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

Abstract

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.Shiga toxins (stx) are the major virulence factors in enterohemorrhagic Escherichia coli infections, causing such diseases as hemorrhagic colitis, infantile diarrhea, and hemolytic uremic syndrome. In virtually all known Shiga toxin-encoding E. coli strains, the genes encoding Shiga toxins are carried on lambdoid prophages (8,19,31,34,35,45) as part of an operon whose activity is ultimately regulated by the bacteriophage repressor (32,33,46,47).Lambdoid phage genomes contain two operator regions, O L and O R , each of which includes promoters whose expression is controlled by the binding of the bacteriophage repressor to multiple binding sites found in each operator region. Efficient functioning of the genetic switch between lysis and lysogeny depends on the ability of the repressor to bind with different affinities to each of the individual sites within O R and O L (40). The repressor directs the establishment and maintenance of the lysogenic state by simultaneously repressing transcription of the genes needed for lytic phage growth and activating transcription of its own gene, the only gene needed for maintenance of the lysogenic state (40).The E. coli O157:H7 strain EDL933 is considered to be the reference strain for disease-causing O157:H7 isolates. Incubating this strain with agents that trigger induction of resident prophages causes this strain to express the disease-causing stx2 gene product and to produce a lambdoid bacteriophage (38). Sequence analysi...

The CI Repressors of Shiga Toxin-Converting Prophages Are Involved in Coinfection of Escherichia coli Strains, Which Causes a Down Regulation in the Production of Shiga Toxin 2

“…Our experiments also suggest differences in the incorporation of the second phage depending on the CI concentration, but more accurate experiments would be necessary to identify the threshold concentration of CI and/or CI homologues necessary to decide whether the incoming phage will integrate or whether superinfection will occur. These observations are in agreement with other authors who anticipate that 933W prophages would direct the synthesis of a tightly regulated amount of repressor (26). They are also consistent with other authors who suggest that repressor differences may be responsible for the variations in toxin and phage production observed in different lysogens obtained with the same host strain (55).…”

The Lysis-Lysogeny Decision of Bacteriophage 933W: a 933W Repressor-Mediated Long-Distance Loop Has No Role in Regulating 933W P RM Activity

“…Also, we reported earlier that in intact 933W O R , the 933W repressor does not bind its O R 1 and O R 2 sites at an identical concentration (23), suggesting that the 933W repressor is incapable of binding cooperatively to these two adjacent sites. Cooperativity is not essential to the construction a of lysis-lysogeny switch in a lambdoid phage.…”

“…We and others have reported that unlike all other well-studied bacteriophages, bacteriophage 933W contains only two, not three, repressor binding sites in O L (16,23,36) (Fig. 1).…”

Transcription of the phage-encoded Panton–Valentine leukocidin of Staphylococcus aureus is dependent on the phage life-cycle and on the host background

“…A linker region connects the two structural domains of the repressor. It contains a specific Ala (or Cys, Leu)-Gly cleavage site that is the target of Cterminal-domain-catalysed proteolysis (Koudelka et al, 2004;Little et al, 1980). Analysis of the cI-like repressor from wSa2mw revealed a structural organization dissimilar to that of the classical lambdoid repressors, making it difficult to envisage a RecA-dependent inactivation.…”