Formation of SXT Tandem Arrays and SXT-R391 Hybrids

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   FIG. 1.

   Tandem arrays of SXTs. (A) Southern blot analysis of five pairs of Tet^r Sxt^r exconjugants resulting from five independent mating experiments between E. coli MG1655 prfC::SXT and E. coli CAG18439. The donor and recipient strains were mated for 6 h at 37°C on LB agar medium without selection. The purified total genomic DNAs of the Tet^r Sxt^r exconjugants were digested with EcoRI/EcoRV and analyzed by Southern blot hybridization with the 600-bp BstBI fragment of pVI8A containing attP from SXT as a probe. The molecular sizes in kilobases correspond to the fragments containing the attL (6.5 kb, EcoRV), attP (4.0 kb, EcoRI/EcoRV), and attR (1.2 kb, EcoRI/EcoRV) junction fragments. (B) Contour-clamped homogenous PFGE of SalI or SpeI restriction patterns of E. coli CAG18439 (Sxt^s), HW220 (Sxt^r), VI186 (Sxt^r), and VI218 (Sxt^r). The gel electrophoresis was carried out for 30 h at 5 V/cm. The size marker (Marker) was SmaI-digested chromosomal DNA of Staphylococcus aureus NCTC8325. The molecular sizes indicated to the right of the panel are in kilobases. The numbers indicated to the left of the bands on the HW220, VI186, and VI218 restriction patterns correspond to the copy numbers of SXT in each fragment. The size of the fragments 1 to 5+ observed in the VI186 restriction pattern are, respectively, 125, 224.5, 324, 424.4, and 522.9 kb or more for SalI, and 165.9, 265.4, 364.8, 464.3, and 563.8 kb or more for SpeI. (C) Southern blot analysis of 10 Kan^r Sxt^r exconjugants (VI336 to VI345) resulting from a mating between V. cholerae MO10 and V. cholerae E4. E. coli VI186 was used as a control. The fragments containing the V. cholerae attL and attR are shown.
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   FIG. 2.

   Tandem arrays in recA and ΔprfC mutant strains of E. coli. Southern blot analyses of Tet^r Sxt^r exconjugants resulting from mating experiments involving a recA⁺ ΔprfC strain (VI31) (A) or a recA ΔprfC strain (VI47) (B) as the recipient. E. coli VI166 (BW25113 prfC::SXT) was used as a donor strain in both experiments. The donor and recipient strains were mated for 6 h at 37°C on LB agar medium without selection. The purified total genomic DNAs of the Tet^r Sxt^r exconjugants were digested with EcoRI/EcoRV and analyzed by Southern blot hybridization with the 600-bp BstBI fragment of pVI8A containing attP from SXT as a probe. The molecular sizes in kilobases correspond to the fragments containing the attL (6.5 kb, EcoRV), attP (4.0 kb, EcoRI/EcoRV), and attR (1.2 kb, EcoRI/EcoRV) junction fragments in E. coli HW220 (CAG18439 prfC::SXT) used as a control strain.
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   FIG. 3.

   Effect of the tandem arrays on the transfer frequency of SXT Δxis. The frequencies of exconjugant formation per donor were obtained by dividing the number of exconjugants (Nal^r Sxt^r CFU) by the number of donor cells (Tc^r CFU). In all of the mating experiments, the recipient strain was E. coli BI533 (MG1655 Nal^r). The donors were E. coli exconjugants obtained from mating VI200 (MG1655 prfC::SXT Δxis pXis) × CAG18439 (□) or from mating VI166 (BW25113 prfC::SXT) × CAG18439 (▪). The expression of xis by pXis in the donor strain VI200 was induced with 0.02% arabinose during the mating experiments on LB agar plates. Real-time quantitative PCR was used to determine the percentage of attP sequences resulting from SXT excision and tandem SXT array formation. Triplicate measurements were performed on each sample, and the means and standard deviations (horizontal bars) are presented for each assay. DNA templates were prepared from overnight LB broth cultures.
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   FIG. 4.

   Analysis of hybrids formed by recombination between SXT and R391. (A) Schematic representation of the structures of the tandem arrays SXT-R391 and R391-SXT and predicted hybrid ICEs formed by the oriT-to-oriT replication-based mechanism. (B) Schematic representation of the structures of SXT and R391. mer, aph, sulII, and dfr18 encode, respectively, resistances to mercury, kanamycin, sulfamethoxazole, and trimethoprim. The asterisks indicate the positions of the oriT in SXT and the probable oriT in R391. (C) Amplification of sequences specific to SXT and R391. The fragments A (452 bp) and C (449 bp) are specifically amplified from the left part of SXT and R391, respectively, with the primer sets VISLF-VISLR3 and VISLF-VISLR2 (Table 2). The fragments B (509 bp) and D (511 bp) are specifically amplified from the right part of SXT and R391, respectively, with the primer sets VISRF-VIRR and VISRF-VISRR2. mer is a 1,371-bp fragment amplified from R391 withMER104A and MER103B. It covers the middle of the mer operon of R391. The fragments s037 (523 bp) and s052 (242 bp) are specific to SXT and were amplified with the primer sets 10SF13-SXT1-13 and YND2-ORF16, respectively. The sizes of the ladder (L) are given in kilobases. (D) Schematic representation of the structures of the isolated hybrid ICEs. The structure of the hybrids was deduced from the antibiotic resistance phenotype and from the pattern of SXT- and R391-specific fragments detected by PCR, as shown in panel A. The positions of the amplified fragments and the genes encoding the antibiotic resistance are indicated. The precise position of the point of recombination in each hybrid is not known, but it is deduced from the presence or absence of the markers tested. The gradient from white to dark gray indicates the fragment within which the recombination probably took place to generate the hybrids. Δ, Δxis mutation.

• American Society for Microbiology