) in the 114 replicons with DRs of AGCAA are classified into those with an inverted internal structure (n = 52) and those without (n = 62) ( Figure 1a and 4c ). This inversion could be explained by TnpR-mediated site-specific recombination at the res sites. Because TIME244 contains a putative res site, the transposition of TIME244-bounded TIME-COMPs with inversely oriented TIME244 copies should follow a two-step process: cointegrate formation and cointegrate resolution in the presence of Tn 21 -related transposons ( Figure 4d ). Cointegrate resolution can occur by RecA-dependent homologous recombination or site-specific recombination involving Tn 21 -related TnpR and res sites. Prevalence of TIMEs in RefSeq Enterobacteriaceae genomes The NCBI core_nt database, which we used in the online blastn analysis, contains a mixture of complete genomes and sequences that are not derived from complete genomes (e.g., plasmid sequences without the host chromosomal sequences). This prevents the accurate estimation of the prevalence of MITEs associated with ARGs (i.e., TIME244, IS 101 , MITESen1, and IMU, all of which are TIMEs) in Enterobacteriaceae genomes. Thus, to circumvent this problem, we downloaded RefSeq complete Enterobacteriaceae genomes (n = 11,572) and detected these TIMEs in the retrieved genomes. The frequency of genomes carrying each TIME was as follows: TIME244 (n = 109, 0.94%), IS 101 (n = 49, 0.42%), MITESen1 (n = 264, 2.28%), and IMU (n = 7, 0.06%) ( Figure 5 and Table S3 ). The frequency of genomes with a replicon(s) carrying multiple copies of each TIME was as follows: TIME244 (n = 71, 0.61%), IS 101 (n = 4, 0.03%), MITESen1 (n = 54, 0.47%), and IMU (n = 2, 0.02%). These TIMEs were mainly detected in clinically important genera, namely Klebsiella , Escherichia , Enterobacter , and Citrobacter . However, these taxa are more likely to be sequenced due to their clinical importance, and care should be taken to interpret the results. In total, 408 genomes (3.53%) harbored at least one of these TIMEs, indicating that these elements, while rare, are present at a detectable frequency. Interestingly, these TIMEs were predominantly detected in plasmids (n = 444, 97.16%) and very rarely in chromosomes (n = 13, 2.84%). Download figure Open in new tab Figure 5. Numbers of RefSeq Enterobacteriaceae genomes carrying TIMEs (TIME244, IS 101 , MITESen1, and IMU) among the downloaded genomes (n = 11,572). “Total” indicates the total number of genomes carrying each TIME. “Multiple copies” indicates the number of genomes with a replicon(s) carrying multiple copies of each TIME. Genera detected in >10 genomes are shown in different colors, while genera detected in ≤ 10 genomes are grouped as “Others” and shown in gray. See also Table S3 for information on individual genomes. Study limitations This study has limitations. First, the self-against-self blastn approach, which allows de novo identification of MITEs that form TIME-COMP-like structures, was performed only on small plasmids (<20 kbp). Thus, novel MITEs potentially present in other types of replicons might have been missed. Performing the self-against-self blastn analysis also on large plasmids and chromosomes may enable the identification of more MITE elements; however, this approach is unwieldly because it will return exponentially more blastn hits, which makes manual curation of the results (e.g., confirmation of DRs at the ends) almost impossible. A direction for future work would be to develop software for automated detection of TIME-COMP-like structures. A second limitation of this study is that we analyzed only the genomes of Enterobacteriaceae. Although rare, there seem to be TIME-COMP-like structures containing ARGs in other bacteria as well, such as Acinetobacter spp. 39 Extending the approach employed in the present study to other bacteria may uncover additional TIME-COMP-like elements. Conclusions The present study identified multiple previously unreported TIME-COMPs containing ARGs. These novel TIME-COMPs are bounded by three types of TIMEs (i.e., TIME244, IS 101 , and MITESen1). The sequences of these three TIMEs and also IMU are divergent and seem to have independently emerged from distinct transposons of the Tn 3 family. Interestingly, the same TIMEs were associated with different ARGs, and the same ARGs were associated with different TIMEs. This study revealed that TIMEs contribute to the intracellular mobilization of ARGs and highlights the importance of taking these elements into account when analyzing the genetic contexts of ARGs. Funding information This work was supported by JSPS KAKENHI (grant number JP22K18038 to RG, JP25K01942 to HY) and the Environment Research and Technology Development Fund (JPMEERF20235R01 to RG) of the Environmental Restoration and Conservation Agency, provided by the Ministry of the Environment of Japan. Author contributions Conceptualization: R.G. Methodology: R.G. Formal analysis: R.G. and H.Y. Data Curation: R.G. Visualization: R.G. and H.Y. Project administration: R.G. Funding acquisition: R.G. and H.Y. Writing - Original Draft: R.G. and H.Y. Writing - Review & Editing: R.G. and H.Y. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgments We thank Dr. Naofumi Handa at University of California, Davis for helpful discussions. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. Funder Information Declared JSPS KAKENHI , JP22K18038 , JP25K01942 Environment Research and Technology Development Fund , JPMEERF20235R01 Footnotes Figures and main text were updated. REFERENCES 1. ↵ Partridge SR , Kwong SM , Firth N , Jensen SO . Mobile genetic elements associated with antimicrobial resistance . Clin Microbiol Rev 2018 ; 31 : e00088 – 17 . OpenUrl CrossRef PubMed 2. ↵ Lerminiaux NA , Cameron ADS . Horizontal transfer of antibiotic resistance genes in clinical environments . Can J Microbiol 2019 ; 65 : 34 – 44 . OpenUrl CrossRef PubMed 3. ↵ Delihas N . 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Share Tn 3 -derived inverted-repeat miniature elements (TIMEs) that mobilize antibiotic resistance genes Ryota Gomi , Hirokazu Yano bioRxiv 2025.11.05.686661; doi: https://doi.org/10.1101/2025.11.05.686661 Share This Article: Copy Citation Tools Tn 3 -derived inverted-repeat miniature elements (TIMEs) that mobilize antibiotic resistance genes Ryota Gomi , Hirokazu Yano bioRxiv 2025.11.05.686661; doi: https://doi.org/10.1101/2025.11.05.686661 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7633) Biochemistry (17680) Bioengineering (13889) Bioinformatics (41927) Biophysics (21445) Cancer Biology (18585) Cell Biology (25491) Clinical Trials (138) Developmental Biology (13373) Ecology (19897) Epidemiology (2067) Evolutionary Biology (24308) Genetics (15606) Genomics (22494) Immunology (17736) Microbiology (40385) Molecular Biology (17175) Neuroscience (88583) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4822) Physiology (7641) Plant Biology (15149) Scientific Communication and Education (2045) Synthetic Biology (4293) Systems Biology (9822) Zoology (2271)