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981800.9083ISCR elements: novel gene-capturing systems of the 21st century? "Common regions" (CRs), such as Orf513, are being increasingly linked to mega-antibiotic-resistant regions. While their overall nucleotide sequences show little identity to other mobile elements, amino acid alignments indicate that they possess the key motifs of IS91-like elements, which have been linked to the mobility ent plasmids in pathogenic Escherichia coli. Further inspection reveals that they possess an IS91-like origin of replication and termination sites (terIS), and therefore CRs probably transpose via a rolling-circle replication mechanism. Accordingly, in this review we have renamed CRs as ISCRs to give a more accurate reflection of their functional properties. The genetic context surrounding ISCRs indicates that they can procure 5' sequences via misreading of the cognate terIS, i.e., "unchecked transposition." Clinically, the most worrying aspect of ISCRs is that they are increasingly being linked with more potent examples of resistance, i.e., metallo-beta-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia. Furthermore, if ISCR elements do move via "unchecked RC transposition," as has been speculated for ISCR1, then this mechanism provides antibiotic resistance genes with a highly mobile genetic vehicle that could greatly exceed the effects of previously reported mobile genetic mechanisms. It has been hypothesized that bacteria will surprise us by extending their "genetic construction kit" to procure and evince additional DNA and, therefore, antibiotic resistance genes. It appears that ISCR elements have now firmly established themselves within that regimen.200616760305
981710.9081Common regions e.g. orf513 and antibiotic resistance: IS91-like elements evolving complex class 1 integrons. The ability of bacteria to procure, sometimes rearrange, and evince acquired DNA continues to impress us-even more so if this genetic plasticity involves the sequestering of antibiotic resistance genes. The acquisition of genes in bacteria is often facilitated by transposons, integrons and archetype insertion elements. Recently however, a new element, 'orf513', has been increasingly associated with class 1 integrons. Moreover, these 'complex' class 1 integrons can potentially mediate resistance to chloramphenicol, trimethoprim, aminoglycosides and tetracycline and may carry a range of beta-lactamase genes as well as the qnrA gene. Elements such as 'orf513' demonstrate IS91-like characteristics and will mobilize adjacent DNA via a process called rolling circle replication, and thus we have renamed them 'insertion sequence CRs' (ISCRs) to appropriately reflect their structure-function properties. In this article, we provide a brief description of these new and clinically important mobile elements, and how they are able to mobilize antibiotic resistance genes.200616751201
300120.9043IS26 and the IS26 family: versatile resistance gene movers and genome reorganizers. SUMMARYIn Gram-negative bacteria, the insertion sequence IS26 is highly active in disseminating antibiotic resistance genes. IS26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS26 can also enhance expression of adjacent potential resistance genes. IS26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS26. TU formed by homologous recombination between the bounding IS26s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS26 is the best characterized IS in the IS26 family, which includes IS257/IS431, ISSau10, IS1216, IS1006, and IS1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens.202438436262
997730.9043IncC conjugative plasmids and SXT/R391 elements repair double-strand breaks caused by CRISPR-Cas during conjugation. Bacteria have evolved defence mechanisms against bacteriophages. Restriction-modification systems provide innate immunity by degrading invading DNAs that lack proper methylation. CRISPR-Cas systems provide adaptive immunity by sampling the genome of past invaders and cutting the DNA of closely related DNA molecules. These barriers also restrict horizontal gene transfer mediated by conjugative plasmids. IncC conjugative plasmids are important contributors to the global dissemination of multidrug resistance among pathogenic bacteria infecting animals and humans. Here, we show that IncC conjugative plasmids are highly resilient to host defence systems during entry into a new host by conjugation. Using a TnSeq strategy, we uncover a conserved operon containing five genes (vcrx089-vcrx093) that