Many virulence factors associated with bacterial pathogenesis are carried on genetic elements that facilitate their horizontal transfer amongst bacteria of the same or even different species. Bacterial viruses represent one class of natural vectors capable of transferring virulence determinants. This proposal is directed toward characterizing coliphage H-19B, which carries genes encoding the Shiga-like toxin Stx1. Following infection, the H-19B genome with its toxin genes can integrate into the bacterial chromosome as a prophage, in this way transferring stx genes to a new host. Such Escherichia coli derivatives expressing high levels of Stx have been isolated from stool samples of patients with acute bloody diarrhea and are referred to as enterohemorrhagic E. coli or EHEC. These infections can progress to cause severe kidney damage leading to death. Infections with EHEC have been associated with contaminated food and their increasing worldwide occurrence highlights the danger they present to public health. Because H-19B is a member of the well characterized lambdoid family of phages, this bacterial virus is ideal for studying the role of transferable genetic elements in pathogenic processes. Individual members of the lambdoid family are mosaics composed of what appears to be a relatively large pool of alternative functional cassettes. The experiments outlined in this proposal are directed toward understanding how the various cassettes of functional activity composing H-19B contribute to the phage's ability to express and release Stx. The studies will exploit the extensive body of knowledge of and highly advanced technology associated with lambdoid phages. In particular, the role of specific phage-encoded functions in stx expression will be studied by directing mutations to genes encoding functions critical in phage growth and gene expression. Once the details of phage control of stx expression are elucidated in well-characterized laboratory strains of E. coli, the work will focus on applying this knowledge to determining the nature of the control of stx expression and toxin release in a specially prepared EHEC strain. Information obtained from the in vitro studies will eventually be used to facilitate studies on the role of these phage factors in the expression of Stx in vivo.