Biology subjects

Chandler, M. G.

Publications and source records attributed to Chandler, M. G..

4 recordsLinked to original sources

bioRxiv2026

A replication hijacking mechanism for Tn3-family replicative transposition

Transposition of all classes of transposable elements generates DNA intermediates that must be processed by the host to be effective. However, the mechanisms whereby transposons communicate with cellular DNA-processing machineries remain poorly investigated. Here, we provide convergent genetic and biochemical evidence that replicative transposition of the Tn3-family transposon Tn4430 is strictly coupled to replication of the target. Blocking target replication abolishes transposition, while blocking replication of the transposon donor molecule has no effect. Furthermore, the insertion preference of Tn4430 was found to be altered by the direction of replication and potential replication impediments within the target, suggesting a functional link between the integration mechanism and replication fork progression. In vitro, the transposase TnpA was found to specifically bind to fork-like DNA structures that mimic replication intermediates. Compared to linear DNA fragments, these structures are efficient substrates for TnpA-catalysed end joining. Strand transfer occurred immediately downstream of the fork, poising the transposon for replication. Together, the data suggest a mechanism in which the transposon targets DNA replication intermediates to directly recruit the host replication machinery at the time of transposition. This "replication hijacking" mechanism contrasts with classical "replication hiring" mechanisms during which replication is recruited after strand transfer. SIGNIFICANCE STATEMENTBacterial transposons of the Tn3 family constitute a threat for human health, being continuously involved in the emergence and spread of new antimicrobial resistances amongst pathogens. The success of these elements is based on their replicative mode of transposition allowing them to produce a new copy of themselves whenever they move. Here, we provide evidence that, rather than recruiting the host replication machinery after integration as is proposed in textbook models for replicative transposition, Tn3-family transposons directly transpose into ongoing replication intermediates. We propose that this "replication hijacking" mechanism provides a means of synchronizing transposition with cell replication activity, thus optimizing the movement of transposons with the expansion of bacterial populations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/728999v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@c04da0org.highwire.dtl.DTLVardef@1285b5aorg.highwire.dtl.DTLVardef@859c5corg.highwire.dtl.DTLVardef@1dc9ce8_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

The Insertion Sequence Excision Enhancer (IEE): a PrimPol-based system for Immobilizing Transposon-Transmitted Antibiotic Resistance Genes?

We provide an overview of a protein, IEE (Insertion Sequence Excision Enhancer), which was originally observed to facilitate high levels of excision of the IS3 family member, IS629, from clinically important Escherichia coli O157:H7. IEE was subsequently shown to affect a large class of bacterial insertion sequences which all transpose by producing a circular intermediate and presumably use the copy-out-paste-in transposition mechanism. Excision is dependent on both IEE and transposase indicating that it is associated with the transposition process itself. We propose that IEE serves to immobilize genes carried by compound transposons by removing the flanking IS copies, an activity which would explain the presence of certain of these genes without associated IS copies in plasmids and chromosomes. The biochemical activities of IEE as a primase with the capacity to recognize microhomologies, together with the observation that its effect appears to be restricted to those IS families which probably use the copy-out-paste-in transposition pathway, suggests a strand switch mechanism during the copy-out step leading to abortive transposition. This reinforces the proposal made for understanding the loss of the IS30 family member, ISApl1, which flanks the mcr-1 gene in the compound transposon Tn6330.

microbiology↗

bioRxiv2022

A subclass of the IS1202 family of bacterial insertion sequences targets XerCD recombination sites

