Transposition Events Research Articles

Active Transposition of Insertion Sequences by Oxidative Stress in Deinococcus geothermalis.

Radiation-resistant bacterium Deinococcus geothermalis has a total of 73 insertion sequences (ISs) in genomes, and some of them are actively transposed to other loci with replicative mode due to oxidative stress of hydrogen peroxide treatment. Here, we detected two transposition events in wild-type (WT) strain and LysR family member gene disrupted strain (Δdgeo_2840). Similar to our previous report (Lee et al., 2019), phytoene desaturase (dgeo_0524), a key enzyme of carotenoid biosynthesis, was disrupted by the integration of IS element, thereby detected a single phenotypically non-pigmented colony in each WT and Δdgeo_2840 strain. Two separate types of IS element have been integrated into non-pigmented clones: ISDge11 for WT and ISDge6 for Δdgeo_2840 strain. Surprisingly, Δdgeo_2840 mutant strain revealed higher resistance to oxidative stress than WT strain at late exponential growth phase. From the qRT-PCR analysis, OxyR (dgeo_1888) was highly up-regulated to 30-fold by oxidative stress through hydrogen peroxide treatment in both WT and Δdgeo_2840 mutant strains. However, the oxidative stress response enzyme, catalase or superoxide dismutase, was not significantly induced by overexpressed OxyR. Thus, a putative LysR family regulator Dgeo_2840 controlled the expression of ISDge6 type transposase and the induction of OxyR under oxidative condition. There is LysR family DNA-binding protein dependent active transposition of specific type IS and the up-regulated OxyR has not positively controlled ROS scavenger enzymes in D. geothermalis.

The rise and fall of globins in the amphibia

The globin gene repertoire of gnathostome vertebrates is dictated by differential retention and loss of nine paralogous genes: androglobin, neuroglobin, globin X, cytoglobin, globin Y, myoglobin, globin E, and the α- and β-globins. We report the globin gene repertoire of three orders of modern amphibians: Anura, Caudata, and Gymnophiona. Combining phylogenetic and conserved synteny analysis, we show that myoglobin and globin E were lost only in the Batrachia clade, but retained in Gymnophiona. The major amphibian groups also retained different paralogous copies of globin X. None of the amphibian presented αD-globin gene. Nevertheless, two clades of β-globins are present in all amphibians, indicating that the amphibian ancestor possessed two paralogous proto β-globins. We also show that orthologs of the gene coding for the monomeric hemoglobin found in the heart of Rana catesbeiana are present in Neobatrachia and Pelobatoidea species we analyzed. We suggest that these genes might perform myoglobin- and globin E-related functions. We conclude that the repertoire of globin genes in amphibians is dictated by both retention and loss of the paralogous genes cited above and the rise of a new globin gene through co-option of an α-globin, possibly facilitated by a prior event of transposition.

Genomic Analysis Revealed a Convergent Evolution of LINE-1 in Coat Color: A Case Study in Water Buffaloes (Bubalus bubalis).

Visible pigmentation phenotypes can be used to explore the regulation of gene expression and the evolution of coat color patterns in animals. Here, we performed whole-genome and RNA sequencing and applied genome-wide association study, comparative population genomics and biological experiments to show that the 2,809-bp-long LINE-1 insertion in the ASIP (agouti signaling protein) gene is the causative mutation for the white coat phenotype in swamp buffalo (Bubalus bubalis). This LINE-1 insertion (3′ truncated and containing only 5′ UTR) functions as a strong proximal promoter that leads to a 10-fold increase in the transcription of ASIP in white buffalo skin. The 165 bp of 5′ UTR transcribed from the LINE-1 is spliced into the first coding exon of ASIP, resulting in a chimeric transcript. The increased expression of ASIP prevents melanocyte maturation, leading to the absence of pigment in white buffalo skin and hairs. Phylogenetic analyses indicate that the white buffalo-specific ASIP allele originated from a recent genetic transposition event in swamp buffalo. Interestingly, as a similar LINE-1 insertion has been identified in the cattle ASIP gene, we discuss the convergent mechanism of coat color evolution in the Bovini tribe.

