Colibactin synthesis cannot be sustained by EntD <i>in vitro</i>.

<p>Colibactin production by <i>Escherichia coli</i> strain M1/5 and derivatives determined by megalocytosis (<b>A</b>) and by quantification of DNA double strand breaks (<b>B</b>) in infected HeLa cells. <b>A.</b> Live <i>E. coli</i> wild type strain M1/5, mutants and complemented derivatives were added directly onto HeLa cells [multiplicity of infection (MOI) = 100], cocultivated for 4 h, then washed as described in Nougayrède et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003437#ppat.1003437-Nougayrede1" target="_blank">[21]</a>. The cells were incubated for 72 h with gentamicin before protein staining with methylene blue. The quantification of staining was measured at OD 660 nm. **: <i>P</i><0.01, *: <i>P</i><0.05, ns: not significant. <b>B.</b> Quantification of DNA double strand breaks through the quantification of phosphorylated H2AX (γ-H2AX) using In Cell Western method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003437#ppat.1003437-Audebert1" target="_blank">[27]</a>. HeLa cells were infected 4 h with strain M1/5 and derivatives [MOI = 50 to 6] fixed, and examined 8 h post infection for quantification of γ-H2AX.