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Journal of Bacteriology, November 2004, p. 7659-7669, Vol. 186, No. 22
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.22.7659-7669.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of the Repressor of the Shiga Toxin-Encoding Bacteriophage 933W: DNA Binding, Gene Regulation, and Autocleavage

Department of Biological Sciences, University at Buffalo, Buffalo, New York

Received 14 May 2004/ Accepted 11 August 2004

The genes encoding Shiga toxin (stx), the major virulence factor of Shiga toxin-encoding Escherichia coli (STEC) strains, are carried on lambdoid prophages resident in all known STEC strains. The stx genes are expressed only during lytic growth of these temperate bacteriophages. We cloned the gene encoding the repressor of the Shiga toxin-encoding bacteriophage 933W and examined the DNA binding and transcriptional regulatory activities of the overexpressed, purified protein. Typical of nearly all lambdoid phage repressors, 933W repressor binds to three sites in 933W right operator (O_R). Also typical, when bound at O_R, 933W repressor functions as an activator at the P_RM promoter and a repressor at the P_R promoter. In contrast to other lambdoid bacteriophages, 933W left operator (O_L) contains only two repressor binding sites, but the O_L-bound repressor still efficiently represses P_L transcription. Lambdoid prophage induction requires inactivation of the repressor's DNA binding activity. In all phages examined thus far, this inactivation requires a RecA-stimulated repressor autoproteolysis event, with cleavage occurring precisely in an Ala-Gly dipeptide sequence that is found within a "linker " region that joins the two domains of these proteins. However, 933W repressor protein contains neither an Ala-Gly nor an alternative Cys-Gly dipeptide cleavage site anywhere in its linker sequence. We show here that the autocleavage occurs at a Leu-Gly dipeptide. Thus, the specificity of the repressor autocleavage site is more variable than thought previously.

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