confer a novel host defence evasion (hde) phenotype. We show that vcrx089-vcrx090 promote resistance against type I restriction-modification, whereas vcrx091-vcxr093 promote CRISPR-Cas evasion by repairing double-strand DNA breaks via recombination between short sequence repeats. vcrx091, vcrx092 and vcrx093 encode a single-strand binding protein, and a single-strand annealing recombinase and double-strand exonuclease related to Redβ and λExo of bacteriophage λ, respectively. Homologous genes of the integrative and conjugative element R391 also provide CRISPR-Cas evasion. Hence, the conserved hde operon considerably broadens the host range of large families of mobile elements spreading multidrug resistance.202032556263
842140.9020Dynamic stepwise opening of integron attC DNA hairpins by SSB prevents toxicity and ensures functionality. Biologically functional DNA hairpins are found in archaea, prokaryotes and eukaryotes, playing essential roles in various DNA transactions. However, during DNA replication, hairpin formation can stall the polymerase and is therefore prevented by the single-stranded DNA binding protein (SSB). Here, we address the question how hairpins maintain their functional secondary structure despite SSB's presence. As a model hairpin, we used the recombinogenic form of the attC site, essential for capturing antibiotic-resistance genes in the integrons of bacteria. We found that attC hairpins have a conserved high GC-content near their apical loop that creates a dynamic equilibrium between attC fully opened by SSB and a partially structured attC-6-SSB complex. This complex is recognized by the recombinase IntI, which extrudes the hairpin upon binding while displacing SSB. We anticipate that this intriguing regulation mechanism using a base pair distribution to balance hairpin structure formation and genetic stability is key to the dissemination of antibiotic resistance genes among bacteria and might be conserved among other functional hairpins.201728985409
299950.9018Integrative and conjugative elements in streptococci can act as vectors for plasmids and translocatable units integrated via IS1216E. Mobile genetic elements (MGEs), such as integrative and conjugative elements (ICEs), plasmids and translocatable units (TUs), are important drivers for the spread of antibiotic resistance. Although ICEs have been reported to support the spread of plasmids among different bacteria, their role in mobilizing resistance plasmids and TUs has not yet been fully explored. In this study, a novel TU bearing optrA, a novel non-conjugative plasmid p5303-cfrD carrying cfr(D) and a new member of the ICESa2603 family, ICESg5301 were identified in streptococci. Polymerase chain reaction (PCR) assays revealed that three different types of cointegrates can be formed by IS1216E-mediated cointegration between the three different MGEs, including ICESg5301::p5303-cfrD::TU, ICESg5301::p5303-cfrD, and ICESg5301::TU. Conjugation assays showed that ICEs carrying p5303-cfrD and/or TU successfully transferred into recipient strains, thereby confirming that ICEs can serve as vectors for other non-conjugative MGEs, such as TUs and p5303-cfrD. As neither the TU nor plasmid p5303-cfrD can spread on their own between different bacteria, their integration into an ICE via IS1216E-mediated cointegrate formation not only increases the plasticity of ICEs, but also furthers the dissemination of plasmids and TUs carrying oxazolidinone resistance genes.202336933870
34960.9015Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.19902172217
670.9012YprA family helicases provide the missing link between diverse prokaryotic immune systems. Bacteria and archaea possess an enormous variety of antivirus immune systems that often share homologous proteins and domains, some of which contribute to diverse defense strategies. YprA family helicases are central to widespread defense systems DISARM, Dpd, and Druantia. Here, through comprehensive phylogenetic and structural prediction analysis of the YprA family, we identify several major, previously unrecognized clades, with unique signatures of domain architecture and associations with other genes. Each YprA family clade defines a distinct class of defense systems, which we denote ARMADA (disARM-related Antiviral Defense Array), BRIGADE (Base hypermodification and Restriction Involving Genes encoding ARMADA-like and Dpd-like Effectors), or TALON (TOTE-like and ARMADA-Like Operon with Nuclease). In addition to the YprA-like helicase, ARMADA systems share two more proteins with DISARM. However, ARMADA YprA homologs are most similar to those of Druantia, suggesting ARMADA is a 'missing link' connecting DISARM and Druantia. We show experimentally that ARMADA protects bacteria against a broad range of phages via a direct, non-abortive mechanism. We also discovered multiple families of satellite phage-like mobile genetic elements that often carry both ARMADA and Druantia Type III systems and show that these can provide synergistic resistance against diverse phages.202541000832