Autosomal sdY Pseudogenes Explain Discordances Between Phenotypic Sex and DNA Marker for Sex Identification in Atlantic Salmon

Despite the key role that sex-determination plays in evolutionary processes, it is still poorly understood in many species. In salmonids, which are among the best studied fishes, the master sex-determining gene sexually dimorphic on the Y-chromosome (sdY) has been identified. However, sdY displays unexplained discordance to the phenotypic sex, with a variable frequency of phenotypic females being reported as genetic males. Multiple sex determining loci in Atlantic salmon have also been reported, possibly as a result of recent transposition events in this species. We hypothesized the existence of an autosomal copy of sdY, causing apparent discordance between phenotypic and genetic sex, that is transmitted in accordance with autosomal inheritance. To test this, we developed a qPCR methodology to detect the total number of sdY copies present in the genome. Based on the observed phenotype/genotype frequencies and linkage analysis among 2,025 offspring from 64 pedigree-controlled families of accurately phenotyped Atlantic salmon, we identified both males and females carrying one or two autosomal copies of sdY in addition to the Y-specific copy present in males. Patterns across families were highly consistent with autosomal inheritance. These autosomal sdY copies appear to have lost the ability to function as a sex determining gene and were only occasionally assigned to the actual sex chromosome in any of the affected families.

An Overview of Water‐Mediated Alkyne Functionalization by Neighboring Group Participation of Carbonyl Groups

AbstractReviewed herein, the progress employing the assistance of a neighboring carbonyl group for alkyne functionalization reactions that are mediated by water. Proper arrangement of these transformations is synthetically worthwhile because it will allow chemists to design straightforward synthetic routes for the construction of complex architectures with perfect regio‐ and chemoselectivity. Researchers must understand the mechanism before aiming a new idea based on these types of transformations as they proceed through a different way in comparison to the strategies that employ carbonyl groups as a directing group for C−H functionalization reactions. In general, an oxygen transposition event occurs in the presence of an alkyne activator. In an aim to expose the molecular diversity that can now be accessed employing intramolecular carbonyl assistance, all the precedents are organized systematically for the first time and can be expected to serve as a comprehensive reference work. Therefore, the key arrangement of this review is schemes accompanied by a concise explanatory text describing both advantages and limitations along with the mechanism. Different transformations are ordered in sections according to the chemical structure of the assisted groups. The overall objective of this review is to promote the application of carbonyl‐assisted reactions beyond the methodologies dedicated to the regioselective synthesis of carbonyl derivatives and hence, serving it as a valuable reaction archetype in modern‐day research for the synthesis of carbo‐ and heterocycles.magnified image

Pooled Plasmid Sequencing Reveals the Relationship Between Mobile Genetic Elements and Antimicrobial Resistance Genes in Clinically Isolated Klebsiella pneumoniae

Plasmids remain important microbial components mediating the horizontal gene transfer (HGT) and dissemination of antimicrobial resistance. To systematically explore the relationship between mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), a novel strategy using single-molecule real-time (SMRT) sequencing was developed. This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant (MDR) Klebsiella pneumoniae from a tertiary referral hospital over a 9-month period. The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates. The plasmid pool was then subjected to SMRT sequencing, and 82 assembled plasmid fragments were obtained. In total, 124 ARGs (responsible for resistance to β-lactam, fluoroquinolone, and aminoglycoside, among others) and 317 MGEs [including transposons (Tns), insertion sequences (ISs), and integrons] were derived from these fragments. Most of these ARGs were linked to MGEs, allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements. Key elements involved in resistance transposition were identified, including IS26, Tn3, IS903B, ISEcp1, and ISKpn19. As the most predominant IS in the network, a typical IS26-mediated multicopy composite transposition event was illustrated by tracing its flanking 8-bp target site duplications (TSDs). The landscape of the pooled plasmid sequences highlights the diversity and complexity of the relationship between MGEs and ARGs, underpinning the clinical value of dominant HGT profiles.

A high-contiguity Brassica nigra genome localizes active centromeres and defines the ancestral Brassica genome

It is only recently, with the advent of long-read sequencing technologies, that we are beginning to uncover previously uncharted regions of complex and inherently recursive plant genomes. To comprehensively study and exploit the genome of the neglected oilseed Brassica nigra, we generated two high-quality nanopore de novo genome assemblies. The N50 contig lengths for the two assemblies were 17.1 Mb (12 contigs), one of the best among 324 sequenced plant genomes, and 0.29 Mb (424 contigs), respectively, reflecting recent improvements in the technology. Comparison with a de novo short-read assembly corroborated genome integrity and quantified sequence-related error rates (0.2%). The contiguity and coverage allowed unprecedented access to low-complexity regions of the genome. Pericentromeric regions and coincidence of hypomethylation enabled localization of active centromeres and identified centromere-associated ALE family retro-elements that appear to have proliferated through relatively recent nested transposition events (<1 Ma). Genomic distances calculated based on synteny relationships were used to define a post-triplication Brassica-specific ancestral genome, and to calculate the extensive rearrangements that define the evolutionary distance separating B. nigra from its diploid relatives.

