DDE Transposase Research Articles

IS26 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.

A systematic approach to classify and characterize genomic islands driven by conjugative mobility using protein signatures.

Genomic islands (GIs) play a crucial role in the spread of antibiotic resistance, virulence factors and antiviral defense systems in a broad range of bacterial species. However, the characterization and classification of GIs are challenging due to their relatively small size and considerable genetic diversity. Predicting their intercellular mobility is of utmost importance in the context of the emerging crisis of multidrug resistance. Here, we propose a large-scale classification method to categorize GIs according to their mobility profile and, subsequently, analyze their gene cargo. We based our classification decision scheme on a collection of mobility protein motif definitions available in publicly accessible databases. Our results show that the size distribution of GI classes correlates with their respective structure and complexity. Self-transmissible GIs are usually the largest, except in Bacillota and Actinomycetota, accumulate antibiotic and phage resistance genes, and favour the use of a tyrosine recombinase to insert into a host's replicon. Non-mobilizable GIs tend to use a DDE transposase instead. Finally, although tRNA genes are more frequently targeted as insertion sites by GIs encoding a tyrosine recombinase, most GIs insert in a protein-encoding gene. This study is a stepping stone toward a better characterization of mobile GIs in bacterial genomes and their mechanism of mobility.

Chromosome-level genome assembly of Bactrocera dorsalis reveals its adaptation and invasion mechanisms

Bactrocera dorsalis is an invasive polyphagous pest causing considerable ecological and economic damage worldwide. We report a high-quality chromosome-level genome assembly and combine various transcriptome data to explore the molecular mechanisms of its rapid adaptation to new environments. The expansions of the DDE transposase superfamily and key gene families related to environmental adaptation and enrichment of the expanded and unique gene families in metabolism and defence response pathways explain its environmental adaptability. The relatively high but not significantly different expression of heat-shock proteins, regardless of the environmental conditions, suggests an intrinsic mechanism underlying its adaptation to high temperatures. The mitogen-activated protein kinase pathway plays a key role in adaptation to new environments. The prevalence of duplicated genes in its genome explains the diversity in the B. dorsalis complex. These findings provide insights into the genetic basis of the invasiveness and diversity of B. dorsalis, explaining its rapid adaptation and expansion.

Genome-Wide Identification, Characterization, and Expression Analysis of DDE_Tnp_4 Family Genes in Eriocheir sinensis.

DDE transposase 4 (DDE_Tnp_4) family is a large endonuclease family involved in a wide variety of biological processes. However, little information is available about this family in crustaceans. In this study, we used HMMER to identify 39 DDE_Tnp_4 family genes in Eriocheir sinensis genome, and the genes were classified into four subfamilies according to phylogenetic analysis. Gene expansions occurred among E. sinensis genome, and synteny analysis revealed that some DDE_Tnp_4 family genes were caused by tandem duplication. In addition, the expression profiles of DDE_Tnp_4 family genes in E. sinensis indicated that subfamily I and II genes were up-regulated in response to acute high salinity and air exposure stress. E. sinensis is a kind of economical crustacean with strong tolerance to environmental stress. We confirmed the expansion of DDE_Tnp_4 family genes in E. sinensis and speculated that this expansion is associated with strong tolerance of E. sinensis. This study sheds light on characterizations and expression profiles of DDE_Tnp_4 family genes in E. sinensis and provides an integrated framework for further investigation on environmental adaptive functions of DDE_Tnp_4 family in crustaceans.

An ISMap02-like insertion sequence in Mycobacterium spp. interferes with specific detection of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease or paratuberculosis (PTB), which is a chronic debilitating disease in ruminants, that is characterized by incurable enteritis and persistent diarrhea. ISMap02 is one of the major targets of PCR because it is present in multicopies (six copies) and known to be specific to MAP. However, in the present study, non-MAP mycobacteria were shown to be positive by ISMap02 targeting PCR. Two bacterial isolates (Sample ID: BO-038 and BO-042) were cultured from bovine fecal samples that produced positive results in three of two ISMap02 targeting PCR analyses with negative results in IS900 real-time PCR. Species identification using 16S rRNA gene sequencing and hsp65 gene partial sequencing revealed that strains BO-038 and BO-042 were M. virginiense and M. nonchromogenicum, respectively, which both belong to the M. terrae complex (MTC). Moreover, the two isolates shared a novel insertion sequence (IS) with high similarity to some parts of nucleotide sequences of ISMap02, and IS was presumed to be identical to that present in M. heraklionense. Both the novel IS and ISMap02 were characterized as IS1182 family members, and several sequences similar to ISMap02 were identified by BLAST analysis. In addition, the DDE transposase of the novel IS showed great similarity in the N-terminal portion with the IS5/1182 DDE transposase of other mycobacteria. These results suggest that ISMap02 has a conserved region with similarity to other ISs, and that the diagnostic value of the primer sets targeting that region should be re-addressed.

