Dual regulatory role of IS91-encoded Orf121 in IS91 transposition

Dual regulatory role of IS91-encoded Orf121 in IS91 transposition | 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 Dual regulatory role of IS 91 -encoded Orf121 in IS 91 transposition View ORCID Profile Aurélien Fauconnier , View ORCID Profile Sandra Da Re , Margaux Gaschet , View ORCID Profile Thomas Jové , View ORCID Profile Marie-Cécile Ploy , View ORCID Profile Cécile Pasternak doi: https://doi.org/10.1101/2025.01.24.634351 Aurélien Fauconnier 1 Univ. Limoges, INSERM, CHU Limoges, RESINFIT, U 1092 , F-87000 Limoges, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aurélien Fauconnier Sandra Da Re 1 Univ. Limoges, INSERM, CHU Limoges, RESINFIT, U 1092 , F-87000 Limoges, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sandra Da Re Margaux Gaschet 1 Univ. Limoges, INSERM, CHU Limoges, RESINFIT, U 1092 , F-87000 Limoges, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Thomas Jové 1 Univ. Limoges, INSERM, CHU Limoges, RESINFIT, U 1092 , F-87000 Limoges, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Thomas Jové Marie-Cécile Ploy 1 Univ. Limoges, INSERM, CHU Limoges, RESINFIT, U 1092 , F-87000 Limoges, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marie-Cécile Ploy For correspondence: marie-cecile.ploy{at}unilim.fr Cécile Pasternak 1 Univ. Limoges, INSERM, CHU Limoges, RESINFIT, U 1092 , F-87000 Limoges, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Cécile Pasternak Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Prokaryotic insertion sequences (IS) are pivotal in the propagation of bacterial multidrug resistance, with IS 91 notably linked to virulence and antibiotic resistance genes. However, the precise mechanism by which IS 91 contributes to gene dissemination remains elusive. Unique among its family, IS 91 features a small open reading frame (orf) upstream of the tnpA transposase gene, potentially encoding a 121-amino acid protein, orf121 , which may be translationally coupled with tnpA . Using a genetic system based on the mating-out assay in Escherichia coli , we explored the role of orf121 in the in vivo transposition of IS 91 . Our findings indicate that the overlap between orf121 and tnpA is crucial for tnpA transcription, with both being primarily transcribed as bicistronic mRNAs from the P orf121 promoter. Additionally, the expression of orf121 (whether in cis or trans ) significantly reduces the frequency of IS 91 transposition and the rate of one-ended transposition. Furthermore, only the single-stranded DNA circles of IS 91 intermediates can integrate into new target sequences, and Orf121 negatively influences this insertion step. In summary, orf121 acts as a negative regulator of transposition while ensuring the expression of tnpA , thus providing insights into the complex mechanisms underlying IS 91 -mediated gene dissemination and its potential role in antibiotic resistance propagation. Introduction Mobile genetic elements (MGEs) are pivotal in the propagation of multidrug-resistant genes, presenting a formidable threat to global health across human, animal, and environmental domains (for recent reviews, see 1 , 2 ). These elements encompass genetic entities that facilitate intercellular transfer (e.g., transmissible plasmids and integrative conjugative elements) as well as transposable elements (TE) that drive intracellular mobility, including insertion sequences (IS), transposons, and gene cassettes from integrons. TEs not only enhance the dissemination but also activate proximal antibiotic resistance genes (ARGs). Among these, the IS 91 family is particularly atypical, with only four members confirmed to be actively transposing: the canonical IS 91 3 , IS 801 4 , IS 1294 5 , and IS 1294b 6 , closely related to IS 1294 ). They encode transposases that belong to the HUH superfamily of single-strand nucleases 7 , which are presumed to transpose via a rolling-circle (RC) mechanism 8 , yet their exact role in gene dissemination remains elusive. Unlike traditional IS that feature terminal inverted repeats (IR) flanking the transposase gene tnpA , IS 91 family members possess two functionally distinct ends 9 : the ori IS sequence, initiating transposition, and the ter IS end, terminating transposition. However, in IS 91 9 and IS 1294 5 , termination often fails at a frequency ranging from 1 to 10% for IS 91 and from 1.7% to 14% for IS 1294b 6 , leading to the mobilization of adjacent DNA fragments, a process known as one-ended transposition (OET). Consequently, as IS 91 -like elements are often linked with virulence and antibiotic resistance genes, they could significantly influence the spread of such genes 9 – 11 . IS 91 family members do not generate target site duplications (TSD) upon insertion but display target sequence specificity. The recognized target for the insert is a tetranucleotide which is closely related to the elements active in transposition, IS 91 (5’-CTTG) 12 , IS 801 (5’- GTTC) 4 , IS 1294 (5’-GTTC) 5 and IS 1294b (5’-GTCC) 6 . The ori IS region of IS 801 and IS 91 contains two imperfect subterminal palindromes, whereas IS 1294 and IS 1294b feature a single presumed palindrome ( Fig. 1 ). The ter IS region contains a single imperfect subterminal palindrome adjacent to the cleavage site, differing between IS 91 (5’-CTCG) and the other three elements (5’-GTTC). Download figure Open in new tab Figure 1. Genetic organization of members of the IS 91 family (IS 801 , IS 1294, IS 1294b and IS 91 ) active for transposition ter IS and ori IS ends carrying the palindromic sequences are depicted as red and blue boxes, cleavage positions at ter IS and ori IS ends are shown by red and blue vertical arrows, respectively. The cleavage site at the ter IS end and the tetranucleotide target site adjacent to ori IS are indicated. orf121 and tnpA encoding sequences are shown as orange and green arrows, respectively. Previous investigations have documented the in vivo formation of both single-stranded (ss) and double-stranded (ds) IS 91 circular intermediates during transposition, with both forms bearing a junction where ori IS 91 and ter IS 91 are juxtaposed 13 . However, it remains to be determined which intermediates play a functional role in the IS 91 transposition pathway. Distinct from other IS 91 family members, the IS 91 features an additional open reading frame called orf121 upstream of the tnpA transposase gene, which encodes a 121 amino acid polypeptide. The termination codon of orf121 overlaps with the initiation codon of tnpA , implying a potential coupling in their expression. Prior research indicated that mutations in orf121 do not impact transposition frequency but do alter the insertion target choice 14 . The specific role of the orf 121 in IS 91 transposition has yet to be elucidated. We aimed to determine the involvement of orf121 in IS 91 transposition within Escherichia coli . Using a genetic system