35080.9002Random transposon vectors pUTTns for the markerless integration of exogenous genes into gram-negative eubacteria chromosomes. A set of random transposon vectors pUTTns that facilitates the markerless integration of new functions into the chromosome of gram-negative bacteria has been developed. The vectors, which are derived from mini-Tn5 transposons, are located on a R6K-based suicide delivery plasmid that provides the IS50(R) transposase tnp gene in cis, but they are external to the mobile element. The vectors' conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. Internal to the mini-Tn5 element is a cassette that contains a selectable antibiotic resistance marker (kanamycin, chloramphenicol, or tetracycline resistance gene), a counter-selectable marker (sacB), a 430-bp repeat of the sacB gene 3' end acted as the directly-repeated (DR) sequence, and modified multiple cloning sites (MCS). After two total rounds of transposon integration and recombination between the two DRs, only the exogenous DNA inserted into the MCS (passenger genes) and a single 430-bp scar sacBDR fragment remained in the chromosome after excision. The utility of these vectors was demonstrated by integrating the organophosphorus insecticide hydrolase gene (mpd) into the chromosome of Escherichia, Pseudomonas, Sphingomonas, and Paracoccus species. Sequential integration of another organophosphorus insecticide hydrolase gene (oph) into the previously engineered bacteria, without bringing any selectable markers, was also successful. These engineered bacteria were relatively stable. Cell viability and original degrading characteristics were not affected compared with the original recipients. This shows that the developed system is very useful for the markerless integration of exogenous genes into the chromosome of gram-negative eubacteria.200919778558
34890.8999Conjugative DNA transfer in Streptomyces by TraB: is one protein enough? Antibiotic-producing soil bacteria of the genus Streptomyces form a huge natural reservoir of antibiotic resistance genes for the dissemination within the soil community. Streptomyces plasmids encode a unique conjugative DNA transfer system clearly distinguished from classical conjugation involving a single-stranded DNA molecule and a type IV protein secretion system. Only a single plasmid-encoded protein, TraB, is sufficient to translocate a double-stranded DNA molecule into the recipient in Streptomyces matings. TraB is a hexameric pore-forming ATPase that resembles the chromosome segregator protein FtsK and translocates DNA by recognizing specific 8-bp repeats present in the plasmid clt locus. Mobilization of chromosomal genes does not require integration of the plasmid, because TraB also recognizes clt-like sequences distributed all over the chromosome.201223082971
417100.8997Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons. Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA- background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5' CTAAAACAAAGTTA 3' (hs2). The double-site-specific recombination occurred with a frequency of 10(-5)-10(-6). The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a transacting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.19911654505
535110.8996Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Improved broad-host-range plasmid vectors were constructed based on existing plasmids RSF1010 and RK404. The new plasmids pDSK509, pDSK519, and pRK415, have several additional cloning sites and improved antibiotic-resistance genes which facilitate subcloning and mobilization into various Gram-negative bacteria. Several new polylinker sites were added to the Escherichia coli plasmids pUC118 and pUC119, resulting in the new plasmids, pUC128 and pUC129. These plasmids facilitate the transfer of cloned DNA fragments to the broad-host-range vectors. Finally, the broad-host-range cosmid cloning vector pLAFR3 was improved by the addition of a double cos casette to generate the new plasmid, pLAFR5. This latter cosmid simplifies vector preparation and has permitted the rapid cloning of genomic DNA fragments generated with Sau3A. The resulting clones may be introduced into other Gram-negative bacteria by conjugation.19882853689
