The genes encoding Shiga toxin (Stx), the major virulence factor of Shiga toxin-producing Escherichia coli, are carried in the genomes of bacteriophages that belong to the lambdoid family of phages. Previous studies demonstrated that induction of prophages encoding stx significantly enhances the production and/or release of Stx from the bacterium. Therefore, factors that regulate the switch between lysogeny and lytic growth, e.g., repressor, operator sites, and associated phage promoters, play important roles in regulating the production and/or release of Stx. We report the results of genetic and biochemical studies characterizing these elements of the Stx-encoding bacteriophage 933W. Like , 933W has three operator repeats in the right operator region (O R ), but unlike and all other studied lambdoid phages, which have three operator repeats in the left operator region (O L ), 933W only has two operator repeats in O L . As was observed with , the 933W O R and O L regions regulate transcription from the early P R and P L promoters, respectively. A lysogen carrying a 933W derivative encoding a noncleavable repressor fails to produce Stx, unlike a lysogen carrying a 933W derivative encoding a cleavable repressor. This finding provides direct evidence that measurable expression of the stx genes encoded by a 933W prophage requires induction of that prophage with the concomitant initiation of phage gene expression.Shiga toxin-producing Escherichia coli (STEC) strains are a group of food-borne pathogens that can infect humans, causing a range of illnesses including hemorrhagic colitis and lifethreatening sequelae such as hemolytic uremic syndrome (26, 50). Shiga toxins (Stx), a group of cytopathic toxins first identified in Shigella dysenteriae (reviewed in reference 39), are essential virulence factors of STEC, eliciting many of the pathological features associated with STEC infections (42). Stx holotoxins, classified as AB 5 toxins, are composed of one enzymatic A subunit and five receptor-binding B subunits (reviewed in reference 38). The A subunit is an rRNA N-glycosidase, catalyzing the removal of a specific adenine residue from the 28S rRNA of the 60S ribosomal subunit, resulting in inhibition of protein synthesis in the target cell (48). STEC produce two types of Stx proteins: Stx1, which is essentially identical to Stx of S. dysenteriae, and Stx2, which differs in sequence but not in function from Stx1 (38).In most identified STEC strains, the toxin genes, stxAB, are located in the genomes of prophages that resemble the coliphage (7,22,24,27,40,58). Lambdoid phages share similar genome organization and schemes of transcription control (3,20). The prophage remains in a quiescent state due to binding of the cI-encoded repressor protein to the right and left operator sites, in this way inhibiting transcription from the phage early promoters P R and P L , respectively (47). Prophage induction, which leads to lytic growth, results from removal of repression. This event occurs primarily through RecA-mediated represso...

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