based on the mating-out assay, we observed that orf121 expression induction reduces IS 91 transposition in vivo . Our findings indicate that orf121 and tnpA are co-expressed, with their overlapping regions being critical for tnpA transcription. Furthermore, we established that only single-stranded DNA circular intermediates are inserted into new target sequences, and Orf121 negatively influences this step. Additionally, orf121 expression significantly reduces the OET rate, suggesting that Orf121 is necessary for the precise recognition and cleavage of the ter IS 91 end. Results orf121 is a conserved feature in the IS 91 element To study if orf121 plays a specific role in IS 91 transposition, we first wanted to address if orf121 was conserved in different isoforms of IS 91 , the paradigm of the IS 91 family. As there was no available database analysis to assess the diversity and characteristics of IS 91 , we performed an in silico analysis using the amino acid sequence of the TnpA transposase encoded by IS 91 (accession number CAD30457.1) as a reference for the first described isoform. Sequences were filtered to retain those with an amino acid identity of 95% or higher and not truncated, identifying 134 distinct isoforms. Most of the isoforms were mainly identified in Enterobacterales, and as single copies on plasmids, with over half of the identified plasmids belonging to the IncFII type. For all isoform sequences described and analyzed in this study, refer to Supplementary File 1 and File 2. The overlap between the orf121 and tnpA genes was highly conserved, in 79% of cases. In other isoforms, the overlap loss was due to insertions or deletions within the homo- polymeric regions of the orf121 sequence. The target insertion sites showed a consistent preference for 5’-GTTC or 5’-CTTG, followed by 5’-CTCG, with a T consistently in the second position. The predominant cleavage site tetranucleotide was 5’-CTCG. This result is in aligning with previous reports 9 , 12 . P orf121 drives expression of both orf121 and tnpA genes As mentioned above, the termination codon of orf121 overlaps with the initiation codon of the tnpA gene in IS 91, suggesting that the two genes could be expressed from the same promoter. For this reason, we have analyzed the transcriptional expression level of orf121 and tnpA in their natural configuration within the IS 91 element. Using BPROM ( http://www.softberry.com ) 15 , we identified putative σ70 promoters and Shine-Dalgarno (SD) sequences for both genes. Notably, the putative tnpA promoter P tnpA was located within the coding sequence of orf121 and P orf121 in the ter IS 91 region of the IS 91 element ( Fig. 2a ). The activity of the predicted promoters P tnpA and P orf121 was assessed by generating transcriptional fusions between each putative promoter and the lacZ gene, including the lacZ ribosome binding site (RBS) ( Fig. 2b ). β-galactosidase assays revealed that both promoters are functional, with P orf121 exhibiting significantly higher activity than P tnpA (62.8-fold; Fig. 2c ). Download figure Open in new tab Figure 2. Sequence of the IS 91 from ter IS 91 to the start of the tnpA gene and estimation of the P orf121 and P tnpA promoters activity. a) Sequence of IS 91 accession number X17114.5 . The ter IS region, the orf121 and tnpA genes are indicated by a red rectangle, an orange arrow and a green arrow above the nucleotides, respectively. Red arrows represent the ter IS terminal palindrome. The cleavage tetranucleotide is in bold with a red arrow indicating the cleavage site. The potential -35, -10, SD and start codon regions are shown in bold (orange for P orf121 and green for P tnpA ). b) Scheme of the P orf121 and P tnpA lacZ -transcriptional fusion constructs described in Table 1 . c) Β-galactosidase activities of the transcriptional fusions between lacZ and the two predicted promoters: P orf121 and P tnpA with lacZ RBS. d) Β-galactosidase activities of the transcriptional fusions between lacZ and P tnpA with its native RBS in the presence or absence of the +1 overlap and between lacZ and P orf121 with its native RBS and orf121 gene in the presence or absence of the +1 overlap. Beta-galactosidase activity is expressed as Miller units. Empty: empty transposase expression vector. The results are the average of at least 3 independent experiments. Ns = non-significant; p = 0,0332 (*) and p <0.0001 (****). View this table: View inline View popup Download powerpoint Table 1. Bacterial strains and plasmids Δ indicates a deletion; Ω indicates an insertion; :: indicates a novel joint; * indicates a mutation and 6his indicates localization of His-tag. To investigate the expression of orf121 and tnpA in their native configuration to determine whether tnpA expression is coupled to orf121 and to assess the significance of the overlap between the orf121 stop codon and the tnpA start codon. We created two new lacZ transcriptional fusions with P tnpA , including its native RBS, either retaining or removing the +1 overlap ( Table 1 , Fig. 2b ). Additionally, we developed two translational fusions between lacZ and the orf121 gene with its native promoter, P orf121 , resulting in P orf121 - orf121 (P tnpA ):: lacZ , with and without the +1 overlap ( Table 1 , Fig. 2b ). Globally, the estimated β-galactosidase activities were much lower in constructs compared to the ones with the lacZ RBS. The activity of P tnpA was completely abolished when maintaining its native RBS compared to the construct with the lacZ RBS (compare Fig. 2c and Fig. 2d ). Although still very low, the activity of P tnpA increased significantly, by a factor of 4.1, when the native +1 overlap was removed. The β-galactosidase activity of P orf121 - orf121 (P tnpA ):: lacZ was low, indicating that P orf121 has a weak activity. In contrast to P tnpA , the removal of the orf121 / tnpA overlap resulted in a 2.9-fold decrease in β-galactosidase activity. Although P tnpA activity was minimal (<1 MU), it increased when the P orf121 promoter was present upstream (P orf121 - orf121 (P tnpA )) in both configurations, with and without the orf121 - tnpA overlap ( Fig. 2d ). In the absence of the overlap, the activity of P orf121 - orf121 (P tnpA )Δ-1 was reduced by 2.91-fold compared to the construct with the overlap. To further investigate the contributions of the P orf121 and P tnpA promoters to tnpA expression, we engineered mutants of these promoters within the P orf121 - orf121 (P tnpA ):: lacZ construct. Specifically, we mutated P orf121 in the -10 box (TATAAA to CGCGAA) and P tnpA in the -35 box (TTGCCG to TGGCGG) without altering the amino acid sequence of Orf121. The activities of the mutated promoters showed significant reductions, experiencing a 18-fold and a a 3-fold decrease respectively, compared to P orf121 P tnpA ( Fig. 2d ). Altogether, these results suggest that tnpA is predominantly expressed from the P orf121 promoter and that its expression is