352120.8995Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. A simple procedure for cloning and stable insertion of foreign genes into the chromosomes of gram-negative eubacteria was developed by combining in two sets of plasmids (i) the transposition features of Tn10 and Tn5; (ii) the resistances to the herbicide bialaphos, to mercuric salts and organomercurial compounds, and to arsenite, and (iii) the suicide delivery properties of the R6K-based plasmid pGP704. The resulting constructions contained unique NotI or SfiI sites internal to either the Tn10 or the Tn5 inverted repeats. These sites were readily used for cloning DNA fragments with the help of two additional specialized cloning plasmids, pUC18Not and pUC18Sfi. The newly derived constructions could be maintained only in donor host strains that produce the R6K-specified pi protein, which is an essential replication protein for R6K and plasmids derived therefrom. Donor plasmids containing hybrid transposons were transformed into a specialized lambda pir lysogenic Escherichia coli strain with a chromosomally integrated RP4 that provided broad-host-range conjugal transfer functions. Delivery of the donor plasmids into selected host bacteria was accomplished through mating with the target strain. Transposition of the hybrid transposon from the delivered suicide plasmid to a replicon in the target cell was mediated by the cognate transposase encoded on the plasmid at a site external to the transposon. Since the transposase function was not maintained in target cells, such cells were not immune to further transposition rounds. Multiple insertions in the same strain are therefore only limited by the availability of distinct selection markers. The utility of the system was demonstrated with a kanamycin resistance gene as a model foreign insert into Pseudomonas putida and a melanin gene from Streptomyces antibioticus into Klebsiella pneumoniae. Because of the modular nature of the functional parts of the cloning vectors, they can be easily modified and further selection markers can be incorporated. The cloning system described here will be particularly useful for the construction of hybrid bacteria that stably maintain inserted genes, perhaps in competitive situations (e.g., in open systems and natural environments), and that do not carry antibiotic resistance markers characteristic of most available cloning vectors (as is currently required of live bacterial vaccines).19902172216
9819130.8994Site-specific recombination and shuffling of resistance genes in transposon Tn21. Many multidrug-resistant transposons found in natural isolates of Gram-negative bacteria are close relatives of Tn21. Thus, the Tn21 subgroup of the Tn3 family of transposable elements is the most successful homogeneous group in acquiring resistance to newly introduced antibiotics. This paper summarizes the mode of acquisition of resistance genes by these elements.19911660178
9832140.8993Interplay between the Xer recombination system and the dissemination of antibioresistance in Acinetobacter baumannii. Antibiotic-resistant infections are a pressing clinical challenge. Plasmids are known to accelerate the emergence of resistance by facilitating horizontal gene transfer of antibiotic resistance genes between bacteria. We explore this question in Acinetobacter baumannii, a globally emerging nosocomial pathogen responsible for a wide range of infections with a worrying accumulation of resistance, particularly involving plasmids. In this species, plasmids of the Rep_3 family harbor antibiotic resistance genes within variable regions flanked by potential site-specific recombination sites recognized by the XerCD recombinase. We first show that the Xer system of A. baumannii functions as described in Escherichia coli, resolving chromosome dimers at the dif site and recombining plasmid-carried sites. However, the multiple Xer recombination sites found in Rep_3 plasmids do not allow excision of plasmid fragments. Rather, they recombine to cointegrate plasmids, which could then evolve to exchange genes. Cointegrates represent a significant fraction of the plasmid population and their formation is controlled by the sequence of recombination sites, which determines the compatibility between recombination sites. We conclude that plasmids in A. baumannii frequently recombine by Xer recombination, allowing a high level of yet controlled plasticity in the acquisition and combination of antibiotic resistance genes.202539777461
