Table 1.
Strains and plasmids used in this study.
Table 2.
Oligonucleotides used in this study.
Figure 1.
The dose response to EDTA on recA-independent induction.
The effect of EDTA was evaluated in two laboratory E. coli lysogens of 933 W: DH5α(933 W) (A) and C600(933 W) (B), in E. coli O157:H7 EDL933 (C) in a 933 W lysogen recA isogenic mutant C600(933 W)ΔrecA::tet alone and complemented with pGEM::recA (D). The effect of EDTA on 933 W in terms of phage induction and the number of viable cells (ml−1) was evaluated after 4 h of incubation at 37°C. Data presented are the mean of three experiments. Error bars indicate the standard deviation.
Figure 2.
The effect of mitomycin C and EDTA on Stx2 phage induction.
Bacteria and 933 W phage evaluation in lysogen cultures incubated in LB broth, LB broth with mitomycin C, LB broth with EDTA and LB broth with EDTA and mitomycin C (E&M) in three strains: (A) DH5α(933 W), (B) C600(933 W) and (C) O157:H7 EDL 933. At the indicated times the numbers of viable bacterial and free Stx phages per ml were determined. Data presented are the mean of three experiments. Error bars indicate the standard deviation.
Figure 3.
The different inducing capacities of EDTA and mitomycin C in wild-type STEC strains.
The differences in Stx2 phage production (ml−1) in control cultures and in cultures incubated with mitomycin C or EDTA.
Figure 4.
Evaluation of the effect of RcsA and DsrA on Stx2 phage induction.
(A) shows the position and direction of the transcription of the rcsA and dsrA genes and the deletions used to generate mutant DH5α(933 W)ΔrcsAΔdsrA::tet. The map was drawn based on the distribution in E. coli strain K12 MG1655 (GenBank accession n° U00096.2). Their close position on the genome permitted the effect of both genes to be evaluated using a single mutation via the Red Recombinase system. The orientation and positions of primers used to construct mutants with deletions in rcsA and drsA genes by introducing a tet cassette are marked by small arrows. The extent of each deletion is shown but is not drawn to scale. (B) shows the number of 933 W phage and viable bacterial counts of the wild-type strain and the mutant with and without EDTA induction. Data presented are the mean of three experiments. Error bars indicate the standard deviation.
Figure 5.
Evaluation of envelope stress pathways in phage induction.
The figure shows the position and direction of transcription of the genes composing the (A) RcsBC, (B) CpxAR and (C) BaeSR pathways, and the deletions used to generate mutants DH5α(933 W) ΔrcsBΔrcsC::tet (A), DH5α(933 W) ΔcpxAΔcpxR::tet (B), and DH5α(933 W) ΔbaeSΔbaeR::tet (C). Schematic representations of the genes mutated for construction of mutants lacking the sensor kinase and response regulator in the selected envelope stress response signalling pathways were drawn based on E. coli strain K12 MG1655 (GenBank accession n° U00096.2). The extent of each deletion is shown but is not drawn to scale. The chart (D) shows the number of 933 W phage and viable bacterial counts of the wild-type strain and the three mutants with and without EDTA induction. Data presented are the mean of three experiments. Error bars indicate the standard deviation.
Table 3.
Effect of chelation of 933 W induction.
Figure 6.
Effect of pH on Stx2 phage induction.
Number of Stx2 phage and viable bacterial counts in EDL933 (A, B and C) and DH5α (933 W) (D, E and F). Cultures were evaluated without inducing agent (A and D), and with inducing agents: mitomycin C (B and E) and EDTA (C and F). The number of phage and viable bacterial counts in cultures were determined after incubation at 37°C for 4 h. Data presented are the mean of three experiments. Error bars indicate the standard deviation.