Molecular analysis of the sequences surrounding blaOXA-45 reveals acquisition of this gene by Pseudomonas aeruginosa via a novel ISCR element, ISCR5

Abstract

The bla(OXA-45) gene of Pseudomonas aeruginosa 07-406 is driven by a promoter found within a truncated ISPme1 insertion sequence. The gene is located between nonidentical repeats of a new ISCR element, ISCR5, which is likely responsible for its acquisition. Sequence analysis indicates that ISCR5 is a chimera of ISCR3 and ISCR16.

FIG. 1.

Schematic of the genetic locus of bla_OXA-45. ORFs are depicted as boxes with arrows indicating the direction of transcription of the ORF. The truncated ISPme1 element is depicted as a checkered box indicating the truncated ORF together with vertical parallel lines representing the right-hand end inverted repeat sequence. The promoter driving the transcription of bla_OXA-45 is drawn as a bent horizontal arrow. Solid black boxes indicate the repeated 376-bp section of DNA found upstream of both ISCR5 elements. The oriIS of ISCR5A and ISCR5B are depicted as dotted vertical lines found downstream of the ISCR5 ORFs.

FIG. 2.

Alignment of transposases of ISCR elements displaying the highest identity to ISCR5A and ISCR5B. Residues found in the majority of sequences are shown on a gray background. The residues that are found conserved in all IS91 family transposases are indicated by black stars above the sequences. Two key residues that differ from those in the transposases of the IS91 family are indicated by open circles above the sequences. The first residue, Q85 in the ISCR5 sequence, is a histidine residue in all IS91 family elements, and the lysine residue K333 (ISCR5) is a tyrosine in all IS91 family elements and an arginine in ISCR1, ISCR2, and ISCR4. (see reference for a discussion of these differences). Gaps introduced to maximize alignment of the sequences are indicated by the dashes.

FIG. 3.

(A) Alignment of the 5′ DNA sequences of ISCR elements displaying the highest identity to ISCR5. Nucleotides that are identical to those in the sequence of ISCR3 are shown on a gray background. The GTG start codons of the various ISCR element transposase genes are indicated by asterisks, and nucleotides are numbered upstream starting at this codon. Sequences were collected from the following data banks and accession numbers: GenBank AF261825 (ISCR3), CP000604 (ISCR16), GenBank AY341249 (ISCR4), and AM849110 (ISCR5B [this study]). The ISCR5 5′-terminal sequence displays 100% identity to the respective sequence from ISCR3 until 98 bp upstream of the start codon where identity to ISCR3 and other elements is abruptly lost. The same sequence displays 86% identity to the equivalent sequence from ISCR16. A short 4-bp inverted repeat sequence at this position is indicated by inverted arrows above the nucleotide sequence which is similar to that found in the terIS of IS91. (B) Alignment of the 3′-terminal DNA sequences of ISCR elements displaying the highest identity to ISCR5. Nucleotides that are identical to those in the sequence of ISCR5 are shown on a gray background. The stop codons of ISCR5, ISCR16, and ISCR3 transposase genes are indicated by black stars, and the oriIS sequence found at the extreme terminus of the various ISCR elements is boxed. The vertical arrow indicates the junction between the ISCR elements and carrier DNA. The ISCR5 3′-terminal sequence displays the highest identity (98%) to ISCR16 but only 80% identity to the respective sequence from ISCR3.

FIG. 4.

(A) Pulsed-field gel of genomic DNA from P. aeruginosa strains 07-406 and 07-408. Lane 1, lambda ladder pulsed-field gel markers; lanes 2, 4, and 6, strain 07-406 DNA digested with SpeI, I-Ceu1, and S1, respectively; lanes 3, 5, and 7, strain 07-408 DNA digested with SpeI, I-Ceu1, and S1, respectively. (B) Autoradiograph of the gel shown in panel A probed with bla_OXA-45.