Asakura et al. 10.1073/pnas.0706695104. |
Fig. 4. Genetic map of cdtI-converting phage. (A) Relative locations of the different ORFs. Functional modules are indicated below the genetic map and are based on sequence and experimental analyses. Regions of significant contiguous similarity to other sequences are described in SI Table 4. (B) The bar indicates the scale of the CDT-1F genome.
Fig. 5. Comparison of genetic maps between CDT-1F and Stx2-IF. Relative locations of the different ORFs in CDT-1F and those in Stx2-IF are shown. Functional modules are indicated with the genetic map and are based on sequence and experimental analyses. Bars connected between CDT-1F and Stx2-IF genetic maps indicate homologous ORFs. Green, orange, and pink colors indicate 40-60%, 60-80%, and 80-100% homology, respectively.
Fig. 6. Southern hybridization of genomic DNA of cdtI gene-positive EPEC strains and CDT-1F. The genomic DNA digested with HindIII was applied to: lane 1, E. coli O127 strain GB1807; lane 2, E. coli strain C600 lysogenized with CDT-1F; lane 3, E. coli O127 strain NT3363; lane 4, CDT-1F; lane 5, E. coli O142 strain VTE1456; and lane 6, E. coli O86 strain GB1371. The DNA analyzed by gel electrophoresis was blotted to Hybond N+ nylone membrane and probed with 32P-labeled cdtI-converting phage DNA probe corresponding to head and tail (A), virulence (B), integrase (C), unknown (D), regulation (E), and lysis (F).
Table 3. Relevant characteristics of bacterial strains used in this study
Strains | Relevant characteristics | Source |
NT3363 | CdtI-positive O127:H7 EPEC strain | 1 |
GB469 | CdtI-positive O127:H7 EPEC strain | 1 |
GB1807 | CdtI-positive O127:H7 EPEC strain | 1 |
VTE1456 | CdtI-positive O142:H6 EPEC strain | 1 |
GB1371 | CdtI-positive O86a:H34 EPEC strain | 1 |
VTE1488 | CdtI-positive O86a:H34 EPEC strain | 1 |
DO5492 | CdtI positive O86a:H34 EPEC strain | 1 |
C600 | E. coli , supE44 hsdR thi-1 thr-1 leuB6 lacY1 tonA21 | Collection |
MW47 | C600 lamB20::Tn5 | 2 |
RAM191 | Δ ompC178 zei-198::Tn10 | 2 |
RAM191 (pMA1) | RAM191 transformed with a cloned ompC gene | This study |
RK4768 | ompF ::Tn5 | 2 |
MW72 | C600 fadL71::Tn10 | 2 |
C600 (CDT-1Φ) | C600 lysogenized with CDT-1Φ | This study |
C600 (Stx2Φ-I) | C600 lysogenized with Stx2Φ-I | This study |
C600 (Stx2Φ-II) | C600 lysogenized with Stx2Φ-II | This study |
1. Pandey M, Khan A, Das SC, Sarkar B, Kahali S, Chakraborty S, Chattopadhyay S, Yamasaki S, Takeda Y, Nair GB, Ramamurthy T (2003) J Clin Microbiol 41:5277-5281.
2. Watarai M, Sato T, Kobayashi M, Shimizu T, Yamasaki S, Tobe T, Sasakawa C, Takeda Y (1998) Infect Immun 66:4100-4107.
Table 6. Mitomycin C-induced CDT activity of different E. coli strains carrying CDT-1 prophage
Strain/serotype | CD50 (HeLa cells assay)a | |||
Cells associatedb | Supernatant | |||
With mitomycin C | Without mitomycin C | With mitomycin C | Without mitomycin C | |
NT3363 (O127:H7) | 8 | 32 (4) | 4 | 32 (8) |
C600 (CdtI-phage) | 1 | 64 (64) | 1 | 8 (8) |
GB1807 (O127:H7) | 64 | 64 (1) | 1 | 4 (4) |
VTE1456 (0142:H6) | 64 | 16 (0.25) | 8 | 512 (64) |
GB1371 (O86a:H34) | 128 | 256 (2) | 8 | 512 (64) |
C600 | ND | ND | ND | ND |
ND, not detected. The numbers in parentheses indicate fold increase in toxin activity.
a
The highest dilution of toxin that caused cell distension in 50% of the HeLa cell monolayer.b
Supernatant obtained by centrifugation of cell sonicate of 6-h culture.c
Supernatant of a 6-h culture.d
Culture was prepared in the absence or presence of mytomicin C (0.5 µg/ml).