Small RNA-Mediated De Novo Silencing of Ac/Ds Transposons Is Initiated by Alternative Transposition in Maize.

Although transposable elements (TEs) comprise a major fraction of many higher eukaryotic genomes, most TEs are silenced by host defense mechanisms. The means by which otherwise active TEs are recognized and silenced remains poorly understood. Here we analyzed two independent cases of spontaneous silencing of the active maize Ac/Ds transposon system. This silencing is initiated by alternative transposition, a type of aberrant transposition event that engages the termini of two nearby separate TEs. Alternative transposition during DNA replication can generate Composite Insertions that contain inverted duplications of the transposon sequences. We show that the inverted duplications of two Composite Insertions are transcribed to produce double-stranded RNAs that trigger the production of two distinct classes of small interfering RNAs: a 24-nt class complementary to the TE terminal inverted repeats and noncoding subterminal regions, and a 21- to 22-nt class corresponding to the TE transcribed regions. Plants containing these small interfering RNA-generating Composite Insertions exhibit decreased levels of Ac transcript and heritable repression of Ac/Ds transposition. Further, we demonstrate that Composite Insertions can heritably silence otherwise active elements in trans This study documents the first case of transposon silencing induced by alternative transposition and may represent a general initiating mechanism for silencing of DNA transposons.

Hypothetical evolutionary models of the variable lymphocyte receptor genes in jawless vertebrates

Abstract Both humoral and cellular arms of an adaptive immune system are common to jawed and jawless vertebrates. However, whereas antigen recognition in the jawed vertebrates is mediated by immunoglobulin domain-based B cell receptors and T cell receptors, antigen recognition in jawless vertebrates is mediated by leucine-rich repeat (LRR) sequences in variable lymphocyte receptors (VLRs). Three types of VLR genes (VLRA, VLRB and VLRC) have been characterized in the extant jawless vertebrates, lampreys and hagfishes. VLRA- and VLRC-producing cells are T cell-like, while VLRB-producing cells are B cell-like. The VLRs are assembled by copying neighboring LRR sequences into the incomplete germline genes. In searching for the evolutionary origin of the VLRs we identified hypothetical VLR-like progenitors in amphioxus and tunicates (VLRPs). Molecular cladistics markers and phylogenetic analysis suggest the VLRPs are structurally more similar than other ancestral candidates to assembled VLRs. Based on our further analysis we propose alternative models for the origin of VLRs: (A) Among the VLRs, VLRB is most similar to the ancestral state and two subsequent duplication events gave rise to VLRC and VLRA-like genes; each of the three ancestral VLR genes in a common jawless vertebrate ancestor was then modified by cut and paste transposition of the internal DNA region encoding multiple LRR modules. (B) A cut and paste transposition event in an ancestral VLRB-like gene was followed by sequential duplication events which gave rise to VLRC and VLRA. In both models, subsequent duplications and fragmentations have generated a large array of genomic donor cassettes. Thus, after the emergence of the three VLR loci, each has undergone independent evolution.

Genome organization changes in GM and non-GM soybean [Glycine max (L.) Merr.] under salinity stress by retro-transposition events

Genetically modified (GM) crops are developed and cultivated worldwide, providing protection against insects and diseases, or tolerance to herbicides. However, despite the advantages that GM crops, they pose many concerns not only to human and animal health but also to the environment. In the present study, we perform inter-retrotransposon amplified polymorphism (IRAP)-PCR to reveal SIRE1, Bagy2 and Nikita transposition events in soybean [Glycine max (L.) Merr.] and GM soybean under increasing salt stress conditions (0 mM as control, 80 mm, 120 mM, 160 mM and 200 mM NaCl). Salinity dramatically affected soybean and GM soybean in a similar way that stem lengths, root lengths and leaf areas were reduced compared to control. According to IRAP-PCR results, polymorphism ratios of SIRE1, Bagy2 and Nikita retrotransposons among all samples were found to be 22–100%, 0–73% and 37–100%, respectively. These results are the first direct report to demonstrate the relationship between TEs polymorphisms and salinity stress in soybean and GM soybean. TEs, which may explain part of the new phenotypes, play essential roles in genome and gene evolution. Such information can be useful to understanding of genome organization differences derived from TEs between soybean and GM soybean.