heavily dependent on the overlap between orf121 and tnpA . Inhibitory role of orf121 in IS 91 transposition After assessing the expression of orf121 and tnpA genes within the IS 91 element, we explored the potential regulatory role of orf121 in IS 91 transposition in vivo . For this purpose, we utilized a genetic system using a mating-out assay in E. coli containing three plasmids to assess the mobility of IS 91 derivatives into the pOX38Km conjugative plasmid, upon expression of tnpA alone or with orf121 in trans (relative to IS 91 ) (see Fig. 3 , and Materials and Methods). Two different IS 91 derivatives were cloned into a high-copy number plasmid. Both derivatives include a sequence of 89 bp from the left-end of IS 91 at the ter IS 91 cleavage site (5’-CTCG) and the final 82 bp of the right-end, shown to be sufficient for functional transposition 8 , encompassing the ori IS 91 as well as the essential 5’-CTTG tetranucleotide adjacent to the ori IS 91 end ( Fig. 3a ). These ter IS 91 and ori IS 91 sequences flanked a Cm R cassette (for selection of transposition events) in the IS 91 derivative termed ter IS 91 - cm R . Another derivative, ter IS 91 :: orf121 - cm R , included the Cm R cassette along with orf121 expressed in cis relative to IS 91 ( Table 1 and Fig. 3a ). TnpA was expressed in trans from a compatible plasmid under the inducible P lac promoter, either alone called P lac :: tnpA or with orf121 termed P lac :: orf121 - tnpA (in its native configuration with overlapping orf121 - stop/ tnpA -start) ( Table 1 , Fig. 3a ). Transposition frequencies of IS 91 derivatives into pOX38Km were evaluated as illustrated in Fig. 3b . With orf121 expressed in trans , we observed a dramatic reduction in transposition frequency (8045-fold) with ter IS 91 - cm R upon IPTG induction when both orf121 and tnpA were expressed (P lac :: orf121 - tnpA ), compared to expression of tnpA alone ( Fig. 3c ). In contrast, under basal conditions of tnpA expression (i.e., without IPTG induction) from P lac :: tnpA , expression in cis of orf121 from its native promoter ( ter IS 91 :: orf121 - cm R ) resulted in a significant 11.1-fold decrease in transposition frequency compared to ter IS 91 - cm R . However, in the presence of IPTG, no significant impact on transposition frequency was observed when orf121 was expressed in cis from its native promoter ( ter IS 91 :: orf121 - cm R ) compared to the construct lacking orf121 ( ter IS 91 - cm R ) in cells expressing only tnpA (P lac :: tnpA ). Download figure Open in new tab Figure 3. Expression of orf121 decreases IS 91 transposition frequency (a) The IS 91 transposase donor plasmids ( expression in trans ) feature the P lac promoter (blue), which controls the tnpA gene (green) with the upstream presence of orf121 (orange). When the two genes are expressed under the control of two promoters, the orf121 gene is under the control of the P LtetO-1 promoter (purple). These plasmids are selected by the sp R resistance gene. IS 91 substrate plasmids are derivatives of the pUC18 and confer ampicillin resistance ( amp R ). They carry the mini-IS composed of the ori IS 91 (blue) and ter IS 91 (red) flanking the cm R mobility reporter gene (Chloramphenicol resistance; yellow) for ter IS 91 - cm R and, the ter IS 91 :: orf121 - cm R composed of the ori IS 91 (blue) and ter IS 91 :: orf121 region (native IS 91 configuration, red and orange) flanking the cm R mobility reporter genes, used for detecting the OET mechanism. (b) Schematic of the mating-out assays, the donor E. coli JS219 (black) carries three plasmids: the IS donor plasmid, a derivative of pUC18 carrying ter IS 91 - cm R ; a second plasmid with an origin of replication p15A and expresses the IS 91 transposase ( tnpA ) under the control of the IPTG-inducible P Lac promoter, called P lac :: tnpA . The conjugative pOX38Km (a derivative of plasmid F) is the target for transposition. We use conjugation to isolate transposition events on the pOX38Km plasmid in the E. coli MC240 recipient cell. (c) Transposition frequency of IS 91 derivatives ( ter IS 91 - cm R and ter IS 91 :: orf121 - cm R ) estimated by mating-out assays (see (b)) when tnpA is expressed in trans alone or with orf121 from the same or independent promoters (box red) or with orf121 expressed in cis from ter IS 91 :: orf121 - cm R (box blue). The – and + indicate the presence or absence of inducers, IPTG (0.5 mM) and aTc (50 ng/ml). Data are represented as box-plot. Experiments were performed at least 5 time. P = 0,0332 (*); p = 0,0002 (***) and p <0.0001 (****). #, undetectable transposition (< 3.35 x 10 −8 ). Then, we explored whether separate expression of orf121 and tnpA genes could impact the mobility of the IS 91 derivative, hypothesizing that Orf121 might modulate transposition frequency. We assessed the transposition frequency of the ter IS 91 - cm R derivative in E. coli , where tnpA and/or orf121 were expressed independently from the inducible promoters P lac and P LtetO-1 , respectively, designated as orf121 ::P LtetO-1 -P lac :: tnpA ( Fig. 3c ). In the absence of IPTG and anhydrotetracycline (aTc) inducers, the basal transposition frequency of ter IS 91 - cm R was observed in cells containing orf121 ::P LtetO-1 -P lac :: tnpA , due to promoter leakage. With aTc leading to the expression of orf121 from P LtetO-1 we observed a significantly decrease of the transposition frequency by 2.3-fold compared to the condition without aTc. When tnpA expression was induced with IPTG alone, transposition frequency increased by 45.1-fold relative to the basal level. However, co-expression of orf121 with tnpA in presence of both inducers aTc and IPTG, led to an 8.6-fold reduction in transposition frequency relative to IPTG alone. This result reveals that, aside from the inherent configuration of the orf121 and tnpA genes impacting element mobility, the Orf121 protein modulates transposition frequency, independently of the orf121 - tnpA overlap. Impact of orf121 on tnpA transcription and translation In conjunction with the mating-out assay, we quantified the transcripts of orf121 and tnpA . Our findings revealed that co-expressing orf121 in trans with tnpA , whether from the same or independent promoters with ter IS 91 - cm R , resulted in a significant reduction in the mRNA copy number of tnpA by 6.9-fold and 1.8-fold, respectively ( Fig. 4a ). Similar trends were observed when orf121 was co-expressed in cis ( ter IS 91 :: orf121 - cm R ) with tnpA (P lac :: tnpA ), with a 2-fold decrease in tnpA transcript levels compared to ter IS 91 - cm R . The decline in tnpA transcripts was inversely proportional to the number of orf121 transcripts. There is no impact of tnpA expression on the mRNA copy number of orf121 ( Fig. 4b ). These observations suggest that orf121 exerts a negative regulatory effect on tnpA transcript levels. Download figure Open in new tab Figure 4. orf121 -driven changes in tnpA transcription