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.

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

(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

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.