2997150.8991Genomic Characterization of Multidrug-Resistant Escherichia coli BH100 Sub-strains. The rapid emergence of multidrug-resistant (MDR) bacteria is a global health problem. Mobile genetic elements like conjugative plasmids, transposons, and integrons are the major players in spreading resistance genes in uropathogenic Escherichia coli (UPEC) pathotype. The E. coli BH100 strain was isolated from the urinary tract of a Brazilian woman in 1974. This strain presents two plasmids carrying MDR cassettes, pBH100, and pAp, with conjugative and mobilization properties, respectively. However, its transposable elements have not been characterized. In this study, we attempted to unravel the factors involved in the mobilization of virulence and drug-resistance genes by assessing genomic rearrangements in four BH100 sub-strains (BH100 MG2014, BH100 MG2017, BH100L MG2017, and BH100N MG2017). Therefore, the complete genomes of the BH100 sub-strains were achieved through Next Generation Sequencing and submitted to comparative genomic analyses. Our data shows recombination events between the two plasmids in the sub-strain BH100 MG2017 and between pBH100 and the chromosome in BH100L MG2017. In both cases, IS3 and IS21 elements were detected upstream of Tn21 family transposons associated with MDR genes at the recombined region. These results integrated with Genomic island analysis suggest pBH100 might be involved in the spreading of drug resistance through the formation of resistance islands. Regarding pathogenicity, our results reveal that BH100 strain is closely related to UPEC strains and contains many IS3 and IS21-transposase-enriched genomic islands associated with virulence. This study concludes that those IS elements are vital for the evolution and adaptation of BH100 strain.202033584554
9071160.8991RAC: Repository of Antibiotic resistance Cassettes. Antibiotic resistance in bacteria is often due to acquisition of resistance genes associated with different mobile genetic elements. In Gram-negative bacteria, many resistance genes are found as part of small mobile genetic elements called gene cassettes, generally found integrated into larger elements called integrons. Integrons carrying antibiotic resistance gene cassettes are often associated with mobile elements and here are designated 'mobile resistance integrons' (MRIs). More than one cassette can be inserted in the same integron to create arrays that contribute to the spread of multi-resistance. In many sequences in databases such as GenBank, only the genes within cassettes, rather than whole cassettes, are annotated and the same gene/cassette may be given different names in different entries, hampering analysis. We have developed the Repository of Antibiotic resistance Cassettes (RAC) website to provide an archive of gene cassettes that includes alternative gene names from multiple nomenclature systems and allows the community to contribute new cassettes. RAC also offers an additional function that allows users to submit sequences containing cassettes or arrays for annotation using the automatic annotation system Attacca. Attacca recognizes features (gene cassettes, integron regions) and identifies cassette arrays as patterns of features and can also distinguish minor cassette variants that may encode different resistance phenotypes (aacA4 cassettes and bla cassettes-encoding β-lactamases). Gaps in annotations are manually reviewed and those found to correspond to novel cassettes are assigned unique names. While there are other websites dedicated to integrons or antibiotic resistance genes, none includes a complete list of antibiotic resistance gene cassettes in MRI or offers consistent annotation and appropriate naming of all of these cassettes in submitted sequences. RAC thus provides a unique resource for researchers, which should reduce confusion and improve the quality of annotations of gene cassettes in integrons associated with antibiotic resistance. DATABASE URL: http://www2.chi.unsw.edu.au/rac.201122140215
