Figure 2.

Induction of RclR response by HOCl and chloramines. (A, B) HOCl was added at the indicated concentrations to aliquots of E. coli cultured to mid-log phase in MOPS minimal medium. After 10 min at 37°C, bacteria were harvested for measurement of rclB expression by qRT-PCR (A) and analysis of cell survival by colony formation (B). Means ± S.E.M. from three experiments are plotted in (A) and a mixed test using restricted maximal likelihood (without Geisser-Greenhouse correction) and Tukey multiple comparison showed HOCl at 10, 100 and 1000 µM caused significantly increased rclB expression (P < 0.03). (B) shows a representative survival assay, also from three replicates. See methods sections for more details. (C) RclR induction in E. coli after 20 min at 37°C in Hank's buffer containing HOCl (5 µM), NH₂Cl (25 µM), GlyCl (100 µM) and TauCl (150 µM) at 5 × 10⁷ bacteria/mL. Reactions were stopped by adding 1 mM methionine and RNA extracts were tested for rclB expression by qRT-PCR, determined as fold induction relative to untreated E. coli. Results are from three independent experiments and show significant induction by NH₂Cl and GlyCl (P < 0.001) by one way ANOVA with Tukey test for multiple comparison) but no significant effect of TauCl. Note that because the oxidant to bacteria ratio determines lethality, the lower bacterial density than for the experiments described in the text required less oxidant to retain viability and give activation.