Functional characterization of IS1999, an IS4 family element involved in mobilization and expression of beta-lactam resistance genes

Abstract

IS1999 and a point mutant derivative, IS1999.2, have been described inserted upstream of emerging antibiotic resistance genes bla(VEB-1) and bla(OXA-48). 5' Rapid amplification of cDNA ends experiments revealed that expression of these beta-lactamase genes was driven by the outward-directed promoter, P(out), located in the IS1999 elements. These findings led us to study IS1999-mediated gene mobilization. Thus, the transposition properties of IS1999 and of IS1999-based composite transposons, made of two copies of IS1999 in different orientations, were investigated. IS1999 or IS1999-based composite transposons were capable of transposing onto the conjugative plasmid pOX38-Gen. Sequence analysis of the insertion sites revealed that IS1999 inserted preferentially into DNA targets containing the consensus sequence NGCNNNGCN. Transposition was more efficient when at least one left inverted repeat end was located at an outside end of the transposon. The transposition frequency of IS1999.2 was 10-fold lower than that of IS1999, and transposition frequencies of the putative natural transposon, Tn1999, were below detection limits of our transposition assay. This reduced transposition frequency of IS1999.2-based elements may result from a lower transcription of the transposase gene, as revealed by reverse transcription-PCR analyses.

FIG. 1.

Schematic map of the constructs used in this study. All constructs were cloned into the multiple cloning site of the pBBR1MCS.3 shuttle vector. Restriction sites that were used for cloning are indicated. The coding regions are shown as boxes, with an arrow indicating the orientation of their transcription. The broken arrows indicate the P_lac promoter. The IS1999 left and right inverted repeats are shown by empty and filled triangles, respectively. IS1999 and iso-IS1999 elements are represented as dark gray boxes and light gray boxes, respectively. Ω Kan^r is an omega fragment conferring resistance to kanamycin. The environments of IS1999 in P. aeruginosa 1 and of Tn1999 on the plasmid pA-1 are shown by dark and gray lines, respectively. Dashed lines represent the cloning vector. The 9-bp sequences located on each side of IS1999.2-left and of IS1999.2-right are indicated on the Tn1999 representation. The five nucleotide substitutions in the IS1999.2 sequence are symbolized with five stars. The position of the primers IS1999B2INV (1) and IS1999IRRext2 (2), used in inverse PCR experiments, are symbolized by small arrows on the TnLL representation. The location of the P_out promoter responsible for the bla_OXA-48 transcription is represented by a small broken arrow on the Tn1999 representation.

FIG. 2.

Target sites of IS1999 insertions. (A) Map positions of IS1999.Kan and TnLL insertions in plasmid pOX38-Gen. Insertions of the tagged insertion sequence (I1 to I11) and of the composite transposons (T1 and T2) are indicated by a vertical arrow. 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 on the pOX38-Gen representation. Nucleotide sequence alignment of the 11 pOX38-Gen::IS1999.Kan transconjugants (B), of the P. aeruginosa 1 attI1::IS1999 junctions (C), of two representative pOX38-Gen::TnLL insertions (D), and of the Tn1999 environment (E) are shown. Nucleotide sequences of the end regions of IS1999.Kan, IS1999, TnLL, and Tn1999 are boxed. Target site sequences duplicated after transposition are indicated by boldface letters. Gray boxes indicate conserved nucleotides in the environment of IS1999.Kan and IS1999 insertions. Orientation of the insertion sequences of IS1999.Kan and TnLL in plasmid pOX38-Gen are indicated by pluses and minuses.

FIG. 3.

Hybridizations of EcoRV-digested whole-cell DNAs of E. coli DH10B (pA-1) (lane 1), K. pneumoniae 11978 (lane 2), E. coli DH10B (lane 3), and P. aeruginosa 1 (lane 4). (A) bla_OXA-48-specific hybridization. (B) IS1999-specific hybridization.

FIG. 4.

Nucleotide sequence of IS1999. The deduced amino acid sequence is designated in a single-letter code below the nucleotide sequence. The start and stop codons of the transposase gene are underlined. The transposase gene name, followed by an arrow indicating its translational orientation, is indicated above the initiation codon. IS1999 left and right inverted repeats (IRL and IRR) are boldfaced. The boxes represent the putative IHF binding site (bp 30 to 42). The complementary inverted repeat sequences are shown by gray boxes. The promoter sequences −10 and −35 of P_out and P_in promoters are double underlined and indicated above the sequence (GenBank accession no. AF133697). Nucleotide changes relative to IS1999.2 are indicated above each nucleotide and are boxed.

FIG. 5.

A. Nucleotide sequence alignments of the promoter regions of IS1999, IS1999.2, and IS10. The start codon of the transposase gene is boldfaced. The boxes represent the conserved boxes of P_out and P_in. The complementary inverted repeated sequences are shown by gray boxes. The promoter sequences −10 and −35 of P_out and P_in promoters are boxed and indicated above the sequence. B. RT-PCR results. M, molecular size marker (100 bp; Invitrogen). Lane 1, no RNA (negative control); lane 2, RNA from IS1999.2-containing cells; lane 3, RNA from IS1999-containing cells. Panel a represents IS1999-specific primers, and panel b represents the gap-specific primers.