In Vitro Study of IS Apl1-Mediated Mobilization of the Colistin Resistance Gene mcr-1

Abstract

The plasmid-mediated mcr-1 gene encodes a phosphoethanolamine transferase that confers resistance to polymyxins. The mcr-1 gene is associated with insertion sequence ISApl1 (IS30 family). In vitro mobilization assays demonstrated the functionality of the composite transposon structure ISApl1-mcr-1-ISApl1 Transposition generated a 2-bp duplication and occurred in AT-rich DNA regions. This is the first report demonstrating the mobility of the mcr-1 gene by transposition.

Keywords: ISApl1; composite transposon; mcr-1; plasmid; polymyxins; transposition.

FIG 1

Schematic map of the different constructs performed for the transposition study. (A) corresponds to the original transposon ISApl1-mcr-1-orf-ISApl1 identified in clinical isolates; (B) shows the different fragments generated by PCR (with corresponding restriction sites indicated) and used as templates for ligation and subsequent genesis of (C) ISApl1-mcr-1–bla_TEM-1-orf-ISApl1 or (D) ISApl1-mcr-1–bla_TEM-1-orf genetic structures. Locations of primers used for the inverse PCR strategy (as listed in Table 2) are indicated by small half arrows. Restriction sites of endonucleases used for cloning are indicated (NheI, SacI, EcoRI, and BamHI).

FIG 2

Target sites of the ISApl1-mcr-1–bla_TEM-1-orf-ISApl1 composite transposon. (A) Map positions of ISApl1-mcr-1–bla_TEM-1-orf-ISApl1 composite transposon in plasmid pOX38-Gen. Insertions of the tagged insertion sequence (Ins-1 to −4) are indicated by a vertical arrow. (B) Nucleotide sequence alignment of the three ISApl1-mcr-1–bla_TEM-1-orf-ISApl1 transposon events identified into pOX38-Gen. Nucleotide sequences of the end regions of transposon are boxed. Boldfaced letters indicate target site sequences duplicated upon transposition. Orientations of the insertion sequences are indicated by (+) and (−).