Spontaneous generation of AI-2 activity in ribulose-5-phosphate solutions. (A) AI-2 activity in pentose phosphate solutions. Reaction buffer (10 mM sodium phosphate, pH 7.7) was incubated alone (control) or containing either 5 mM MHF, 5 mM ribose-5-phosphate (Rib-5-P), 5 mM xylulose-5-phosphate (Xyl-5-P), or 5 mM ribulose-5-phosphate (Rul-5-P). After incubation for 24 h at 37°C, the solutions were analysed for the presence of bioluminescence-inducing activity using the V. harveyi BB170 and BB886 bioassays. V. harveyi BB170 (white bars) is specifically activated by AI-2 and BB886 (grey bars) by AI-1. (B) AI-2 activity in 5 mM ribose-5-phosphate solutions incubated with phosphoriboisomerase (10 U/ml). Samples were taken immediately after the start of the experiment (white bars, 0 h) or after 2 h incubation at 37°C (grey bars). Controls contained reaction buffer only (buffer), no enzyme (buffer + Rib-5-P), no substrate (buffer + enzyme), or heat-inactivated enzyme (buffer + Rib-5-P + enzyme (inactive)). (C) Kinetics of AI-2 formation in Rul-5-P solutions. 0.5 mM Rul-5-P in reaction buffer was incubated at 37°C and samples removed and snap-frozen in liquid nitrogen at the times indicated. The samples were then analysed for AI-2 activity using V. harveyi BB170. Results represent the mean (± SD) of three independent experiments. AI-2 activity is determined by the fold-induction of light emission relative to that of the negative reaction buffer control. For comparison, approximately 400-fold induction was observed with E. coli MG1655 culture supernatants.