Integrase-mediated recombination of the veb1 gene cassette encoding an extended-spectrum β-lactamase

Abstract

The veb1 gene cassette encodes the extended spectrum β-lactamase, VEB-1 that is increasingly isolated from worldwide Gram-negative rods. Veb1 is commonly inserted into the variable region of different class 1 integrons in which it is always associated with a downstream-located aadB gene cassette encoding an aminoglycoside adenylyltransferase. In Pseudomonas aeruginosa, the majority of veb1-containing integrons also carry an insertion sequence, IS1999 that is inserted upstream of the veb1 gene cassette and disrupts the integron specific recombination site, attI1. Investigation of the recombination properties of the sites surrounding veb1 revealed that insertion of IS1999 reduces significantly the recombination frequency of attI1 and that veb1 attC is not efficient for recombination in contrast to aadB attC. Subsequent sequence optimisation of veb1 attC by mutagenesis, into a more consensual attC site resembling aadB attC, successfully improved recombination efficiency. Overall, this work gives some insights into the organisation of veb1-containing integrons. We propose that IS1999 and the nature of veb1 attC stabilize the veb1 gene cassette environment likely by impairing recombination events upstream or downstream of veb1, respectively.

Figure 1. Schematic representations of the veb1-containing integron from P. aeruginosa 14 and of the recombination sites

. (A) veb1-containing integron from P. aeruginosa 14. The 5′ and the 3′-CSs are underlined. ORFs are shown as boxes with an arrow indicating the orientation of the coding sequence. The promoter P_c is indicated by a broken arrow. The black diamond represents attI1 and circles represent attC sites. (B–D) Recombination sites. Sequences related to the 7-bp core site are boxed; their relative orientations are indicated with arrows. The crossover points are marked by vertical arrows. The region derived from the downstream-located cassette at the recombination point is in lower case. The extra-helical bases (EHBs) as defined by Bouvier et al. are marked with an asterisk. (B) attI1: the nucleotides belonging to attI1 are indicated in white on a black background. The experimentally determined strong and weak IntI1-binding sites and the pair of direct repeats, DR1 and DR2, as well as the location of the IS1999 insertion in attI1 are indicated. (C) veb1 attC and (D) aadB attC: 7-bp putative core sites (1L, 2L, 2R and 1R) related to the core site consensus as defined by Stokes et al. are shown. The aadB gene cassette is boxed in grey.

Figure 2. Schematic representation of the veb1-containing integron from P. aeruginosa 15 and of the disrupted attI1 recombination site .

(A) veb1-containing integron from P. aeruginosa 15. ORFs are shown as boxes with an arrow indicating the orientation of the coding sequence. Promoters P_c and P_out are indicated by broken arrows. Disruption of attI1 by IS1999 is represented by a split black diamond; circles represent attC sites. The inverted repeats of IS1999 are shown as empty triangles. (B) Sequence of the disrupted attI1 site (ΔattI1). The nucleotides belonging to IS1999 and attI1 are indicated in grey and in white on a black background, respectively. Sequences related to the 7-bp core site are boxed; their relative orientations are indicated with arrows. The crossover point is marked by vertical arrow. The region derived from the downstream-located cassette (veb-1) at the recombination point is in lower case. The 7-bp from IS1999 replacing the fourth integrase binding site from attI1 are shown in a dashed box.

Figure 3. Bla _VEB-1-specific hybridization and PCR amplifications of veb1-containing fragments.

(A) Bla _VEB-1-specific hybridization of small BspEI-digested DNA fragments from E. coli DH10B (p112.Kan) harboring pAttI.veb (1), pAttI.veb.aadB (2), pAttI.IS.veb (3), pAttI.IS.veb.aadB (4), pAttI.veb* (5), pAttI.veb*.aadB (6), pVeb (7), pVeb.aadB (8) and from E. coli DH10B (pTRC99A.Kan) harboring pAttI.veb.aadB (9) Locations of the low (L) 1.1-kb and high (H) 1.6-kb signals are indicated. (B) PCR amplifications of veb1-containing fragments using the outward directed primers VEBINV3-VEBINV2 and ca. 1.1-kb (L) and 1.6-kb (H) DNAs that were gel-extracted based on (A), as template.

Figure 4. Recombinant junction analysis.

(A) Representation of the pAttI.veb.aadB recombinant plasmid. The location of the different crossover points involved in recombination are indicated by a number: (1) 7-bp core site of attI1-veb1; (1α) secondary recombination site found within the bla _VEB-1 coding sequence; (2), (2*) and (3) 7-bp site found at the veb1 attC-aadB, veb1 attC*-aadB and aadB attC-qacEΔ1 junctions, respectively. (B) Sequencing results of the recombinant junctions from experimentally isolated veb1-containing recombination products. The numbers shown on the left indicate the recombination sites that were involved to create the recombinant junctions. Sequences related to the 7-bp core site are boxed. The crossover point is marked by vertical arrow. The nucleotides belonging to the aadB gene cassette are boxed in grey.

Figure 5. Bottom strand secondary structures.

(A) consensus attC structure as of Cambray et al. ; (B) aadB attC; (C) veb1 attC; (D) veb1 attC*; (E) veb1 attC^Δ; and (F) veb1 attC'. The putative IntI1 binding domains are marked with boxes and the inverted repeats 1L-1R and 2L-2R are indicated. The protruding G that determines the recombination strand present in 2L and the protruding T that increases the recombination efficiency, are also boxed. CT, conserved triplet; UCS, unpaired central spacer; EHB, extrahelical base; VTS, variable terminal structure. Folded representations were based on secondary structures obtained thanks to the mfold Web Server. The folded structure of the veb1 attC* site was similar to the folded structure of aadB attC and possessed a free energy of −30.3 Kcal/mol.

Figure 6. veb1-containing recombination products depending on veb1 attC variants.

of veb1-containing fragments. (A) Bla _VEB-1-specific hybridization of small BspEI-digested DNA fragments from E. coli DH10B (p112.Kan) harboring pAttI.veb' (1), pAttI1.veb^Δ (2), pAttI.veb (3), pAttI.veb* (4), and from E. coli DH10B (pTRC99A.Kan) harboring pAttI.veb (5). (B) PCR amplifications of veb1-containing fragments using the outward directed primers VEBINV3-VEBINV2 primers and the ca. 1.1 kb DNAs that were gel extracted from (A) as template.