IS1202, originally isolated from Streptococcus pneumonia in the mid-1990s had been previously tagged as an emerging IS family in ISfinder. While searching for plasmid-associated Xer recombinase recombination sites (xrs) in Acinetobacter baumannii, we observed that some insertion sequences related to IS1202 were repeatedly found abutting these sites in a number of plasmids. The plasmids often carried repeated xrs thought to form a new type of mobile genetic element (MGE) which uses the chromosomally-encoded XerCD recombinase for mobility. The MGE (xrs cassette) consist of xrs flanking one or a small number of genes often including different clinically important carbapenemase-encoding bla-OXA. The IS1202- related IS are inserted with their left, transposase proximal extremity, IRL, five base pairs from xrs and include a characteristic 5bp flanking target duplication. Further searches revealed that many different plasmid- and chromosome-borne xrs can be targeted and that IS1202-xrs combinations are not limited to Acinetobacter baumannii but occur in other bacteria. In addition to 28 IS1202 group ISs in ISfinder and a number which had been subsequently submitted, we undertook a survey of the NCBI (February 2020) and identified 138 additional IS1202-related IS. These could be divided into 3 principal subgroups based on their transposase sequences and on the length of the DR generated on insertion: subgroup IS1202 27-28bp DR); ISTde1 (15-17bp); and ISAba32 (5-6bp). Members of each group which lacked DR were also found. But since other examples of most of these were subsequently identified having DR, those lacking DR may have been generated by intra-replicon recombination. Only members of the group which generate 5bp DR were found to target xrs. These were not only identified in plasmids but also occurred at some individual xrs sites, dif, located at the chromosome replication terminus and involved in post-replication chromosome segregation. Further analysis showed the presence of subgroup-specific indels in their transposases which may be responsible for the differences in their behavior. We propose that this collection of IS be classed as a new insertion sequence family: the IS1202 family composed of at three subfamilies, only one of which specifically targets plasmid-borne xrs. We discuss the implications of xrs targeting for gene mobility.

genomics↗

bioRxiv2021

TnCentral: A Prokaryotic Transposable Element Database and Web Portal for Transposon Analysis

We describe here the structure and organization of TnCentral (https://tncentral.proteininformationresource.org/). a web resource for prokaryotic transposable elements (TE). TnCentral currently contains ~400 carefully annotated TE, including transposons from the Tn3, Tn7, Tn402 and Tn554 families, compound transposons, integrons and associated insertion sequences (IS). These TE carry passenger genes, including genes conferring resistance to over 25 classes of antibiotics and nine types of heavy metal as well as genes responsible for pathogenesis in plants, toxin/antitoxin gene pairs, transcription factors and genes involved in metabolism. Each TE has its own entry page providing details about its transposition genes, passenger genes, and other sequence features required for transposition as well as a graphical map of all features. TnCentral content can be browsed and queried through text and sequence-based searches with a graphic output. We describe three use cases, which illustrate how the search interface, results tables, and entry pages can be used to explore and compare TEs. TnCentral also includes downloadable software to facilitate user-driven identification, with manual annotation, of certain types of TE in genomic sequences. Through the TnCentral homepage, users can also access TnPedia which provides comprehensive reviews of the major TE families including an extensive general section, and specialised sections with descriptions of insertion sequence and transposon families. TnCentral and TnPedia are intuitive resources that can be used by clinicians and scientists to assess TE diversity in clinical, veterinary and environmental samples. ImportanceThe ability of bacteria to undergo rapid evolution and adapt to changing environmental circumstances drives the public health crisis of multiple antibiotic resistance as well as outbreaks of disease in economically important agricultural crops and animal husbandry. Prokaryotic transposable elements (TE) play a critical role in this. Many carry "passenger genes" (not required for the transposition process) conferring resistance to antibiotics or heavy metals or causing disease in plants and animals. Passenger genes are spread by the normal TE transposition activities, by insertion into plasmids which then spread via conjugation within and across bacterial populations. Thus, an understanding of TE composition and transposition mechanisms is key to developing strategies to combat bacterial pathogenesis. Toward this end, we have developed TnCentral, a bioinformatics resource dedicated to describing and exploring the structural and functional features of prokaryotic TE and whose use is intuitive and accessible to users with or without bioinformatics expertise.

genomics↗