(A) Acid tolerance assay. The ability of RDN02 (wild-type M19 strain), RDN306 (isogenic luxS-deficient mutant), EC480 (RDN02 (pEC82)), EC481 (RDN306 (pEC82)) and EC482 (RDN306 (pEC83)) to survive an acid challenge (pH 4) is shown as survival in percent (y axis) at the 6 h time point. The average % survival values and standard deviations of three independent experiments are shown. The asterisk indicates that the difference in survival rates between the luxS-deficient mutant and the wild-type strain was statistically significant (P < 0.05). Similar data were obtained with the RDN29 (M1 type) derivative strains. (B) Effect of low pH on luxS expression. Northern blot analysis was performed with total RNA isolated from RDN02 cultures grown to early-logarithmic phase and then incubated for 90 min with THY medium at pH 5.5, 6.0 and 7.5. Blots were hybridized with probes specific to luxS and rRNA16S (acting as loading control). The estimated sizes of transcripts are indicated. Pixel rates of signals (pixel counts obtained with the luxS probe versus pixel counts obtained with the rRNA16S probe) are shown. The blot shown is representative of three independent experiments. Similar data were obtained with the RDN29 strain. (C) Induction of luminescence in V. harveyi reporter strain BB170 by CM of RDN02. CM was prepared from RDN02 cultures grown to early-logarithmic phase and then incubated for 90 min with THY medium at pH 5.5 (RDN02-pH 5.5), 6.0 (RDN02-pH 6.0) and 7.5 (RDN02-pH 7.5) for 90 min. THY medium at pH 7.5, 6.0 and 5.5 and CM of DH5-α (AI-2 deficient) were used as negative controls; CM of BB170 was used as positive control. The luminescence values expressed in specific light units (SLU = RLU (relative light units)/OD620 nm) (y axis) are plotted versus time (h) (x axis). Data shown are representative of three independent experiments. Similar data were obtained with CM of the RDN29 derivative strains.