Extension of the transposable bacterial virus family: two genomic organisations among phages and prophages with a Tn552-related transposase

Mu-like transposable phages and prophages have been isolated from, or predicted, in a wide range of bacterial phyla. However, related B3-like transposable phages, which differ in their genome organisation and the DDE transposase and transposition activator they code for have thus far been restricted to a very limited set of hosts. Through sequence similarity searches, we have now expanded the number of predicted B3-like prophages and uncovered a third genomic organisation. These new genomes, although only prophages, further illustrate the previously reported mosaicism existing in the proposed “Saltoviridae” family of Caudovirales, and further support the proposal to move morphology criteria (contractile vs. flexible or short tail, i.e. Myo-vs. Sipho- or Podoviridae) from the family to the subfamily level in the taxonomic classification of the Caudovirales.

Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus.

ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013 Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci.IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one of two known families of ICEs in the pathogen Staphylococcus aureus, but its core functions of excision and conjugation are not well studied. Here, we show that ICE6013 depends on its IS30-like DDE transposase for excision, which is unique among ICEs, and we demonstrate the conjugative transfer and integration site preference of ICE6013 A sequence analysis revealed that ICE6013 has diverged into seven subfamilies that are dispersed among staphylococci.

New Insights into the Classification and Integration Specificity of Streptococcus Integrative Conjugative Elements through Extensive Genome Exploration.

Recent genome analyses suggest that integrative and conjugative elements (ICEs) are widespread in bacterial genomes and therefore play an essential role in horizontal transfer. However, only a few of these elements are precisely characterized and correctly delineated within sequenced bacterial genomes. Even though previous analysis showed the presence of ICEs in some species of Streptococci, the global prevalence and diversity of ICEs was not analyzed in this genus. In this study, we searched for ICEs in the completely sequenced genomes of 124 strains belonging to 27 streptococcal species. These exhaustive analyses revealed 105 putative ICEs and 26 slightly decayed elements whose limits were assessed and whose insertion site was identified. These ICEs were grouped in seven distinct unrelated or distantly related families, according to their conjugation modules. Integration of these streptococcal ICEs is catalyzed either by a site-specific tyrosine integrase, a low-specificity tyrosine integrase, a site-specific single serine integrase, a triplet of site-specific serine integrases or a DDE transposase. Analysis of their integration site led to the detection of 18 target-genes for streptococcal ICE insertion including eight that had not been identified previously (ftsK, guaA, lysS, mutT, rpmG, rpsI, traG, and ebfC). It also suggests that all specificities have evolved to minimize the impact of the insertion on the host. This overall analysis of streptococcal ICEs emphasizes their prevalence and diversity and demonstrates that exchanges or acquisitions of conjugation and recombination modules are frequent.

The Diversity of Prokaryotic DDE Transposases of the Mutator Superfamily, Insertion Specificity, and Association with Conjugation Machineries

Transposable elements (TEs) are major components of both prokaryotic and eukaryotic genomes and play a significant role in their evolution. In this study, we have identified new prokaryotic DDE transposase families related to the eukaryotic Mutator-like transposases. These genes were retrieved by cascade PSI-Blast using as initial query the transposase of the streptococcal integrative and conjugative element (ICE) TnGBS2. By combining secondary structure predictions and protein sequence alignments, we predicted the DDE catalytic triad and the DNA-binding domain recognizing the terminal inverted repeats. Furthermore, we systematically characterized the organization and the insertion specificity of the TEs relying on these prokaryotic Mutator-like transposases (p-MULT) for their mobility. Strikingly, two distant TE families target their integration upstream σA dependent promoters. This allowed us to identify a transposase sequence signature associated with this unique insertion specificity and to show that the dissymmetry between the two inverted repeats is responsible for the orientation of the insertion. Surprisingly, while DDE transposases are generally associated with small and simple transposons such as insertion sequences (ISs), p-MULT encoding TEs show an unprecedented diversity with several families of IS, transposons, and ICEs ranging in size from 1.1 to 52 kb.

Analysis of CACTA transposases reveals intron loss as major factor influencing their exon/intron structure in monocotyledonous and eudicotyledonous hosts.

