ISAba825, a functional insertion sequence modulating genomic plasticity and bla(OXA-58) expression in Acinetobacter baumannii

Abstract

ISAba825, an insertion sequence found inactivating Acinetobacter baumannii carO, was tagged with a kanamycin (Kn) resistance cassette. ISAba825::Kn effectively transposed in A. baumannii, showing preference for short, AT-enriched target sequences, generating 6- to 9-bp target duplications. Additionally, we detected the presence of ISAba825 upstream of a plasmid-borne bla(OXA-58) gene, generating a hybrid promoter largely enhancing its expression and leading to carbapenem resistance. Overall, a role for ISAba825 in carbapenem resistance modulation in A. baumannii is proposed.

FIG. 1.

(A) Construction of ISAba825::Kn. First, we generated an EcoRV site immediately downstream of the transposase (Tpase) gene and upstream of the right inverted repeat (IRR) of ISAba825 by PCR using primers 825TF and 825TR (Table 1). As the Tpase stop codon TAA is within the IRR, we introduced another TAA codon upstream of the EcoRV site to maintain both the original transposase sequence and the IRR structure. Afterwards, a kanamycin resistance cassette (Kn^R) bounded by two EcoRV sites was generated by PCR using primers KnEcoRVF and KnEcoRVR (Table 1), employing plasmid pBBR1MCS5 (10) as a template, and was inserted into the equivalent site present in ISAba825-EcoRV. The single TAA site in ISAba825 and the two TAA sites in ISAba825::Kn are underlined. The Tpase gene is boxed and the direction of transcription indicated by an arrow. The IRL and IRR are also underlined. The scheme is not drawn to scale. (B) Target sites of ISAba825::Kn insertions in linearized representations of ATCC 17978 endogenous plasmids pAB1 (GenBank accession number NC_009083) and pAB2 (GenBank accession number NC_009084). Genes and their corresponding directions of transcription are indicated by horizontal arrows. The locus tag of each gene is indicated in parentheses below the gene description. Dark arrowheads indicate ISAba825::Kn insertions at the corresponding nucleotide positions in the plasmids. Arrowheads above and below lines indicate IS insertions in opposite orientations. The scheme is not drawn to scale. (C) ISAba825::Kn and ISAba825 target sequences. Target site duplications (bold letters) and flanking regions of ISAba825::Kn insertions in pAB1 and pAB2 and ISAba825 insertions in carO and tnpA are shown. IR regions are boxed, and arrows indicate the Tpase transcription direction.

FIG. 2.

ISAba825 insertion generated a hybrid promoter driving bla_OXA-58 overexpression in the clinical A. baumannii strain Ab880. (A) Schematic representation of the genetic structure resulting from ISAba825 insertion within the ISAba3-like element located 5′ upstream of the bla_OXA-58 gene present in plasmid pAb880. Genes and their corresponding transcription orientations are indicated by horizontal arrows. The boundaries of the sequenced fragment are indicated by vertical bars at the edges. The figure is not drawn to scale. (B) Promoter regions for bla_OXA-58 in the arrangement described above. The −35 and −10 motifs inferred for each of the different promoters are boxed, and the transcription initiation site (G in bold) resulting from the hybrid promoter (as determined by 5′ RACE-PCR) is indicated by +1. Promoter prediction was done using BPROM (SoftBerry). The different ATG codons for bla_OXA-58 and tnpA are indicated in bold, and the corresponding directions of transcription are shown by arrows. The inverted repeats of the different IS elements are shaded gray. cont., continued.