Documentation

The Importance of Transposable Elements

Transposable elements (TE) are key facilitators of bacterial adaptation and therefore are central players in the emergence of multiple antibacterial resistances such as resistance to antibiotics, heavy metals and to transmission of pathogenic traits. TE capture passenger genes using a number of mechanisms and transmit them to larger mobile genetic elements, plasmids, where they accumulate and are then transferred within and between bacterial populations. TE also contribute significantly to the ongoing reorganization of bacterial genomes, giving rise to new strains that are more and more adept at proliferating both in the environment and in hospitals. Understanding TE nature, distribution and action is therefore an indispensable part of the struggle to cope with the public health crisis of multiple antimicrobial resistance (AMR) (1,2). To understand the impact of TE on bacterial populations, to follow the flow of genes important in public health both in clinical and environmental settings and to provide some measure of understanding which might allow prediction of resistance transmission, it is essential to provide a detailed description and catalog of TE structures and diversity. This has already been undertaken for the simplest TE, the insertion sequences (IS), in the form of the online knowledge base ISfinder (https://www-is.biotoul.fr/index.php) (3,4), an international resource for IS currently including over 5000 individual examples. The ISfinder platform also includes a set of software tools, ISsaga, allowing semi-automatic genome annotation for IS using the ISfinder database (5). Although movement of IS has a profound and continuous impact on genome organization and function due to their ability to rearrange DNA, regulate neighboring genes and generate mutations (6–9), they do not themselves generally carry integrated passenger genes.

There are a large number of significantly more complex TE, arguably even more important in the global emergence of AMR. These are generically called transposons and may carry multiple passenger genes, including some of the most clinically important antibiotic resistance genes. They are grouped into a number of distinct families with characteristic organizations (6). Like IS, their transposition activities facilitate the rapid spread of groups of antibiotic resistance genes and promote their horizontal transfer to other bacterial strains, species and genera via natural vectors such as conjugal plasmids and bacterial viruses. Yet another important aspect of their impact is their ability to assemble passenger genes into resistance clusters (10,11). While there appears to be wide-spread appreciation that mobile plasmids are responsible for the spread of antibiotic resistance, fewer people are aware that IS and transposons are the conduit that transfers this information between chromosomes and plasmids.

It is crucial to stress the importance of educating the clinical world concerning transposition mechanisms in an easy-to-use way. Most scientists consider that IS/Tn all behave in the same way and believe that cataloging them is simply “busy-work”. However, a thorough understanding of how IS/Tn assemble antimicrobial resistance genes and effect rapid changes in plasmid vector structure is critical to understanding the increasingly efficient AMR spread observed today and combatting future AMR outbreaks (see Figure 1).

Figure 1. This figure represents the relationship between a group of plasmids, many isolated from patients during an outbreak of carbapenem resistant bacteria at the NIH Clinical Center (references 10 and 11). The DNA sequences in brackets show small direct target repeats generated during transposon movement and act as an important guide to the way in which the plasmids have been rearranged during the course of the infections. This type of information will be crucial in developing the fourth stage of the project: semiautomatic annotation of plasmid groups of interest such as those isolated from clinical outbreaks.

TnCentral

There has been a need for a database for transposons providing similar comprehensiveness, transparency and usability that ISfinder provides for IS. TnCentral is a pilot transposon database conceived as a resource for transposons, their associated passenger genes, and their host organisms.

TnCentral: Content

Mobile Element Groups Covered

TnCentral initially focuses on two transposon groups—the Tn3 transposon family (Figures 2, 4 and 5) and the composite (or compound) transposons composed of two IS flanking a variety of passenger genes --because they include some of the most clinically important AMR transposons (Figure 3 and 6).

The Tn3 family

Tn3 family members form a tightly knit group. The basic Tn3 family transposition module is composed of transposase and resolvase genes and two ends with related terminal inverted repeat DNA sequences, the IRs, of 38-40bp or sometimes even longer (12). There is a large (~1000 aa) DDE transposase, TnpA, significantly longer than the DDE transposases normally associated with Insertion Sequences (IS) (see (7)). TnpA catalyzes the DNA cleavage and strand transfer reactions necessary for formation of a cointegrate transposition intermediate during replicative transposition. The cointegrate is composed of fused donor (with the transposon) and target (without the transposon) circular DNA molecules fused into a single circular molecule and separated by two directly repeated transposon copies, one at each junction (13).

A second feature of members of this transposon family is that they carry short (~100-150bp) DNA segments, res (for resolution) or rst (for resolution site tnpS tnpT – see below; (14)) at which site-specific recombination between each of the two Tn copies occurs to “resolve” the cointegrate into individual copies of the transposon donor and the target molecules each containing a single transposon copy (see (15)). This highly efficient recombination system is assured by a transposon-specified sequence-specific recombinase enzyme: the resolvase.

There are at present three known major resolvase types: TnpR, TnpI, and TnpS+TnpT, distinguished, among other things, by the catalytic nucleophile involved in DNA phosphate bond cleavage and rejoining during recombination: TnpR, a classic serine (S)-site-specific recombinase (e.g. (16)); TnpI, a tyrosine (Y) recombinase similar to phage integrases (17)(see (15); and a heteromeric resolvase combining a tyrosine recombinase, TnpS, and a divergently expressed helper protein, TnpT, with no apparent homology to other proteins (14,18). The resolvase genes can be either co-linear, generally upstream of tnpA or divergent. In the former case the res site lies upstream of tnpR and in the latter case, between the divergent tnpR and tnpA genes. For relatives encoding TnpS and TnpT, the corresponding genes are divergent and the res (rst) site lies between tnpS and tnpT. Examples of these architectures are shown in Figure 1. Each res includes a number of short DNA sub-sequences which are recognised and bound by the cognate resolvases. These are different for different resolvasesystems (Figure 1) But where analysed, res sites also include promoters which drive both transposase and resolvase expression. Indeed, TnpR from Tn3 was originally named for its ability to repress transposase expression by binding to these sites (19,20). A number of Tn3 members do not include a resolvase gene and therefore, although cointegrates are formed during transposition of these transposons, no efficient sequence-specific recombination occurs to resolve these structures. Instead, “resolution” depends on the homologous recombination system of the host which uses the directly repeated transposon copies as a substrate.

The complexity of these Tn resides in the diversity of other mobile elements incorporated into their structures (such as, Insertion Sequences (IS) and integrons as well as other Tn3 family members – see (15)) (Figure 4) and other passenger genes. The most notorious of these genes are those for antibiotic and heavy metal resistance although other genes involved in organic catabolite degradation and virulence functions for both animals and plants (Figure 2) also form part of the Tn3 family arsenal of passenger genes.