BackgroundCACTA elements are DNA transposons and are found in numerous organisms. Despite their low activity, several thousand copies can be identified in many genomes. CACTA elements transpose using a ‘cut-and-paste’ mechanism, which is facilitated by a DDE transposase. DDE transposases from CACTA elements contain, despite their conserved function, different exon numbers among various CACTA families. While earlier studies analyzed the ancestral history of the DDE transposases, no studies have examined exon loss and gain with a view of mechanisms that could drive the changes.ResultsWe analyzed 64 transposases from different CACTA families among monocotyledonous and eudicotyledonous host species. The annotation of the exon/intron boundaries showed a range from one to six exons. A robust multiple sequence alignment of the 64 transposases based on their protein sequences was created and used for phylogenetic analysis, which revealed eight different clades. We observed that the exon numbers in CACTA transposases are not specific for a host genome. We found that ancient CACTA lineages diverged before the divergence of monocotyledons and eudicotyledons. Most exon/intron boundaries were found in three distinct regions among all the transposases, grouping 63 conserved intron/exon boundaries.ConclusionsWe propose a model for the ancestral CACTA transposase gene, which consists of four exons, that predates the divergence of the monocotyledons and eudicotyledons. Based on this model, we propose pathways of intron loss or gain to explain the observed variation in exon numbers. While intron loss appears to have prevailed, a putative case of intron gain was nevertheless observed.

Modular Evolution of Tn GBS s, a New Family of Integrative and Conjugative Elements Associating Insertion Sequence Transposition, Plasmid Replication, and Conjugation for Their Spreading

Integrative and conjugative elements (ICEs) have a major impact on gene flow and genome dynamics in bacteria. The ICEs TnGBS1 and TnGBS2, first identified in Streptococcus agalactiae, use a DDE transposase, unlike most characterized ICEs, which depend on a phage-like integrase for their mobility. Here we identified 56 additional TnGBS-related ICEs by systematic genome analysis. Interestingly, all except one are inserted in streptococcal genomes. Sequence comparison of the proteins conserved among these ICEs defined two subtypes related to TnGBS1 or TnGBS2. We showed that both types encode different conjugation modules: a type IV secretion system, a VirD4 coupling protein, and a relaxase and its cognate oriT site, shared with distinct lineages of conjugative elements of Firmicutes. Phylogenetic analysis suggested that TnGBSs evolved from two conjugative elements of different origins by the successive recruitment of a transposition module derived from insertion sequences (ISs). Furthermore, TnGBSs share replication modules with different plasmids. Mutational analyses and conjugation experiments showed that TnGBS1 and TnGBS2 combine replication and transposition upstream promoters for their transfer and stabilization. Despite an evolutionarily successful horizontal dissemination within the genus Streptococcus, these ICEs have a restricted host range. However, we reveal that for TnGBS1 and TnGBS2, this host restriction is not due to a transfer incompatibility linked to the conjugation machineries but most likely to their ability for transient maintenance through replication after their transfer.

Ginger DNA transposons in eukaryotes and their evolutionary relationships with long terminal repeat retrotransposons

BackgroundIn eukaryotes, long terminal repeat (LTR) retrotransposons such as Copia, BEL and Gypsy integrate their DNA copies into the host genome using a particular type of DDE transposase called integrase (INT). The Gypsy INT-like transposase is also conserved in the Polinton/Maverick self-synthesizing DNA transposons and in the 'cut and paste' DNA transposons known as TDD-4 and TDD-5. Moreover, it is known that INT is similar to bacterial transposases that belong to the IS3, IS481, IS30 and IS630 families. It has been suggested that LTR retrotransposons evolved from a non-LTR retrotransposon fused with a DNA transposon in early eukaryotes. In this paper we analyze a diverse superfamily of eukaryotic cut and paste DNA transposons coding for INT-like transposase and discuss their evolutionary relationship to LTR retrotransposons.ResultsA new diverse eukaryotic superfamily of DNA transposons, named Ginger (for 'Gypsy INteGrasE Related') DNA transposons is defined and analyzed. Analogously to the IS3 and IS481 bacterial transposons, the Ginger termini resemble those of the Gypsy LTR retrotransposons. Currently, Ginger transposons can be divided into two distinct groups named Ginger1 and Ginger2/Tdd. Elements from the Ginger1 group are characterized by approximately 40 to 270 base pair (bp) terminal inverted repeats (TIRs), and are flanked by CCGG-specific or CCGT-specific target site duplication (TSD) sequences. The Ginger1-encoded transposases contain an approximate 400 amino acid N-terminal portion sharing high amino acid identity to the entire Gypsy-encoded integrases, including the YPYY motif, zinc finger, DDE domain, and, importantly, the GPY/F motif, a hallmark of Gypsy and endogenous retrovirus (ERV) integrases. Ginger1 transposases also contain additional C-terminal domains: ovarian tumor (OTU)-like protease domain or Ulp1 protease domain. In vertebrate genomes, at least two host genes, which were previously thought to be derived from the Gypsy integrases, apparently have evolved from the Ginger1 transposase genes. We also introduce a second Ginger group, designated Ginger2/Tdd, which includes the previously reported DNA transposon TDD-4.ConclusionsThe Ginger superfamily represents eukaryotic DNA transposons closely related to LTR retrotransposons. Ginger elements provide new insights into the evolution of transposable elements and certain transposable element (TE)-derived genes.