and translation dynamics (a and b) Transcript levels of tnpA (a) and orf121 (b) in mating-out experiments. Transcript levels of tnpA and orf121 were normalized to the housekeeping gene dxs . Data are the average of transcripts levels measured from at least 5 independent mating-out experiments. Error bars indicate the SD. Asterisks indicate significant difference: ns = non-significant; p = 0,0002 (***) and p <0.0001 (****). Symbols and constructs are detailed above ( Fig 4a ). (c) Transposition frequency of the IS 91 derivative ( ter IS 91 - cm R ) estimated by mating assays when tnpA is expressed in trans alone or with orf121 from the same (box green) or independent (box yellow) promoters, when tnpA and orf121 are tagged with a His-tag (C-term for tnpA * or N-term for * orf121 ) compared construction without tagged (box black). (d and e) Detection of TnpA (d) and Orf121 (e) protein by western immunoblotting. His- tagged TnpA (C-term; tnpA*; expected size 47 kDa) and His-tagged Orf121 (N-term; *orf121; expected size 17 kDa) were extracted from the mating-out experiment culture. The - and + indicate the presence or absence of inducers, IPTG (0.5 mM) and aTc (50 ng/ml). *, His-tag position; AU: Arbitrary Unit of quantity of protein. Symbols and constructs are detailed above. In parallel, we assessed the protein levels of Orf121 and TnpA to complement the mating assay results that highlighted the impact of Orf121 on IS 91 mobility. Using C-terminal His- tagged TnpA ( tnpA 6his ) and N-terminal His-tagged Orf121 ( 6his orf121 ) proteins in mating-out assays, we performed Western-immunoblotting ( Table 1 ). It was confirmed that His-tags did not influence transposition frequencies ( Fig. 4c ). Orf121 was exclusively detected in the soluble fraction of the bacterial lysate, whereas TnpA was present in the pellet as inclusion bodies. The identities of 6his Orf121 and TnpA 6his were verified via mass spectrometry. As shown in Fig. 4d , the quantity of TnpA remained stable, around 20 AU (Arbitrary Units), regardless of whether tnpA was expressed alone or with orf121 when their expression was independent ( orf121 6his ::P LtetO-1 -P lac :: tnpA 6his ). However, when tnpA and orf121 were expressed from the same promoter (P lac :: orf121 - tnpA 6his ), the quantity of TnpA was 4.1-fold lower. Orf121 production remained similar across different expression conditions ( Fig. 4e ). These findings indicate that the reduced production of the TnpA protein is primarily due to the coupled transcription of orf121 and tnpA . This correlates with the decreased number of tnpA transcripts observed (P lac :: orf121 - tnpA ). Collectively, the transcript and His-tag analyses suggest that the regulation of coupled orf121 and tnpA expression operates at both transcriptional and translational levels. Effect of Orf121 on integration of IS 91 circular intermediate forms in E. coli It has been previously established that two distinct DNA species, single-stranded (ss) and double-stranded (ds) IS 91 circular intermediates, are independently formed in vivo post- induction of transposase expression, with ori91 and ter91 fusion 13 . Nevertheless, the functional intermediates facilitating IS 91 insertion have yet to be clarified. To address this, we employed a suicide mating assay via transformation or conjugation of the ter91 - ori91 or ori91 - ter91 junction into E. coli DH5α strains harboring various tnpA and/or orf121 expression plasmids (P lac :: tnpA or P lac :: orf121 - tnpA ). The sequence of the ter91 - ori91 junction and the ds or ss insertion assays are presented in Fig. 5a and Fig. 5b ( Table 1 ). Download figure Open in new tab Figure 5. Only ssDNA circular intermediates are successfully inserted into a new target sequence. (a) Sequence of the ori91 - ter91 or ter91 - ori91 junction present in the substrates used for insertion analyses. ter IS and ori IS ends are depicted as red and blue, cleavage positions are shown by blue vertical arrows, respectively. The cleavage site at the ter IS is indicated (bold red underline). (b) Principe of suicide mating assay via transformation or conjugation of the ter91 - ori91 or ori91 - ter91 junction into E. coli DH5α strains to test ds- and ss-circular intermediates. (c) Transformation assays assessing ter91 - ori91 or ori91 - ter91 dsDNA-circular IS 91 intermediates insertion into E. coli chromosome when TnpA was expressed alone or with Orf121 from the same promoter. The pUC18 plasmid was used as a positive control of transformation efficiency. Experiments were performed 4 times. #, undetectable transformants (< 2.63 x 10 −9 ). (d and e) Suicide conjugation assays assessing ter91 - ori91 or ori91 - ter91 ssDNA-circular IS 91 intermediates insertion into E. coli chromosome when TnpA was expressed alone or with Orf121 from the same promoter (d) or with Orf121 from independent promoters (e). Experiments were performed 4 times. p = 0,0021 (**) and p <0.0001 (****). #, undetectable exconjugants or transformants (< 2.93 x 10 −9 ). For dsDNA experiments, the positive control transformation with the pUC18 plasmid yielded the expected transformation frequency with 100 ng of pUC18 ( Fig. 5c ). However, no transformants were detected with any ds-circular ter91 - ori91 or ori91 - ter91 junction intermediate, suggesting the dsDNA intermediate is not a viable substrate. In contrast, for ssDNA experiments, the absence of Cm R exconjugants was observed with donor cells carrying the empty plasmid pSW23T (lacking ter91 - ori91 or ori91 - ter91 junction) and recipient cells expressing P lac :: tnpA or P lac :: orf121 - tnpA as excepted ( Fig. 5d ). Likewise, the frequency of Cm R exconjugants was undetectable when the donors possessed the DNA junction in the ter91 - ori91 orientation. Conversely, when the donor cells carried the ori91 - ter91 junction, we observed a significant reduction in transposition frequency in conditions without IPTG (promoter leakage) when both orf121 and tnpA were expressed from the same promoter (P lac :: orf121 - tnpA ) compared to tnpA alone (P lac :: tnpA ) ( Fig. 5d ). Upon IPTG- induced expression, ori91 - ter91 junction transposition frequency was detectable in cells expressing tnpA alone but became undetectable in cells expressing both orf121 and tnpA , with a substantial 12347-fold reduction. Further experiments with recipient cells expressing tnpA and orf121 under distinct inducible promoters ( orf121 ::P LtetO-1 -P lac :: tnpA ) verified the negative impact of Orf121 on ori91 - ter91 junction in insertion ( Fig. 5e ). Molecular analysis via arbitrarily primed PCR (AP-PCR) of numerous exconjugant clones confirmed all Cm R exconjugants originated from ori91 - ter91 junction insertion into the recipient cell’s chromosome (Supplementary Table 2). Our results indicate that the single-stranded circular intermediate is the exclusive form capable of initiating insertion, and that only the bottom strand is engaged