A sensitive and simple plaque formation method for the Stx2 phage of Escherichia coli O157:H7, which does not form plaques in the standard plating procedure

Bacteriophages are fascinating genetic elements that play key roles in the evolution and diversification of bacterial genomes. Shiga toxin (Stx)-transducing phages are important genetic elements that disseminate the stx genes among enterohemorrhagic Escherichia coli (EHEC). They are generally regarded as lambda-like phages, but their biological and genetic properties have not been fully elucidated. This is partly due to a serious obstacle in obtaining visible plaques. Here, we describe a modified double agar overlay method that allows us to easily detect and accurately enumerate plaques of Sp5, the Stx2 phage of the EHEC O157 Sakai strain, which otherwise does not produce plaques in the standard plating procedure. In the modified method, the top agar was supplemented with mitomycin C (MMC) and Ca2+ (or Mg2+). MMC appears to prevent the lysogenization of Sp5 and/or compel Sp5 to follow the lytic cycle by inducing the SOS response in the host cells. The divalent cations significantly improve phage adsorption to the host cells and thus yield a synergistic effect in combination with MMC. We further applied this method to a receptor analysis of Sp5 and obtained findings that suggest that the YaeT (BamA) protein serves as the receptor of Sp5. This method would be a very useful tool in studies of Stx2 phages and studies of other phages from various bacteria, in which researchers often encounter problems with plaque formation.

► Sp5, an Stx2 phage of EHEC produces no visible plaque by the standard procedure. ► We developed a modified double agar overlay method. ► Mitomycin C and CaCl2 (or MgCl2) were included in the top agar. ► The modified method yielded clearly visible Sp5 plaques, allowing its enumeration. ► By applying the method, we identified the YaeT protein as a possible receptor of Sp5.