Mobile antibiotic resistance encoding elements promote their own diversity

Abstract

Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter-ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage lambda Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.

Figure 1. Evidence suggesting that recombination occurs between SXT/R391 ICEs.

(A) The middle gray box represents the set of genes (arrows) conserved in the 4 SXT/R391 genomes shown. Gray ORFs represent genes of unknown function, white ORFs represent genes of known function ,,, and black ORFs correspond to s065 and s066. Below, variable ICE regions are shown with colors according to the elements in which they were originally described: SXT (blue), R391 (red), ICEPdaSpa1 (green), and ICESpuPO1 (purple). (B) A close-up of the attL-s025 region of ICEPdaSpa1 (accession number AJ870986) is shown in the upper left. The variation of percentage of identity was plotted using a Multi-LAGAN pairwise comparison of this ICEPdaSpa1 region with the corresponding regions of SXT (accession number AY055428) and R391 (accession number AY090559) and the mVista visualization module with a sliding window of 100 bp. The minimum width and the minimum percent conservation identity that must be maintained over that width for a region to be considered conserved were set at 100 bp and 70% respectively. The dark gray area highlights the large nearly identical region conserved between SXT and ICEPdaSpa1. (C) A comparison of s065 and s066, which are present in all SXT/R391 ICEs, to the bacteriophage λ Red genes (numbers represent % similarity between S065 and Bet, and S066 and Exo, respectively) is shown in the upper right. drf18 encodes trimethoprim resistance; floR encodes chloramphenicol resistance; strAB encodes streptomycin resistance; sulII encodes sulfamethoxazole resistance; tetAR encodes tetracycline resistance; aph encodes kanamycin resistance; and mer encodes mercury resistance.

Figure 2. Schematic of colony color-based semi-quantitative assay for the detection of hybrid ICE–containing colonies.

Relative positions of resistance markers (trimethoprim (Tm), sulfamethoxazole (Su), kanamycin (Kn)) and phenotypic markers (lacZ and galK) in SXT and R391 are indicated. DNA originating from SXT is shown in blue and DNA originating from R391 is shown in red. The use of a ΔgalK lacZ ⁻ lacI::Tn10 recipient strain allows constitutive expression of the inserted lacZ and galK from the ICEs in the exconjugant colonies. Mating between a donor cell (green) containing an SXT-R391 tandem array and a recipient cell (orange) yields exconjugants that may contain a single element, a hybrid element or a tandem array. MacConkey X-gal D-galactose indicator agar containing trimethoprim, sulfamethoxazole and tetracycline (bottom panel) reveals colonies harboring single parental ICEs (blue colonies), hybrid ICEs (red colonies), and SXT-R391 tandem arrays (purple colonies). Purple colonies may also consist of cells containing an array composed of SXT and a hybrid element on this media (e.g. exconjugant 4). Red and purple colonies are larger on this medium because they can use D-galactose as a carbon source. (A,B) SXT left and right extremities; (C,D) R391 left and right extremities, respectively amplified by primer pairs VISLF/VISLR3, VISRF/VISRR, VISLF/VISLR2, VISRF/VISRR2 .

Figure 3. Involvement of recA, s065, and s066 in the formation of hybrid ICEs.

recA⁺ (A) or recA (panel B) donor strains, which contained either wild-type (WT), Δs065, Δs066, or Δ(s065-s065) SXT-R391 tandem arrays, were used as donors in these assays. The recipient strains were either E. coli VB38 (recA⁺) or E. coli VB47 (recA⁻). D/R + and – indicate the recA genotype of the donor and recipient strains, respectively. SetDC was expressed from a plasmid when recA donors were used. Bars represent the percentage of exconjugants containing hybrid ICEs and were calculated by dividing the number of exconjugants containing hybrid ICEs (red Tc^R Su^R Tm^R CFU) by the total number of exconjugants (Tc^R Su^R Tm^R CFU). The means and standard deviations obtained from at least three independent assays are shown and the number of colonies containing a hybrid ICE counted for each assay is presented in Table S2. Note the differences in the scale of the y-axis in panels A and B. One-way ANOVA with a Tukey-Kramer post-test was used to compare the means of hybrid ICE-containing exconjugant colonies. The confidence interval for the comparisons of mutant tandem arrays relatively to WT tandem arrays was P<0.001, except □ which indicates P<0.05 and • which indicates that the difference was statistically not significant. * indicates that the percentage of exconjugants bearing a hybrid ICE was below the limit of detection (<0.01%).