The CI repressors of Shiga toxin-converting prophages are involved in coinfection of Escherichia coli strains, which causes a down regulation in the production of Shiga toxin 2

Abstract

Shiga toxins (Stx) are the main virulence factors associated with a form of Escherichia coli known as Shiga toxin-producing E. coli (STEC). They are encoded in temperate lambdoid phages located on the chromosome of STEC. STEC strains can carry more than one prophage. Consequently, toxin and phage production might be influenced by the presence of more than one Stx prophage on the bacterial chromosome. To examine the effect of the number of prophages on Stx production, we produced E. coli K-12 strains carrying either one Stx2 prophage or two different Stx2 prophages. We used recombinant phages in which an antibiotic resistance gene (aph, cat, or tet) was incorporated in the middle of the Shiga toxin operon. Shiga toxin was quantified by immunoassay and by cytotoxicity assay on Vero cells (50% cytotoxic dose). When two prophages were inserted in the host chromosome, Shiga toxin production and the rate of lytic cycle activation fell. The cI repressor seems to be involved in incorporation of the second prophage. Incorporation and establishment of the lysogenic state of the two prophages, which lowers toxin production, could be regulated by the CI repressors of both prophages operating in trans. Although the sequences of the cI genes of the phages studied differed, the CI protein conformation was conserved. Results indicate that the presence of more than one prophage in the host chromosome could be regarded as a mechanism to allow genetic retention in the cell, by reducing the activation of lytic cycle and hence the pathogenicity of the strains.

FIG. 1.

Schematic map of the generation of the recombinant phages (cat or tet) harboring the antibiotic resistance genes in the stx gene, indicating the precise positions where the cassettes were incorporated, the sizes of fragments replaced by the antibiotic cassettes, and the primers used to generate the fragments.

FIG. 2.

Generation of Lys933WΔcro::cat and Lys933WΔcIΔcro::tet strains. Schematic map of the E. coli 933W cro-cI locus indicating the positions of the promoters and the cro and cI genes. (A) Schematic diagram of plasmids pGcro, pGcIcro, pGcI and pBAD::cI indicating the orientation of the genes and promoters. (B) Sizes of the genes and substitution by the antibiotic cassettes to construct the mutants Lys933Δcro and Lys933ΔcIΔcro.

FIG. 3.

Evaluation of the sensitivity of lytic versus lysogenic infection (lytic/lysogenic) of each recombinant phage on each E. coli lysogen. Results of lytic infection were obtained by spot test and hybridization with the specific probe. Results of lysogenic infection were obtained by colony blotting and confirmed by PCR. The sensitivity of lytic versus lysogenic infection (lytic/lysogenic) are shown as follows: for lytic infection, −, no lysis; +, lysis; for lysogenic infection, −, no lysogenic conversion in the lysogen or no substitution; +, lysogenic conversion with the recombinant phage. Those cases in which lytic infection was achieved but lysogenic infection was not or vice versa are indicated by gray shading. The different immunity groups (groups 1 to 5) established are shown at the bottom of the figure.

FIG. 4.

Comparison of toxin and phage production between lysogens carrying one Stx prophage and lysogens carrying two prophages (Stx2 phage and a recombinant phage). Phages are shown below the x axis without the initial Φ symbol. Toxin production was evaluated in the supernatant of the cultures after mitomycin C induction. The concentration was calculated with a standard curve performed with purified Stx2. Values on the z axis correspond to the phage counts on S. sonnei strain 866 in the supernatants of the cultures after mitomycin C induction. The results are the averages plus standard deviations (error bars) from three independent trials.

FIG. 5.

Plaque assay of phages induced from strain LysA549/3538 carrying two phages. Most of the plaques generated from lysogenic strains harboring two prophages correspond to the Stx2 phage. Both membranes were transferred from the same plate in the same experiment; one was hybridized with the stxA₂ probe, while the other was hybridized with the cat probe to reveal the differences in plaque formation of each phage present in the E. coli strain harboring both phages.

FIG. 6.

Effects of lysogen (933W) cI and cro genes in the incorporation of a second prophage. (A) Generation of colonies incorporating a second prophage in the wild-type (WT) Lys933W strain compared with the Lys933WΔcro mutant. (B) Same as panel A but comparing the wild-type Lys933W strain with the Lys933WΔcIΔcro mutant. (C) Same as panel B after complementation of the Lys933WΔcIΔcro mutant with plasmid pGcI. Phages are shown below the x axis without the initial Φ symbol. Results are the averages plus standard deviations (error bars) from three independent experiments.

FIG. 7.

Effect of cI expression in the incorporation of a second prophage. (A) Expression of cI in the constructs evaluated by RT-PCR. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing CI protein produced by the Lys933WΔcroΔcI mutant complemented with pBAD::cI in the absence of arabinose or at different final concentrations of arabinose (Ara). The mutant strain harboring pBAD vector without an insert at 0.2% arabinose was the control. (C) Representative experiment showing generation of colonies incorporating a second prophage in the Lys933WΔcIΔcro mutant harboring empty pBAD (with 0.2% arabinose) and pBAD::cI induced with 0.002 or 0.2% arabinose. (D) Representative experiment showing strain C600 harboring pBAD and pBAD::cI, expression induced with 0.2% arabinose. In panels C and D, phages are shown below the x axes without the initial Φ symbol and log CFU/ml is shown on the y axes.

FIG. 8.

(A) Alignment of the CI protein sequences of the phages studied, 933W (933) and ΦA75 (A75). The proteins were aligned using CLUSTALW (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html). Amino acids that were identical in the two phages are indicated by asterisks and gray shading. Symbols in the sequence: −, no amino acids in this fragment in one of the sequences; ., the amino acids of both sequences are noncoincident. Symbols below the sequence: :, amino acids of both chains are strongly similar; ., amino acids of both chains are weakly similar. (B) Domain map of CI. The protein domains are indicated. The white boxes represent the helix-turn-helix XRE region for DNA binding. The gray boxes represent the RecA-mediated peptidase domains which correspond to the self-cleavage of CI mediated by RecA activity. The predicted model for each domain is indicated below the boxes.