in the process. Additionally, Orf121 acts as an inhibitor, reducing the insertion efficiency of the circular intermediate mediated by TnpA. Lack of Orf121 influence on IS 91 target site insertion Earlier research indicated that Orf121 might influence insertion specificity, as orf121 mutations affected target selection without impacting transposition 14 . To confirm that the observed Cm R exconjugants were due to transposition of the IS 91 derivative into the pOX38Km plasmid, rather than spontaneous mutations, AP-PCR analysis was conducted on numerous transposition events to identify target insertion sites. Given our findings, we performed a χ² test to evaluate whether the presence or absence of orf121 , as well as its cis and trans expression, affected the target site preferences at 5’-GTTC and 5’-CTTG. The data revealed that the target site specificity was consistent across conditions, with orf121 expressed either in cis ( ter IS 91 :: orf121 - cm R ) or trans (P lac :: orf121 - tnpA ) and was similar to that observed with tnpA alone (P lac :: tnpA ) ( Table 2 ) (χ² value = 22.20; p-value = 0.45). Additionally, no significant differences in target specificity were detected when orf121 and tnpA were regulated by distinct promoters ( orf121 ::P LtetO-1 - P lac :: tnpA ) ( Table 2 ) (χ² value = 3.77; p-value = 0.29). These results suggest that the expression of Orf121, whether in cis or trans , does not alter the specificity of target selection or insertion sites. View this table: View inline View popup Download powerpoint Table 2. Identification of the tetranucleotide insertion target in the different mating-out assays orf121 is a key factor in precise ter IS 91 end recognition and cleavage We explored the involvement of Orf121 in the specific recognition and cleavage of the ter IS site by analyzing the tetranucleotide cleavage patterns at the ter IS 91 end across multiple transposition events, utilizing AP-PCR. Our findings consistently identified a single cleavage site (5’-CTCG) in all instances, whether the transposase was expressed independently (P lac :: tnpA with ter IS 91 - cm R ; 43 events) or alongside orf121 in trans (P lac :: orf121 - tnpA with ter IS 91 - cm R ; 68 events). Subsequently, we investigated the proportion of one-ended transposition (OET) in different mating-out assays, given the known inefficiency of IS 91 ter IS 91 end. As detailed in Table 3 , expressing tnpA alone (P lac :: tnpA or orf121 ::P LtetO-1 -P lac :: tnpA with IPTG only) led to approximately half of the events being OET. In contrast, the co-expression of orf121 in trans P lac :: orf121 - tnpA and orf121 ::P LtetO-1 -P lac :: tnpA with IPTG and aTc significantly lowered OET rates by 2.94- and 1.5-fold, respectively (relative to the conditions without orf121 ). Similarly, orf121 expression in cis (P lac :: tnpA with ter IS 91 :: orf121 - cm R ) reduced OET incidence to 28%, marking a substantial 1.89-fold decrease compared to the condition without orf121 (P lac :: tnpA with ter IS 91 - cm R ). Notably, even without tnpA induction, the Orf121 negative effect on OET was evident, as shown by a 2.64-fold reduction in OET (11% with aTc; 29% without aTc, Table 3 ) when the P LtetO-1 promoter with orf121 ::P LtetO-1 -P lac :: tnpA (addition of aTc) was derepressed. These results highlight that Orf121 is crucial in ensuring precise recognition and cleavage at the ter IS 91 boundary. View this table: View inline View popup Download powerpoint Table 3. Estimation of the % of OET depending on the presence of Orf121 The n value indicates the number of clones analysed, the - and + indicate the presence or absence of inducer. Impact of orf121 expression in cis or in trans on recognition and cleavage of the terIS site. The exponents * indicate that the difference between the percentages is significant compared to the condition without orf121 expression, i.e. the observed difference cannot be due to random sampling. The exponents £ indicate that the difference between the percentages is significant compared to the condition without inducer, i.e. the observed difference cannot be due to random sampling. The exponents $ indicate that the percentage difference is significant compared to IPTG-only. Discussion IS 91 as the prototype for both the IS 91 family and the related IS CR s family has often been associated with or located near antibiotic resistance genes 16 – 20 , suggesting that these IS elements might play a role in the mobilization of antibiotic resistance. Despite its biological and clinical relevance, this family remains poorly characterized, necessitating further studies into its regulation and transposition mechanisms. In this study, we mechanistically explored the IS 91 transposition and showed the crucial role orf121 plays in the transcriptional and translational regulation of the IS 91 TnpA transposase. Our findings reveal that orf121 is a conserved feature across most IS 91 isoforms, with its stop codon overlapping the start codon of tnpA . This configuration establishes a transcriptional coupling mechanism wherein orf121 ensures tnpA transcription while negatively regulating transposition frequency. The intrinsic P orf121 promoter is significantly stronger than P tnpA , driving the expression of both genes. The transcriptional coupling, supported by the identification of a potential slippage region in orf121 (5’-A 192 AACAAAAA 200 ), aligns with previous models of RNA polymerase-mediated slippage observed in other insertion sequences 21 , 22 . Interestingly, isoforms with disrupted overlap due to homopolymeric insertions or deletions may exhibit altered regulatory dynamics, leading to a truncated orf121 gene and loss of overlap with tnpA , which may alter tnpA expression levels, highlighting the evolutionary flexibility of this mechanism. Further studies are required to analyse in depth the overlap between the two genes and the effect of the potential slippage region in orf121 on tnpA transcription, thereby determining which of the IS 91 isoforms identified can activate transposition. This process involves a -1 frameshift that allows the ribosome to slip one base upstream and continue translating in an alternative reading frame, as shown in other IS elements 23 – 26 . The mechanism of transcriptional coupling observed in orf121 and tnpA resembles that described for other insertion sequences, such as IS 10 27 and IS 50 28 , overlapping regulatory regions introduce modulate translation efficiency. Orf121 demonstrates a dual regulatory function: (i) facilitating tnpA transcription while reducing TnpA protein levels and (ii) limiting transposition frequency and insertion efficiency. Co-expression of orf121 with tnpA significantly decreases transposition frequency, irrespective of whether the genes are expressed in cis or trans . This effect of Orf121 on transposition can be explained by: i) a direct interaction between the Orf121 and TnpA proteins, akin to