Brief Report
21 October 2016

A Model for Transposition of the Colistin Resistance Gene mcr-1 by ISApl1

ABSTRACT

Analysis of mcr-1-containing sequences identified a common ∼2,607-bp DNA segment that in many cases is flanked on one or both ends by ISApl1. We present evidence that mcr-1 is mobilized by an ISApl1 composite transposon which has, in some cases, subsequently lost one or both copies of ISApl1. We also show that mcr-1 can be mobilized in some circumstances by a single upstream copy of ISApl1 in conjunction with the remnants of a downstream ISApl1.

TEXT

Colistin is increasingly relied upon to treat infections caused by multidrug-resistant, carbapenemase-producing Enterobacteriaceae (1). Consequently, there is great concern surrounding plasmid-borne colistin resistance mediated by mcr-1 (2). mcr-1 has now been reported in Enterobacteriaceae from five continents: Asia, Europe, Africa, South America, and North America (reviewed in reference 3). A recent study detected mcr-1 from Escherichia coli collected in the early 1980s, suggesting that the emergence of mcr-1 occurred much earlier than previously thought (4). Given the global dissemination of mcr-1 and the potential clinical consequences, understanding the molecular mechanisms underlying its mobility is critical. Here we provide a detailed examination of the genetic context surrounding mcr-1 and present evidence that mcr-1 is primarily mobilized by an ISApl1 composite transposon.
There is increasing evidence that ISApl1 plays a pivotal role in the mobilization of mcr-1 (5, 6). ISApl1 was first detected in Actinobacillus pleuropneumoniae, an etiological agent of disease in swine (7). It belongs to the IS30 family and possesses features and activities similar to those of IS30 members. It is flanked by 27-bp inverted repeats (designated IRL and IRR, for inverted repeat left and inverted repeat right, respectively) and contains a 927-bp open reading frame (ORF) that encodes a DD(E/D) superfamily transposase protein. Like IS30 and other family members, ISApl1 moves through a covalently closed double-stranded DNA (dsDNA) circular intermediate containing 2 bp of host flanking DNA between the abutted transposon ends and, upon integration, generates 2-bp target site duplications (TSDs) (79). Such circular intermediates are characteristic of a large number of IS families (10) and appear to be produced by a replicative mechanism known as copy out—paste in (11). Furthermore, the ends of these IS elements are often preferred target sites for insertion by the same IS (12).
We employed the Geneious R9.1 software suite (Geneious, Auckland, New Zealand) to analyze 77 mcr-1-containing sequences, 19 of which were too fragmented to be useful (see Table S1 in the supplemental material). This represents every unique sequence present in NCBI as of this writing (August 2016). A common feature in all sequences is an ∼2,607-bp DNA segment containing mcr-1 and a putative 765-bp ORF encoding a protein similar to a PAP2 superfamily protein (Fig. 1). In 7 sequences, this segment is flanked by two copies of ISApl1 in the same orientation, reminiscent of a composite transposon (Fig. 2A). In a further two sequences (pSA-MCR-1 and p100R), the downstream ISApl1 is in the reverse orientation. No TSDs flank these IS elements, but an analysis indicates that this is due to the inversion of a large segment of DNA that encompasses the downstream IS (data not shown). In 14 sequences, a single copy of ISApl1 is located upstream of mcr-1 (Fig. 3), while in the remaining 35 no intact copies of ISApl1 are present (see Fig. S1 in the supplemental material). Notably, in many of the structures lacking one or both ISApl1 copies, deletions and mutations at the extremities bestow some minor variability on the length of the mcr-1 DNA segment. It has been previously shown that IS30 family members exhibit a specific target preference for an imperfect palindrome sequence that has a slight GC bias in the center and an AT-rich distal region (8). An alignment of all sequences containing at least one copy of ISApl1 reveals a significant bias for insertion in just such a structure, with a preponderance of thymine residues and adenine residues located directly upstream and downstream of the insertion site, respectively (Fig. 1).
FIG 1
FIG 1 Consensus of ISApl1 genomic insertion sites generated by the Geneious R9.1 software suite from the flanking sequences of all unique mcr-1-containing segments with one or both copies of ISApl1 (n = 10). Sequences without any flanking ISApl1 were omitted to prevent consensus bias. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each nucleic acid at that position. Open arrows represent coding sequences (white arrows, ISApl1; red arrows, mcr-1; gray arrows, putative ORF) and indicate the direction of transcription. Vertical black lines represent ISApl1 IRL and IRR. The conserved dinucleotides abutting all upstream (AT) and downstream (CG) copies of ISApl1 are highlighted.
FIG 2
