Functional characterization of Tn4401, a Tn3-based transposon involved in blaKPC gene mobilization

Abstract

The carbapenemase gene bla(KPC), which is rapidly spreading worldwide, is located on a Tn3-based transposon, Tn4401. In a transposition-conjugation assay, Tn4401 was able to mobilize bla(KPC-2) gene at a frequency of 4.4 × 10(-6)/recipient cell. A 5-bp target site duplication was evidenced upon each insertion without target site specificity. This study demonstrated that Tn4401 is an active transposon capable of mobilizing bla(KPC) genes at high frequency.

Fig. 1.

Schematic representation of Tn4401a inserted into pOX38-Gen (insertion I1 in Fig. 3). Genes and their corresponding transcription orientations are indicated by horizontal arrows. Tn4401a (isoform a [100-bp deletion as described in reference 13]) is delimited by two inverted repeat sequences, IRR and IRL (grey triangles). Small open triangles represent the inverted repeats of ISKpn6 and ISKpn7. Small arrows below the sequence represent primers used for direct sequencing of the insertion sites of Tn4401. The thick line corresponds to the pOX38-GEN DNA sequence.

Fig. 2.

Plasmid extraction by the Kieser method. (A) transposition experiments performed with the natural plasmid pBC633. Lanes 1 to 3, transconjugants in E. coli J53 Az^r after a transposition assay. E. coli J53 transconjugant 1 (lane 1) possesses both pOX38Gen and pBC633, due to cointegration events, whereas E. coli J53 transconjugants 2 and 3 (lanes 2 and 3) harbor only pOX38Gen::Tn4401, due to true transposition events. Lane 4, E. coli RZ211 harboring pOX38Gen; lane 5, K. pneumoniae KPN633 harboring the natural plasmid pBC633 (11); lane 6, E. coli RZ211 harboring both pOX38Gen and pBC633; lane 7, E. coli J53 Az^r (only the chromosomal band [Chr] is visible); lane 8, E. coli 50192 harboring four plasmids (7, 48, 66, and 154 kb). (B) Transposition experiments performed with the recombinant plasmid pTn4401. Lane 1, E. coli 50192 harboring four plasmids (7, 48, 66, and 154 kb); lane 2, E. coli RZ211 harboring pOX38Gen; lane 3, E. coli DH10B harboring pTn4401; lane 4, E. coli J53 transconjugant possessing both plasmids pOX38Gen and pTn4401 due to a cointegration event; lanes 5 to 8, E. coli J53 transconjugants harboring pOX38Gen::Tn4401, selected as Cm^s colonies; lane 9, E. coli RZ211 harboring both pOX38Gen and pTn4401 (Cm^r); lane 10, subsequent transconjugant from strain 9 to E. coli DH10B Str^r.

Fig. 3.

Target sites of Tn4401 insertions. (A) Map positions of Tn4401 insertions in plasmid pOX38-Gen. Insertions of the tagged transposon (I1 to I7) are indicated by vertical arrows. The origin of replication (oriV), the origin of transfer (oriT), the tra genes required for plasmid transfer, and the gentamicin resistance gene (Gen^r) are indicated. (B and C) Nucleotide sequence alignments of Tn4401 in pOX38-Gen. (B) Seven transconjugants were analyzed from a transposition assay with pBC633 or pTn4401. (C) Tn4401 insertions in natural plasmids. 1, pCOL from P. aeruginosa 2404 (13); 2, pBC2303 from K. pneumoniae KPN2303 (13); 3, pBC633 from K. pneumoniae KPN633 (13); 4, pYC from K. pneumoniae YC (13). Nucleotide sequences of the end regions of Tn4401 are boxed. Target site sequences duplicated after transposition are indicated by boldface letters. The small arrows above the schematic representation of Tn4401 represent primers used for direct sequencing of pOX38Gen::Tn4401 transposants.