Insertion sequence ISEcp1B is involved in expression and mobilization of a bla(CTX-M) beta-lactamase gene

Abstract

The genetic structures (ca. 10-kb DNA fragment) surrounding the plasmid-borne extended-spectrum beta-lactamase bla(CTX-M-19) gene in a Klebsiella pneumoniae clinical isolate were determined. This beta-lactamase gene was part of a 4,797-bp transposon inserted inside orf1 of Tn1721. Inside this transposon, bla(CTX-M-19) was bracketed upstream and downstream by insertion sequences ISE cp1B and IS903D, respectively, and further downstream by a truncated gene encoding an outer membrane protein for iron transport. The single-copy ISEcp1B element was probably involved alone in the mobilization process that led to a 5-bp duplication at the target site of the transposed fragment. This mobilization event probably involved one inverted repeat of ISE cp1B and a second sequence farther away, resembling its second inverted repeat. Additionally, ISEcp1B provided -35 and -10 promoter sequences, contributing to the high-level expression of the bla(CTX-M-19) gene. Southern blot analysis failed to identify a reservoir of ISEcp1-like sequences among a series of gram-negative and gram-positive bacterial species usually found in the skin and intestinal human floras. The ability of ISEcp1-like elements to mobilize and to promote the expression of beta-lactamase genes may explain, in part, the current spread of CTX-M-type enzymes worldwide.

FIG. 1.

Schematic map of a 9,590-bp DNA fragment of the natural plasmid pILT-3 from K. pneumoniae ILT-3 containing ISEcp1B, IS903D, and the bla_CTX-M-19 gene. The 5′ and 3′ conserved sequences (5′-CS and 3′-CS) of class 1 integrons are indicated in the upper left part of the figure. ORFs and genes are shown as boxes with an arrow indicating the transcription orientation. Black dots are for 59-be sequences. IRL and IRR motifs are indicated by vertical arrows. The IRL and IRR of the ISEcp1B-mobilized DNA fragment are boxed, those of the ISEcp1B element are italicized, and those of IS903D are in lowercase; the inverted repeat left of Tn1721 (Irl) is also shown. The cloned sequences of recombinant plasmids are indicated by arrows at both ends, with the corresponding plasmid names indicated on the right.

FIG. 2.

Nucleotide sequence of a 9,590-bp DNA fragment of the natural pILT-3 plasmid of K. pneumoniae ILT-3 containing ISEcp1B, β-lactamase CTX-M-19, and IS903D coding sequences. The deduced amino acid sequence is indicated in single-letter code below the nucleotide sequence. The start codons of the ORFs are indicated by horizontal arrows, and stop codons are indicated by asterisks. The −35 and −10 promoter sequences of the P_c, P_int, and P_a promoters are underlined, as well as the +1 position of the transcriptional start of the bla_CTX-M-19 site. The 5-bp duplicated target sites of the putative insertion site for the DNA fragment resulting from an ISEcp1B-mediated transposition process are doubly underlined. RBS indicates the putative ribosome binding site for the cmlA1-like gene, and the 9-amino-acid leader peptide sequence of the variant of cmlA1 cassette is indicated. Core and inverse core sites located at each cassette boundary are boxed. TnpA, transposase gene; iron, gene encoding a putative outer membrane lipoprotein for iron uptake; and Orf1, the gene encoding the putative methyl-accepting chemotaxis protein of Tn1721.

FIG. 2.

Nucleotide sequence of a 9,590-bp DNA fragment of the natural pILT-3 plasmid of K. pneumoniae ILT-3 containing ISEcp1B, β-lactamase CTX-M-19, and IS903D coding sequences. The deduced amino acid sequence is indicated in single-letter code below the nucleotide sequence. The start codons of the ORFs are indicated by horizontal arrows, and stop codons are indicated by asterisks. The −35 and −10 promoter sequences of the P_c, P_int, and P_a promoters are underlined, as well as the +1 position of the transcriptional start of the bla_CTX-M-19 site. The 5-bp duplicated target sites of the putative insertion site for the DNA fragment resulting from an ISEcp1B-mediated transposition process are doubly underlined. RBS indicates the putative ribosome binding site for the cmlA1-like gene, and the 9-amino-acid leader peptide sequence of the variant of cmlA1 cassette is indicated. Core and inverse core sites located at each cassette boundary are boxed. TnpA, transposase gene; iron, gene encoding a putative outer membrane lipoprotein for iron uptake; and Orf1, the gene encoding the putative methyl-accepting chemotaxis protein of Tn1721.