what has been reported for the IS 911 element 29 , ii) considering the impact of orf121 expression on tnpA mRNA levels, we cannot exclude the possibility of mRNA-mRNA interactions, mRNA-protein interactions, or a role for orf121 in stabilizing tnpA mRNA, similar to what has been suggested for IS 10 27 . Indeed, we could have the presence of a third partner protein in the regulation, the strand opposite the sequence of orf121 and tnpA has never been studied, although the presence of two other potentials ORFs has been suggested 9 . Additionally, our in vivo mating-out assays indicate that Orf121 exerts a negative control over transposition frequency when the two genes are expressed under the control of separate promoters. This observation raises the possibility that the Orf121 protein interacts directly with TnpA, or alternatively, that Orf121 competes with TnpA for binding to the ori IS 91 and ter IS 91 by forming a dimer. An analysis of the Orf121 peptide sequence identified a potential leucine zipper motif (V49-X 5 -L55-X 5 -V61 or V61-X 6 -L68-X 6 -V75) that could be involved in DNA binding, along with a classic zinc finger motif, CxxC, which might facilitate transposase interaction or dimerization (C105-X 2 -C108) ( Fig. 6a ). The element appears to function as a dimer, with cysteines 105 and 108 being crucial for its activity and possibly for the regulation of IS 91 transposition 30 . Further investigation into the Orf121 protein could significantly advance our understanding of its role and the mechanisms underlying IS 91 transposition. Download figure Open in new tab Figure 6. Global diagram of the role of Orf121 in IS 91 mobility. a) Amino acid sequence of Orf121 (accession number CAC14584.1). In blue, bold and highlight or in green, bold and highlight, the first and second potential leucine zipper interacting with DNA. In red, bold and highlight the CxxC domain of zinc fingers enabling dimer formation or interaction with TnpA. b) Global IS 91 interaction model involving Orf121. orf121 and tnpA mRNA are primarily transcribed from the P orf121 promoter. The overlap between the two genes reduces the rate of tnpA transcript via the -1 shift between the STOP codon of orf121 and the start codon of tnpA . Orf121 can interact directly with TnpA or tnpA mRNA, negatively regulating its activity in the element’s mobility capacity (cyan arrow). Orf121 via dimer formation could bind to the ori IS end and compete with TnpA for site binding, reducing element transposition (red arrow). Orf121, through interaction with TnpA or by binding to the ter IS end directly, enables better recognition of the ter IS end and clean excision of the element (green arrow). The 3D structures of Orf121 and TnpA proteins were modeled with AlphaFold3. Moreover, the Western-immunoblotting analysis demonstrated that the quantity of TnpA protein is significantly lower in the natural gene configuration than when the tnpA gene is expressed alone. These results suggest that the natural configuration might sequester translation initiation signals, leading to a reduction in protein production. This hypothesis is supported by studies on IS 10 31 and IS 50 28 , where it has been proposed that secondary mRNA structures could sequester the translation initiation signals of the tnpA gene, reducing protein synthesis. Consequently, while the natural gene configuration is essential for tnpA transcription, it simultaneously acts as a negative regulator of TnpA protein production. Our findings demonstrate that Orf121 plays a critical role in regulating the insertion efficiency of single-stranded circular intermediates during IS 91 transposition. Specifically, Orf121 negatively impacts the transposition process by significantly reducing the efficiency of bottom-strand intermediate insertion. This regulatory effect indicating a robust mechanism of control independent of the gene’s positional context. Mechanistically, this inhibition may involve Orf121 competing with TnpA for binding to the insertion machinery or interfering with the proper recognition of target sites by forming specific protein-DNA or protein-protein interactions. Additionally, the interaction between Orf121 and the transposition complex could restrict the availability or conformation of the bottom-strand circular intermediate, thereby limiting its ability to integrate into the recipient DNA. These findings highlight the dual functionality of Orf121, not only ensuring accurate excision of the circular intermediate but also finely tuning its insertion frequency to prevent potential genomic instability. Orf121 ensures accurate excision and processing of the circular intermediate by facilitating precise cleavage at the ter IS 91 , our data demonstrate that Orf121 enhances the accurate recognition and cleavage of the ter IS 91 , reducing the occurrence of OET and limiting the mobilization of adjacent genes. This precision is crucial, as unchecked OET could facilitate the dissemination of resistance and virulence determinants. Indeed, it has been shown that IS 91 -type elements, such as IS 801 , can mobilize DNA fragments as large as 40 kb 32 . The exact molecular mechanism underlying this regulation remains unknown but may involve Orf121-TnpA interactions or the modulation of target DNA recognition through protein-DNA complexes. This study highlights the pleiotropic role of orf121 in IS 91 transposition, underscoring its triple function ( Fig. 6b ): (i) ensuring transcription of orf121 and tnpA via the P orf121 promoter; (ii) modulating tnpA transcript and protein levels to control transposition frequency; and (iii) enhancing precise cleavage at ter IS 91 , limiting the spread of adjacent genes. These findings not only expand our understanding of IS 91 biology but also offer broader insights into the regulation of transposable elements associated with resistance gene dissemination. Materials and Methods Bacterial strains, media and growth conditions Bacterial strains and plasmids used in this study are listed in Table 1 and supplementary Table 3. All E. coli strains were grown in Lysogeny Broth (LB) at 37°C under agitation at 300 rpm. Media were supplemented, when necessary, with the appropriate antibiotics used at the following concentrations: rifampicin 50 μg/mL, spectinomycin 50 μg/mL, kanamycin 25 µg/mL, chloramphenicol 25 µg/mL, nalidixic acid 25 µg/mL, and ampicillin 100 µg/mL. The expression of IS 91 tnpA alone or the co-expression with orf121 from the P lac promoter was induced by adding 0.5 mM IPTG to the media. The expression of orf121 from the P LTet-O1 promoter was induced by adding 50 nM anhydrotetracycline (aTc) to the media. Transformation of E. coli with plasmid DNA was performed as previously described 39 . All plasmid constructions were verified by DNA sequencing. DNA manipulations PCR reactions were carried out using Phusion DNA Polymerase (ThermoScientific) to amplify the fragments used for cloning and