Investigation of rice (Oryza sativa L.) retrotransposons in different taxa

Retrotransposons are the subclass of transposable elements and they can increase their copy numbers in the genome through their copy-paste transposition mechanism. Throughout evolutionary process, their transposition events result in mutations. Thus, they cause the formation of new gene alleles and, in the long term, speciation. In contrast, their transposition events might have harmful effects. Therefore most of the organisms have been developing various mechanisms for silencing retrotransposons throughout evolutionary processes. By this way they protect their genome from harmful transposition events. In this study, we investigated transposition events of four Oryza sativa LTR-retrotransposons (Hopi, Houba, Osr30 and RIRE1) in Oryza sativa L. (rice), Brachypodium distachyon (L.) P. Beauv., Hordeum vulgare L., (barley) and Triticum aestivum L. (wheat) by PCR based IRAP marker technique. PCR resulted in different band profiles and polymorphism ratios between individuals of each species. In four species, only rice showed significant polymorphisms between individuals. This result indicates that all tested retrotransposons are still active and cause genomic polymorphism between individuals of rice while they were silenced with various mechanisms in other species. Although these four plant species have a common ancestor, rice was distinguished from others more than 40 million years ago. This result might show that the mechanisms that repress the transposition events of retrotransposons were developed after this point.

38. Retrotransposon activity in young patients with oral Squamous Cell Carcinoma (OSCC)

Background: A recent alarming increase in the incidence of OSCC has been seen in young patients in the absence of tobacco or alcohol use, or HPV integration. Retrotransposons are mobile DNA elements which have been shown to have a role in cancer, particularly LINE-1, with transposition events resulting in mutagenesis. Aims: To compare the activity of three retrotransposon families (LINE-1, SVA and Alu) between young (<45) and older patients with OSCC. Methods: 7 young and 16 older non-HPV OSCC patients, whose genomes were sequenced at high depth, were identified from The Cancer Genome Atlas (TCGA). The Mobile Element Locator Tool (MELT) was employed to detect non-reference retrotransposon events in the selected genomes. Results and conclusions: The total count of putative somatic LINE-1 insertions was significantly higher in the young as compared to the older cohort (p=0.004). A significantly higher number of these events were found to occur close to genes (p=0.019), with possible roles in cancer predisposition syndromes (e.g., OR4C45 and DTNB). RNA-Seq data is being analysed to assess the potential impact of the insertions on gene expression. The presence of elevated somatic LINE-1 activity in young patients may shed light on the increasing development of OSCC at an early age.

Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters

BackgroundTransposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage. The papillation assay provides a powerful visual screen for hyperactive transposases. Transposition is revealed by the activation of a promoter-less lacZ gene when the transposon integrates into a non-essential gene on the host chromosome. Transposition events are detected as small blue speckles, or papillae, on the white background of the main Escherichia coli colony.ResultsWe analysed the parameters of the papillation assay including the strength of the transposase transcriptional and translational signals. To overcome certain limitations of inducible promoters, we constructed a set of vectors based on constitutive promoters of different strengths to widen the range of transposase expression. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. The highest rate of transposition was observed with the weakest promoters. We then took advantage of our approach to investigate how the level of transposition responds to selected point mutations and the effect of joining the transposase monomers into a single-chain dimer.ConclusionsWe generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. The use of weak promoters should allow screening for truly hyperactive transposases rather than those that are simply resistant to auto-regulatory mechanisms, such as overproduction inhibition (OPI). We also found that mutations in the Hsmar1 dimer interface provide resistance to OPI in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques.

New Delhi Metallo-β-Lactamase 1-Producing Klebsiella pneumoniae ST719 Isolated from a Neonate in China.