9874170.8991Genomic islands related to Salmonella genomic island 1; integrative mobilisable elements in trmE mobilised in trans by A/C plasmids. Salmonella genomic island 1 (SGI1), an integrative mobilisable element (IME), was first reported 20 years ago, in the multidrug resistant Salmonella Typhimurium DT104 clone. Since this first report, many variants and relatives have been found in Salmonella enterica and Proteus mirabilis. Thanks to whole genome sequencing, more and more complete sequences of SGI1-related elements (SGI1-REs) have been reported in these last few years among Gammaproteobacteria. Here, the genetic organisation and main features common to SGI1-REs are summarised to help to classify them. Their integrases belong to the tyrosine-recombinase family and target the 3'-end of the trmE gene. They share the same genetic organisation (integrase and excisionase genes, replicase module, SgaCD-like transcriptional activator genes, traN, traG, mpsB/mpsA genes) and they harbour AcaCD binding sites promoting their excision, replication and mobilisation in presence of A/C plasmid. SGI1-REs are mosaic structures suggesting that recombination events occurred between them. Most of them harbour a multiple antibiotic resistance (MAR) region and the plasticity of their MAR region show that SGI1-REs play a key role in antibiotic resistance and might help multiple antibiotic resistant bacteria to adapt to their environment. This might explain the emergence of clones with SGI1-REs.202133582118
5180.8990GmRAR1 and GmSGT1 are required for basal, R gene-mediated and systemic acquired resistance in soybean. RAR1, SGT1, and HSP90 are important components of effector-triggered immunity (ETI) in diverse plants, where RAR1 and SGT1 are thought to serve as HSP90 co-chaperones. We show that ETI in soybean requires RAR1 and SGT1 but not HSP90. Rsv1-mediated extreme resistance to Soybean mosaic virus (SMV) and Rpg-1b-mediated resistance to Pseudomonas syringae were compromised in plants silenced for GmRAR1 and GmSGT1-2 but not GmHSP90. This suggests that RAR1- or SGT1-dependant signaling is not always associated with a dependence on HSP90. Unlike in Arabidopsis, SGT1 in soybean also mediates ETI against the bacterial pathogen P. syringae. Similar to Arabidopsis, soybean RAR1 and SGT1 proteins interact with each other and two related HSP90 proteins. Plants silenced for GmHSP90 genes or GmRAR1 exhibited altered morphology, suggesting that these proteins also contribute to developmental processes. Silencing GmRAR1 and GmSGT1-2 impaired resistance to virulent bacteria and systemic acquired resistance (SAR) in soybean as well. Because the Arabidopsis rar1 mutant also showed a defect in SAR, we conclude that RAR1 and SGT1 serve as a point of convergence for basal resistance, ETI, and SAR. We demonstrate that, although soybean defense signaling pathways recruit structurally conserved components, they have distinct requirements for specific proteins.200919061405
9979190.8990Type II and IV toxin-antitoxin systems coordinately stabilize the integrative and conjugative element of the ICESa2603 family conferring multiple drug resistance in Streptococcus suis. Integrative and conjugative elements (ICEs) play a vital role in bacterial evolution by carrying essential genes that confer adaptive functions to the host. Despite their importance, the mechanism underlying the stable inheritance of ICEs, which is necessary for the acquisition of new traits in bacteria, remains poorly understood. Here, we identified SezAT, a type II toxin-antitoxin (TA) system, and AbiE, a type IV TA system encoded within the ICESsuHN105, coordinately promote ICE stabilization and mediate multidrug resistance in Streptococcus suis. Deletion of SezAT or AbiE did not affect the strain's antibiotic susceptibility, but their duple deletion increased susceptibility, mainly mediated by the antitoxins SezA and AbiEi. Further studies have revealed that SezA and AbiEi affect the genetic stability of ICESsuHN105 by moderating the excision and extrachromosomal copy number, consequently affecting the antibiotic resistance conferred by ICE. The DNA-binding proteins AbiEi and SezA, which bind palindromic sequences in the promoter, coordinately modulate ICE excision and extracellular copy number by binding to sequences in the origin-of-transfer (oriT) and the attL sites, respectively. Furthermore, AbiEi negatively regulates the transcription of SezAT by binding directly to its promoter, optimizing the coordinate network of SezAT and AbiE in maintaining ICESsuHN105 stability. Importantly, SezAT and AbiE are widespread and conserved in ICEs harbouring diverse drug-resistance genes, and their coordinated effects in promoting ICE stability and mediating drug resistance may be broadly applicable to other ICEs. Altogether, our study uncovers the TA system's role in maintaining the genetic stability of ICE and offers potential targets for overcoming the dissemination and evolution of drug resistance.202438640137