FIG 2 (A) Alignment of the flanking sequences from seven examples of the putative composite transposons described in this study. Open arrows represent coding sequences (white arrows, ISApl1; red arrows, mcr-1; dark gray arrows, putative ORF; blue, light gray, and green, other ORFs) and indicate the direction of transcription. Vertical black lines represent ISApl1 IRL and IRR. The putative positions of expected target site duplications (TSD) are indicated in bold, with confirmed TSDs underlined. Note that pECJP-59-244 and pS38 share >99% homology, except that the mcr-1 segment is inserted in different locations. Though a putative TSD is present in pS38 (AA), an analysis of this region in pECJP-59-244 indicates that this is not a genuine TSD, as a deletion has occurred removing a large segment of DNA, including part of the downstream ISApl1 (indicated by enclosure in a box). (B) Alignment of pEC2-4 and a homologous region in pB71 lacking the mcr-1 insertion depicting the same dinucleotide (TG) in this location that constitutes the putative TSDs in pEC2-4. (C) Alignment of pECJP-59-244 and the homologous region in pS38 lacking the insertion. The large 4,212-bp deletion may have caused the loss of the downstream TSD (AG) in pECJP-59-244, as indicated.
FIG 3
FIG 3 Depiction of the three distinct variants of the mcr-1 segment with just a single upstream copy of ISApl1. Open arrows represent coding sequences (white arrows, ISApl1; red arrows, mcr-1; gray arrows, putative ORF) and indicate the direction of transcription. Broken arrows in panel B represent 33-bp and 90-bp fragments of ISApl1 (see the text for details). Vertical black lines represent ISApl1 IRL and IRR. (A) Alignment of the nine unique one-ended sequences that lack any remnant of the downstream ISApl1. Deletions and mutations at the 3′ extremity are highlighted in bold. Note that pHNSHP45, pSCS23, pABC149-MCR-1, pAF23, and pA31-12 share >99% homology except for the deletions and mutations at these positions. (B) Alignment of the three one-ended sequences that have either a 33-bp (S51 genome, pVT553) or 90-bp (RL465 genome) remnant of ISApl1, including the 27-bp IRR. Deletions and mutations at the 3′ extremity are highlighted in bold, and the expected positions of TSDs are highlighted in bold green, with confirmed TSDs underlined. The deletion of the downstream TSD in pVT553 is indicated by enclosure in a box.
Of the 7 complete composite transposon sequences analyzed (see Table S1 in the supplemental material), five are flanked by the characteristic 2-bp TSDs of ISApl1 transposition (Fig. 2A). Fortunately, for every example of the composite transposon insertion, there are multiple sequences in NCBI of the corresponding region without the insertion (empty sites). In every example, this region contains the same dinucleotide that forms the TSD (Fig. 2B), as would be expected if mcr-1 were mobilized as a composite transposon. Furthermore, in the two examples where the TSDs are missing (pECJP-59-244 and pS38), a comparison of the sequence with a corresponding region lacking the transposon identified deletions that removed the downstream TSD (Fig. 2C). Taken together, these data provide compelling evidence that the mcr-1 segment is mobilized as part of an ISApl1 composite transposon.
There are 14 examples, involving 4 distinct insertion sites, in NCBI where mcr-1 is delimited by ISApl1 on the 5′ end only (see Table S1 in the supplemental material). However, 2 sequences (PEC2_1-4 and pKP81-BE) were not analyzed further due to a large deletion that removed most of the 3′ end, including the putative ORF. In the remaining 12, we identified 3 variants of the one-ended examples based on differences at the 3′ end. In the first example (Fig. 3A), present in 9 of the 12 one-ended variants, the mcr-1 segment ends where the putative ORF downstream of mcr-1 terminates. There are significant variations in the last 6 bp of this segment, where mutations and deletions have resulted in distinct sequences (Fig. 3A). In the second and third examples, an additional 33 bp and 90 bp, corresponding to the last 33 and 90 bp of ISApl1, respectively, are present immediately downstream of the ORF (Fig. 3B). There are intact 27-bp IRRs of ISApl1 included in these regions. In the latter two structures, 2-bp TSDs flank this second IRR and the IRL of the upstream ISApl1 copy, with the exception of pVT553, where a subsequent deletion removed the downstream TSD (Fig. 3B). Similarly to that observed in the analysis of the composite transposon structures, as shown by comparisons to corresponding empty sites in NCBI, the same dinucleotides as those in the TSD are present. In contrast, only pHNSHP45 has a putative TSD (GA) in the absence of this second IRR. However, an alignment of this region with multiple other homologous sequences deposited in GenBank reveals that this putative TSD is present in some but not others due to a poly(A) tract at this location (data not shown). Hence, it is unclear whether this is a true TSD or a sequencing artifact caused by the poly(A) sequence. Overall, these data indicate that ISApl1-mediated transposition of the mcr-1 segment can also occur when an additional ISApl1 IRR is present downstream of the region without an assembled composite transposon. In contrast, there is scant evidence for the mobilization of mcr-1 in the absence of this additional IRR.