The spread of carbapenem-resistant Klebsiella pneumoniae has become a significant problem for public health in recent years. In this study, we reported a New Delhi metallo-β-lactamase 1 (NDM-1)-producing K. pneumoniae strain KP14003 from a neonate in Beijing, China. Whole-genome sequencing was performed. The strain belonged to sequence type ST719. Coexistence of blaNDM-1 and blaSHV-12 was found on a self-transferable plasmid, which had a typical IncX3 backbone. The horizontal transfer of blaNDM-1 was associated with Tn125 followed by possible transposition events. Other class A extended-spectrum β-lactamase genes (blaSHV-27 and blaTEM-1) were also identified on chromosome or plasmid. The dissemination of NDM-1-producing K. pneumoniae causes great challenges to the treatment of clinical infections. Effective actions need to be taken to control the further spread of this pathogen.

Two Phenotype-Differentiated Acinetobacter baumannii Mutants That Survived in a Meropenem Selection Display Large Differences in Their Transcription Profiles.

37662RM1 and 37662RM2 are two phenotypically different, carbapenem-resistant mutants of Acinetobacter baumannii 37662 isolate following selection with meropenem (MEM) at sub-inhibitory concentrations. 37662RM2 lacks capsule synthesis and shows dramatically increased biofilm formation, while 37662RM1 shows merely impaired capsule synthesis. Here we report that 37662RM1 and RM2 have transcription profiles that are different from those of their starting strain, 37662WT. There were far more differentially expressed genes in 37662RM2 than in 37662RM1. The capsule polysaccharide (CPS) synthesis-required genes (itrA2, gtr5, psaA, psaB, psaC, psaD, psaE, psaF, kpsS2, wzx, wzy, wza, wzb, and wzc) showed reduced transcription levels in 37662RM2, which may at least partially explain the loss of capsule synthesis. The csu operon genes responsible for pili assembly and their regulator genes bfmR-bfmS were over-expressed in 37662RM2. This result together with the established critical roles of these genes in biofilm formation provide solid evidence that up-regulation of csu and bfmR-bfmS should be considered responsible for the enhanced biofilm formation in 37662RM2. ISAba1 was found to insert into the intergenic region between the csu operon and the acrR gene and should be responsible for the significant up-regulation of acrR, which was proposed to be associated with biofilm formation. Genome sequencing revealed that the ISAba1 upstream blaOXA–508 (a new member of blaOXA–51-like) and acrR were duplicated, suggesting a replicative transposition event. Altogether, the phenotype divergence driven by MEM selection mainly occurs at the RNA level and the transposition of ISAba1 plays an important role in modulating gene expression to adapt to the environment.

Detection and Characterization of Transposons in Bacteria.

Bacterial transposons, through their ability to transfer DNA sequences from one position in the genome to another, play a central role in the shape and the evolution of genomes. Extensive studies have been performed during the last five decades to understand the molecular mechanisms involved in the transposition of a variety of elements. Among the methods used, the papillation and the mating out coupled to arbitrary primed PCR assays described in this chapter are widely used as very powerful approaches to detect and characterize transposition events in vivo.

Targeted transposition with Tn7 elements: safe sites, mobile plasmids, CRISPR/Cas and beyond.

Transposon Tn7 is notable for the control it exercises over where transposition events are directed. One Tn7 integration pathways recognizes a highly conserved attachment (att) site in the chromosome, while a second pathway specifically recognizes mobile plasmids that facilitate transfer of the element to new hosts. In this review, I discuss newly discovered families of Tn7-like elements with different targeting pathways. Perhaps the most exciting examples are multiple instances where Tn7-like elements have repurposed CRISPR/Cas systems. In these cases, the CRISPR/Cas systems have lost their canonical defensive function to destroy incoming mobile elements; instead, the systems have been naturally adapted to use guide RNAs to specifically direct transposition into these mobile elements. The new families of Tn7-like elements also include a variety of novel att sites in bacterial chromosomes where genome islands can form. Interesting families have also been revealed where proteins described in the prototypic Tn7 element are fused or otherwise repurposed for the new dual activities. This expanded understanding of Tn7-like elements broadens our view of how genetic systems are repurposed and provides potentially exciting new tools for genome modification and genomics. Future opportunities and challenges to understanding the impact of the new families of Tn7-like elements are discussed.

Transposable elements contribute to dynamic genome content in maize.

