Shiga Toxin 2 Is Specifically Released from Bacterial Cells by Two Different Mechanisms

<jats:title>ABSTRACT</jats:title> <jats:p> Shiga toxin 1 (Stx1) is located in the periplasmic fraction, while Stx2 is found in the extracellular fraction, suggesting that enterohemorrhagic <jats:italic>Escherichia coli</jats:italic> (EHEC) contains a specific Stx2 release system. Both <jats:italic>stx</jats:italic> <jats:sub>1</jats:sub> and <jats:italic>stx</jats:italic> <jats:sub>2</jats:sub> are found within the late operons of Stx-encoding phages. Stx2 production is greatly induced by mitomycin C, suggesting that <jats:italic>stx</jats:italic> <jats:sub>2</jats:sub> can transcribe from the late phage promoter of the Stx2-encoding phage. However, the Stx1 promoter adjacent to <jats:italic>stx</jats:italic> <jats:sub>1</jats:sub> is a dominant regulatory element in Stx1 production. With the deletion of phage lysis genes of the Stx2-encoding phage, Stx2 remains in the bacterial cells. Further, we demonstrate that the Stx2-encoding phage, but not the Stx1-encoding phage, is spontaneously induced at extremely low rates. These results indicate that spontaneously specific Stx2-encoding phage induction is involved in specific Stx2 release from bacterial cells. Furthermore, to examine whether another system for specific Stx2 release is present in EHEC, we analyze the <jats:italic>stx</jats:italic> -replaced mutants. As expected, Stx2 derived from the Stx1 promoter is located in both the extracellular and cell-associated fractions, while mutant Stx2 (B subunit, S31N) derived from the Stx1 promoter is found only in the cell-associated fraction. These results indicate that EHEC has another Stx2 release system that strictly recognizes the serine 31 residue of the B subunit. Overall, we present evidence that specific Stx2 release from bacterial cells is involved in both the Stx2-encoding phage induction system and another Stx2 release system. </jats:p>