There are 35 mcr-1 segments in NCBI that have no flanking ISApl1, 12 of which represent unique sequences (see Table S1 in the supplemental material). In every unit, the segment is delineated at the 3′ end by the putative ORF, though the same mutations and deletions that were present at the 3′ end in the one-ended structures are also present (see Fig. S1 in the supplemental material). Similarly, in 8 of the 12 sequences, the element is delineated on the 5′ end by a conserved trinucleotide (5′-ATA-3′), a sequence found immediately downstream of the IRR of ISApl1 (in both the composite transposon and single-copy ISApl1 variant cases) (see Fig. S1). In the remaining 4, deletions and mutations in this area have resulted in changes at this position (see Fig. S1). Notably, in one sequence (LVOP01000041), a partial copy of an upstream ISApl1, including the IRR, is still present (see Fig. S1).
In addition to providing a working hypothesis on the mobilization of mcr-1 by ISApl1, our analysis also allowed us to construct a plausible hypothesis on how this structure emerged.
An alignment of all composite transposon sequences revealed a conserved dinucleotide between the 3′ end of the IRR of the upstream ISApl1 and the beginning of the mcr-1 segment (AT) and between the end of the mcr-1 segment and the 5′ end of the IRL of the downstream ISApl1 (CG) (Fig. 1). Furthermore, in every structure with just an upstream copy of ISApl1, the same conserved dinucleotide (AT) is also present between the IRR of the upstream IS and the beginning of the mcr-1 segment (Fig. 1). This suggests that the original composite transposon was formed by two separate insertions of ISApl1 flanking the mcr-1 segment, generating characteristic 2-bp TSDs during the process. Subsequently, as this composite transposon moved, it generated new TSDs at each location, thus masking the original flanking TSDs.
Much of the uncertainty surrounding the movement of mcr-1 has centered on the observation that many sequences lack one or both copies of ISApl1 (13). However, Szabó et al. have shown with IS30 that the loss of one or both IS elements in the composite transposon Tn2706 can contribute to replicon stabilization through both transposition and illegitimate recombination (14). In 37% to 57% of the plasmids analyzed by Szabó and colleagues, IS30 was partially or completely removed by a process that generated mismatches and deletions in the area of excision (14). An analysis of mcr-1 segments having one or no copies of ISApl1 also shows a preponderance of deletions and mutations surrounding the area where ISApl1 would have been located (see Fig. 3). For example, an alignment of 5 plasmids with high homology to pHNSHP45 (GenBank accession number KP347127) reveals that, while the plasmids are almost identical, there is a high concentration of mutations and deletions at the extremity of the mcr-1 segment, precisely at the location where the downstream copy of ISApl1 is located in the composite transposon.
Overall, these data provide compelling evidence that mcr-1 is mobilized primarily as a composite transposon composed of directly orientated copies of ISApl1. In view of the established IS30 family transposition mechanism, it seems probable that the entire composite transposon transposes via a circular intermediate in which the left and right ISApl1 copies abut. Over the course of evolution, this composite transposon has lost one or both copies of ISApl1, most likely through a process of illegitimate recombination. ISApl1 loss may have served to increase the stability of mcr-1 in plasmid vectors, facilitating widespread dissemination of this gene. Functional experiments should further clarify the nature of how mcr-1 transposes and its origins, with significant implications for understanding dissemination of this important resistance gene.

ACKNOWLEDGMENTS

We thank Alessandro Varani for valuable assistance in assembling several short read Miseq data sets into longer contigs. Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation. The views expressed in this article are ours and do not reflect the official policy of the Department of the Army, Department of Defense, or the U.S. Government.

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Published In

cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 60Number 11November 2016
Pages: 6973 - 6976
PubMed: 27620479

History

Received: 9 July 2016
Returned for modification: 28 July 2016
Accepted: 3 September 2016
Published online: 21 October 2016

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Contributors

Authors

Erik Snesrud
Multidrug-resistant Organism Repository and Surveillance Network, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA
Susu He
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
Michael Chandler
Laboratoire de Microbiologie et Genetique Moleculaires, Centre National de la Recherche Scientifique, Toulouse, France
John P. Dekker
Department of Laboratory Medicine, Clinical Center, Microbiology Service, National Institutes of Health, Bethesda, Maryland, USA
Alison B. Hickman
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA
Patrick McGann
Multidrug-resistant Organism Repository and Surveillance Network, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA
Fred Dyda
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

Notes

Address correspondence to Fred Dyda, [email protected].
E.S. and S.H. contributed equally to this article.

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