Transposable elements (TEs) are ubiquitous components of eukaryotic genomes and can create variation in genome organization and content. Most maize genomes are composed of TEs. We developed an approach to define shared and variable TE insertions across genome assemblies and applied this method to four maize genomes (B73, W22, Mo17 and PH207) with uniform structural annotations of TEs. Among these genomes we identified approximately 400000 TEs that are polymorphic, encompassing 1.6Gb of variable TE sequence. These polymorphic TEs include a combination of recent transposition events as well as deletions of older TEs. There are examples of polymorphic TEs within each of the superfamilies of TEs and they are found distributed across the genome, including in regions of recent shared ancestry among individuals. There are many examples of polymorphic TEs within or near maize genes. In addition, there are 2380 gene annotations in the B73 genome that are located within variable TEs, providing evidence for the role of TEs in contributing to the substantial differences in annotated gene content among these genotypes. TEs are highly variable in our survey of four temperate maize genomes, highlighting the major contribution of TEs in driving variation in genome organization and gene content. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://github.com/SNAnderson/maizeTE_variation; https://mcstitzer.github.io/maize_TEs.

Y chromosome-linked genes implicated in spermatogenesis in cattle

Summary. The mammalian sex chromosomes evolved from an ordinary pair of autosomes during evolution. Unlike the X chromosome that is highly conserved, the Y chromosome is poorly conserved among mammalian lineages. Several special features set the Y chromosome apart from the rest of genome: male-limited transmission, absence of recombination, abundance of Y-specific repetitive sequences, degeneration of Y-linked genes during evolution, acquisition of autosomal genes, and accumulation and functional cluster of "testis genes" for maleness and reproduction. Since the degeneration process is lineage-dependent, different lineages retain different subsets of genes from the ancestral proto-Y chromosome, resulting in a diverse and lineage-specific Y chromosome gene content. During bovine evolution, a lineage-specific 'autosome-to-Y' transposition event resulted in three bovid-specific Y chromosome gene families, PRAMEY, ZNF280BY and ZNF280AY. Together, the male-specific region (MSY) of the bovine Y chromosome (BTAY) contains ~1200 protein coding genes that can be classified into 12 single copy and 16 multiple copy protein families. The copy number (CN) of these Y-linked gene families varies from 13 for PRAMEY to 236 for ZNF280BY, with significant differences between the taurine and indicine Y lineages. In addition, 367 non-coding RNA families (ncRNAs) were also identified on BTAY. Transcriptome analysis revealed that 95% of the BTAY genes/ncRNAs are expressed predominantly in testis and may be involved in spermatogenesis and male fertility. Though the functional role for the majority of the Y-linked genes needs to be determined, the preliminary data on PRAMEY clearly indicated a role in spermiogenesis. Furthermore, copy number variations (CNVs) of PRAMEY, ZNF280BY, TSPY and HSFY were found to be associated with testis size, sperm quality and fertility in dairy bulls. The authors discuss several challenges that influence male fertility selection associated with the bovine Y chromosome.

The ISApl1 2 Dimer Circular Intermediate Participates in mcr-1 Transposition.

Objectives: The mobile colistin resistance gene mcr-1 is a serious threat to global human and animal health. The composite transposon Tn6330 and its circular intermediate were proposed to be involved in the spread of mcr-1 but their roles remain poorly understood.Methods: To further explore the intermediates during the transposition of Tn6330, we engineered Escherichia coli strains that carry an intact Tn6330 transposon or its deletion derivatives. PCR assays were performed to detect IR-IR junctions and possible circular intermediates. We carried out transposition experiments to calculate transposition frequency. The transposition sites were characterized by whole genome sequence and ISMapper-based analyses.Results: The presence of an intact Tn6330 was demonstrated to be essential for the successful transposition of mcr-1, although both Tn6330 and Tn6330-ΔIR could form circular intermediates. The insertion sequence junction structure was observed in all constructed plasmids but the ISApl1 dimer was only formed in one construct containing an intact Tn6330. The average frequency of mcr-1 transposition in an E. coli strain possessing an intact Tn6330 was ∼10-6 per transformed cell. We identified 27 integration sites for the Tn6330 transposition event. All the transposition sites were flanked by 2 bp target duplications and preferentially occurred in AT-rich regions.Conclusion: These results indicate that mcr-1 transposition relies on the presence of an intact Tn6330. In addition, formation of the tandem repeat ISApl12 could represent a crucial intermediate. Taken together, the current investigations provide mechanistic insights in the transposition of mcr-1.