IDENTIFICATION AND CHARACTERIZATION OF A NEWLY ISOLATED SHIGA TOXIN 2-CONVERTING PHAGE FROM SHIGA TOXIN-PRODUCING ESCHERICHIA-COLI

Shiga toxins 1 (Stx1) and 2 (Stx2) are encoded hv toxin-converting bac teriophages of Stx-producing Escherichia coli (STEC), and so far two S tx1 and one Stx2-converting phages have been isolated from two STEC st rains (A, D, O'Brien, J, W, Newlands, S, F. Miller, R. K, Holmes, H. W . Smith, and S. E. Formal, Science 226: 694-696, 1984), In this study, we isolated two Stx2-converting phages, designated Stx2 Phi-I and Stx 2 Phi-II, from two clinical strains of STEC associated with the outbre aks in Japan in 1996 and found that Stx2 Phi-I resembled 933W, the pre viously reported Stx2-converting phage, in its infective properties fo r E. coli K-12 strain C600 while Stx2 Phi-II was distinct from them. T he sizes of the plaques of Stx2 Phi-I and Stx2 Phi-II in C600 were dif ferent; the former was larger than the latter. The restriction maps of Stx2 Phi-I and Stx2 Phi-II were not identical; rather, Stx2 Phi-II DN A was approximately 3 kb larger than Stx2 Phi-I DNA. Furthermore, Stx2 Phi-I and Stx2 Phi-II showed different phage immunity, with Stx2 Phi- I and 933W belonging to the same group. Infection of C600 by Stx2 Phi- I or 933W was affected by environmental osmolarity differently from th at by Stx2 Phi-II. When C600 was grown under conditions of high osmola rity, the infectivity of Stx2 Phi-I and 933W was greatly decreased com pared with that of Stx2 Phi-II. Examination of the plating efficiency of the three phages for the defined mutations inn C600 revealed that t he efficiency of Stx2 Phi-I and 933W for the fadL mutant decreased to less than 10(-7) compared with that for C600 whereas the efficiency of Stx2 Phi-II decreased to 0.1% of that for C600. In contrast, while th e plating efficiency of Stx2 Phi-II for the lamB mutant decreased to a low level (0.05% of that for C600), the efficiencies of Stx2 Phi-I an d 933W were not changed. This was confirmed by the phage neutralizatio n experiments with isolated outer membrane fractions from C600,fadL mu tant, or lamB mutant or the purified His(6)-tagged FadL and LamB prote ins, Based on the data, we concluded that FadL acts as the receptor fo r Stx2 Phi-I and Stx2 Phi-II whereas LamB acts as the receptor only fo r Stx2 Phi-II.