Integrative and Sequence Characteristics of a Novel Genetic Element, ICE 6013 , in Staphylococcus aureus

A survey of chromosomal variation in the ST239 clonal group of methicillin-resistant Staphylococcus aureus (MRSA) revealed a novel genetic element, ICE6013. The element is 13,354 bp in length, excluding a 6,551-bp Tn552 insertion. ICE6013 is flanked by 3-bp direct repeats and is demarcated by 8-bp imperfect inverted repeats. The element was present in 6 of 15 genome-sequenced S. aureus strains, and it was detected using genetic markers in 19 of 44 diverse MRSA and methicillin-susceptible strains and in all 111 ST239 strains tested. Low integration site specificity was discerned. Multiple chromosomal copies and the presence of extrachromosomal circular forms of ICE6013 were detected in various strains. The circular forms included 3-bp coupling sequences, located between the 8-bp ends of the element, that corresponded to the 3-bp direct repeats flanking the chromosomal forms. ICE6013 is predicted to encode 15 open reading frames, including an IS30-like DDE transposase in place of a Tyr/Ser recombinase and homologs of gram-positive bacterial conjugation components. Further sequence analyses indicated that ICE6013 is more closely related to ICEBs1 from Bacillus subtilis than to the only other potential integrative conjugative element known from S. aureus, Tn5801. Evidence of recombination between ICE6013 elements is also presented. In summary, ICE6013 is the first member of a new family of active, integrative genetic elements that are widely dispersed within S. aureus strains.

A Single-Copy IS5-Like Transposon in the Genome of a Bdelloid Rotifer

In the course of sequencing telomeric chromosomal regions of the bdelloid rotifer Adineta vaga, we encountered an unusual DNA transposon. Unlike other bdelloid and, more generally, eukaryotic transposable elements (TEs), it exhibits similarity to prokaryotic insertion sequences (ISs). Phylogenetic analysis indicates that this transposon, named IS5_Av, is related to the ISL2 group of the IS5 family of bacterial IS elements. Despite the apparent intactness of the single open reading frame coding for a DDE transposase and the perfect identity of its 213-bp terminal inverted repeats (TIRs), the element is present in only one copy per diploid genome. It does not exhibit any detectable levels of transcription, so that its transposase gene appears to be silent in the bdelloid host. Although horizontal transfers of TEs between kingdoms are not known to happen in nature, it appears likely that IS5_Av underwent integration into the A. vaga genome relatively recently, but was not successful in adapting to the new host and failed to increase in copy number. Alternatively, it might be the only known member of a novel eukaryotic DNA TE superfamily which is so rare that its other members, if any, have not yet been identified in eukaryotic genomes sequenced to date.

Atypical association of DDE transposition with conjugation specifies a new family of mobile elements

We describe in Streptococcus agalactiae an atypical family of conjugative transposons named TnGBSs which associates DDE transposition and conjugation. We present evidence that the transposition of TnGBS2, the prototype of this family, is catalysed by a new class of DDE transposases that are widespread in Gram-positive bacteria. Remarkably, transposition occurs in intergenic regions, 15 or 16 bp upstream the -35 sequence of promoters, minimizing the burden on the host cell and suggesting an association between transcription and transposition. Transposition catalyses the formation of a circular intermediate that is substrate for subsequent conjugative intercellular transfer. Conjugation is initiated at an origin of transfer by a transposon-encoded relaxase. Whereas all integrative and conjugative elements described so far encode a phage-related integrase, TnGBS2 is the first example of conjugative transposon whose recombination is mediated by a DDE transposase. The combination of DDE transposition with conjugation implies recombination constraints linked to the physical separation of donor and recipient molecules.