| Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx2e from one human STEC isolate has been reported to be encoded within a toxin-converting phage. In this study, I examined O91:H21 STEC isolate B2F1 that carries two functional alleles (stx2d1 and stx 2d2) for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages and other potential regulators of toxin expression. Mutants of B2F1 that produced one or the other Stx2d toxin were made. The Stx2d1-producing mutant (stx2d2:: cat) was less cytotoxic for Vero cells than the Stx2d2-producing mutant (stx2d1::cat). Consistent with those results, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection, while the Stx2d2-producing mutant was nearly as virulent as wild-type B2F1. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. An stx2d1-containing lysogen was isolated from plaques on strain DH5alpha that had been exposed to lysates of the mutant that produced Stx2d1 only. However, that RecA- lysogen could not be induced for phage nor were culture lysates from it cytotoxic for Vero cells. By contrast, when the lysogen was transformed with a plasmid encoding RecA and induced with mitomycin C, culture extracts were cytotoxic for Vero cells. Furthermore, electron microscopic examination of extracts from the &phis;B2F1-lysogen showed hexagonal particles that resembled the prototypic Stx2-converting phage 933W. These observations provide strong evidence that expression of Stx2d1 is bacteriophage-associated.;The finding that synthesis of Stx2d1 but not Stx2d2 was associated with phage induction led me to investigate regulation of Stx2d2 production. Transposon mutagenesis of DH5alpha revealed genes associated with reduced expression of an stx2d2 promoter::lacZ fusion in a reporter plasmid, observations that suggested the inactivation of potential activators of transcription of stx2d2. (Abstract shortened by UMI.)... |