Quick-Load® Taq 2X Master Mix (NEB) for all other applications. All PCR reactions were purified using MACHEREY-NAGEL’s NucleoSpin Gel and PCR Clean-up according to the manufacturer’s recommendations. IS 91 derivate insertion sites in the pOX38Km reporter plasmid or the E. coli DH5α genome, were mapped by arbitrary priming PCR (AP-PCR). The first PCR round was performed in a final volume of 50 μl containing 0.8 μM of primers (arbitrary primer, ARB1 and chloramphenicol resistance gene-specific, Cm-1005) and 15 μl of template (a single Cm R colony lysed in 20 µl). PCR was performed as follows: 2 min 95°C, 6 cycles of 30 sec 95°C, 30 sec 30°C, 1 min 30 sec 72°C; 30 cycles of 30 sec 95°C, 30 sec 45°C, 2 min 72°C; and finally, 72°C for 5 min. The second PCR cycle was performed in a final volume of 50 μl containing 0.8 μM of primers (arbitrary primer ARB2 and Cm-1049) and 5 μl of the purified PCR product from the first cycle as template. PCR was performed as follows: 2 min 95°C, 30 cycles (30 sec 95°C, 30 sec 57°C, 2 min 72°C) and a final step 72°C for 5 min. The PCR products were directly sequenced using the Ori91-1104 primer. The ter IS cleavage site was mapped using the same procedure with primer pairs ARB1/Cm- 473 for the first cycle of PCR and ARB2/Ter91-337 for the second cycle, and the PCR products were directly sequenced with the Ter91-276 primer. Oligonucleotides used for PCR amplification of DNA fragments required for plasmid construction, diagnostic PCR or sequencing are described in supplementary Table 3. In silico analysis of IS 91 DNA sequences from the public database The amino acid sequence of the TnpA transposase isoforms encoded by IS 91 (accession number CAD30457.1) were blasted with BLASTp (NCBI). The matching sequences of TnpA were filtered to retain only those sequences for which the level of amino acid identity was higher (or equal) than 95%. Recovered nucleotide sequences in which the IS and TnpA were partial or truncated were discarded. For each accession number recovered, we analysed the target insertion and cleavage sites and data related to the bacterial host, the host of the IS and the genetic support. Genetic support was analysed using the PLSDB site 40 , 41 . When the PLSDB site gave no results, we searched for plasmid-specific genes (such as PsiA - B , StbA - B , RepA… ) or chromosome-specific genes (such as DNAa , SeqA , DNAt ) to identify the genetic support. To define each isoform, we used a 95% nucleotide identity threshold over the entire IS with at least one nucleotide that differs. This data extraction was carried out on 2020-10-15 for IS 91 . Estimation of in vivo transposition frequencies of IS 91 derivatives into E. coli by mating- out assay Mating-out assays were performed as previously described 42 . The donor strain was E. coli JS219 43 carrying three plasmids: i) ter IS 91 - cm R or ter IS 91 :: orf121 - cm R (used as IS 91 -Cm R derivatives donor plasmids P IS ; Amp R ), ii) P lac :: tnpA , P lac :: orf121 - tnpA or orf121 ::P LtetO-1 - P lac :: tnpA (used as transposase +/- orf121 expression plasmids; Sp R ) and iii) pOX38Km, the conjugative target plasmid (Km R ) ( Table 1 , Fig. 3 .b and supplementary Table 3). The recipient strain was E. coli MC240 (Rif R , Nal R ). Following mating of the donor with the recipient strain, cells were plated on LB plates supplemented with rifampicin and kanamycin (count of exconjugants i.e. all recipients carrying pOX38Km without and with IS 91 -Cm R derivatives) or nalidixic acid, kanamycin and chloramphenicol (count of transposants i.e. recipients carrying pOX38Km with IS 91 -Cm R derivatives). Transposition frequency was determined by dividing the CFU of transposants (Nal R , Km R and Sp R ) by the CFU of exconjugants (Nal R , Km R ). Transposition event frequencies were estimated from more than 5 independent experiments. These experiments were used to calculate mean values and standard deviations. Estimation of the proportion of one-ended transposition events in mating-out assay Several hundred clones resulting from transposition were subcultured onto both LB plates supplemented with kanamycin, ampicillin and nalidixic acid (numeration of recipient cells with pOX38Km carrying the whole derivatives donor plasmids P IS resulting from OET), and LB plates supplemented with kanamycin, chloramphenicol and nalidixic acid (Total population of recipients carrying pOX38Km with IS 91 -Cm R derivatives). The percentage of OET was determined by dividing the number of (Km R , Amp R and Nal R ) clones by the number of (Km R , Cm R and Nal R ) clones and multiplying by 100. Estimation of in vivo insertion of IS 91 circular intermediates into E. coli To test the double-stranded circular intermediate , E. coli DH5α cells were rendered competent in the presence or absence of the inducer IPTG (0.5 mM) containing the plasmid P lac :: tnpA or P lac :: orf121 - tnpA . These competent cells were transformed with 500 ng of suicide plasmids ori91 - ter91 or ter91 - ori91- containing the junction in the pSW23T derivative ( Table 1 , supplementary Table 3). Cells were heat-shocked at 42°C for 1 min, followed by 5 min on ice. The cells were then placed in 1 ml LB with or without inducer for 1 hour at 37°C with agitation, then plated. Determination of the total number of viable cells was performed on LB plates supplemented with spectinomycin (Sp R ), and insertion of junctions into the E. coli DH5α genome was selected on LB plates supplemented with spectinomycin and chloramphenicol (SpR, Cm R ). To test the single-stranded circular intermediate , overnight cultures with Cm R + DAP (0.3 mM) for donors ( E. coli β 2163 ( pir- ) carrying the suicide plasmid (pSW23T (Empty), ori91 - ter91 or ter91 - ori91 ; Table 1 ) and Sp R for recipients ( E. coli DH5α target strain containing different tnpA and/or orf121 expression plasmids (P lac :: tnpA , P lac :: orf121 - tnpA or orf121 ::P LtetO-1 -P lac :: tnpA ; Table 1 ) were diluted 1:100, without antibiotics, and further grown to OD 600 = 0.3. Expression of tnpA and/or orf121 was then induced by adding respectively 0.5 mM IPTG and/or aTc at 50 ng/ml in the recipient cells cultures. The strains were left for a further 1 h at 37°C with agitation. Filters containing the mixture of donor and recipient strains in a 1:8 ratio were incubated for 3 h at 37°C on LB plates supplemented with 0.3 mM DAP and 0.5 mM IPTG, or 0.3 mM DAP, 0.5 mM IPTG and 50 ng/ml aTc. Cells were then suspended in 2 mL LB by vortexing the filter, and appropriate dilutions were plated on selective LB plates (Sp R ) for recipients and LB plates (Sp R + Cm R ) for transconjugants. The frequency of Cm R transconjugants was calculated as the number of Cm R -tagged recipient cells relative to the total number of recipient cells. The frequencies of the insertion event per viable cell from 4 independent experiments were used to calculate mean values and standard deviations. Quantification of orf121 and tnpA transcripts For mating-out transcripts, overnight cultures were diluted to OD 600 = 0.2 and grown to the mean log phase (OD 600 = 0.5). Cells were pelleted and total RNA was extracted with the NucleoSpin® RNA Extraction Kit (Macherey-Nagel Inc.). Contaminating DNA was removed from RNA samples by using the Turbo DNA-free Kit (Ambion). RNA integrity was check and quantified. cDNAs were synthesized from 1 μg of DNase-treated total RNA by using PrimeScriptTM RT Reagent kit (TaKaRa Clontech) following manufacturer’s instruction. cDNA of genes tnpA , orf121 and dxs was quantified using the PerfeCTa® SYBR® Green FastMix® Kit (Quanta BioSciencesTM) with appropriate oligonucleotides (supplementary Table 3). Relative expression of orf121 (Primers ORF121 F and ORF121 R) and tnpA (Primers TnpA F and TnpA R) genes was estimated by normalizing copy number of transcripts to that of the housekeeping gene dxs (Primer dxs-LC3 and dxs-LC4). The tnpA / dxs or orf121 / dxs ratio was obtained from 3 independent experiments, which were used to calculate mean values and standard deviations. β -galactosidases assays β-galactosidase assays were performed with the E. coli MG1656 strain carrying various plasmids ( Table 1 ; 44 ). Overnight cultures were diluted 1:100, and further grown to mid-log phase (OD 600 = 0.4-0.6). The β-galactosidase assay was performed as previously described 45 . β-galactosidase activity was obtained from 3 independent experiments, which were used to calculate mean values and standard deviations. Estimation of His-tagged ORF121 or TnpA IS91 protein expression by western- immunoblotting Overnight cultures were diluted to OD 600 = 0.2 and grown to mid-log phase (OD 600 = 0.5). Gene expression was induced by adding 0.5 mM IPTG ( tnpA alone or with and orf121 ) and/or 50 nM aTc ( orf121 ) in the media. Cultures were incubated for further 3 h at 37°C, and 1 mL of each culture was centrifuged and stored at -20°C. Cell extracts were prepared using the B- PER bacterial protein extraction kit according to the manufacturer’s recommendations at a rate of 100 μL of reagent per OD unit (Thermo Scientific, Ref. 90078) and inclusion bodies proteins using the solubilization reagent used according to the manufacturer’s instructions (Thermo Scientific, Ref. 78115). Cell extract samples were subjected to 15% SDS-PAGE electrophoresis, followed by a transfer to a PVDF membrane according to the manufacturer’s instructions (Trans-Blot Turbo Transfer System, BioRad). The membrane was incubated in a 1:3000 dilution of mouse anti-His-tag antibody (Invitrogen, Ref. MA121315) overnight at 4°C, then washed 3 times for 10 minutes with PBS. Next, the membrane was incubated in a 1:3000 dilution of anti-Mouse IgG, HRP antibody (Invitrogen, Ref. A28177), then washed 6 times for 5 minutes with what. The membrane was revealed with SuperSignal West Pico PLUS (Thermo Scientific, Ref. 34577). The amount of protein was determined in arbitrary units (AU) by calculating the value of the area under the curve using ImageLab software. Analysis of His-tagged ORF121 or TnpA IS 91 proteins by mass spectrometry Peptides from SDS-PAGE were analyzed by micro-LC-MS/MS using a nanoLC 425 system in micro-flow mode (Eksigent, Dublin, CA, USA) coupled with time-of-flight (TOF) (TripleTOF 5600+ Sciex, Framingham, MA, USA) operating in high-sensitivity mode. Reverse-phase LC was performed via a trap and elute configuration using a trap column (C18 Pepmap100 cartridge, 5 µm pore size; Thermo Fisher Scientific) and an analytical column (ChromXP C18 column, 12 nm, 3 µm pore size, Sciex) with the following mobile phases: loading solvent (water/ACN/TFA 98/2/0.05% (v/v)), solvent A (0.1% (v/v) TFA in water) and solvent B (water/ACN/TFA 5/95/0.1% (v/v)). All samples were loaded, trapped, and desalted using a flow rate of 10 μL/min with loading solvent for 5 minutes. The chromatographic separation was performed at a flow rate of 2 µL/min as follows: initial, 5% solvent B, increased to 25% for 90 minutes, then increased to 95% B for 10 minutes, maintained at 95% for 5 minutes and, finally, decreased to 5% B for re-equilibration. The peptides were then reprocessed via ProteinPilot with the Mascot module using Escherichia coli Uniprot 2022_03. The following parameters were entered: trypsin 1 maximum missed cleavage, cysteine carbamidomethylation (fixed), methionine oxidation (variable), Precursor Tolerance 0.01Da; MS/MS fragment tolerance: 0.05 Da, a p 25 - bold red required and a protein score > 100. Statistical analyses All analyses were performed using R Statistical Software (v4.4.1) and RStudio (v 2024.04.2+764). ’s Statistical analyses include a Mann-Whitney U-test and a Chi-square test to compare percentages and insertion target sites. Graphics were rendered via the ggplot2 R package (v3.5.1) 46 . All figures were prepared using Inkscape 1.2.2 ( https://inkscape.org/ ). Supplementary tables Supplementary Table 1. Host of bacteria carrying IS 91 elements in GenBank® Supplementary Table 2. Analysis of the effect of Orf121 expression on the IS mimicking IS 91 target insertion sites in the E. coli chromosome Supplementary Table 3. Oligonucleotides used in this study Supplementary Files Supplementary File 1. Sequences of the different IS 91 isoforms described in this study. IS 91 -V1 corresponds to the first sequence of the IS 91 element identified (number accession X17114.5 ). Supplementary File 2. Description of the different IS 91 isoforms described in this study. Supplementary File 3. Complete data sets. Acknowledgements A. Fauconnier acknowledges the French ministère de l’Enseignement Supérieur, de la Recherche (MESR) for his doctoral training grant. This work was supported by fundings from the French research institute Inserm. 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Share Dual regulatory role of IS 91 -encoded Orf121 in IS 91 transposition Aurélien Fauconnier , Sandra Da Re , Margaux Gaschet , Thomas Jové , Marie-Cécile Ploy , Cécile Pasternak bioRxiv 2025.01.24.634351; doi: https://doi.org/10.1101/2025.01.24.634351 Share This Article: Copy Citation Tools Dual regulatory role of IS 91 -encoded Orf121 in IS 91 transposition Aurélien Fauconnier , Sandra Da Re , Margaux Gaschet , Thomas Jové , Marie-Cécile Ploy , Cécile Pasternak bioRxiv 2025.01.24.634351; doi: https://doi.org/10.1101/2025.01.24.634351 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 Molecular Biology Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17650) Bioengineering (13871) Bioinformatics (41881) Biophysics (21424) Cancer Biology (18566) Cell Biology (25461) Clinical Trials (138) Developmental Biology (13365) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15590) Genomics (22476) Immunology (17713) Microbiology (40331) Molecular Biology (17148) Neuroscience (88473) Paleontology (666) Pathology (2827) Pharmacology and Toxicology (4816) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)