Effects of AHL signals on anaerobic growth. Cells were grown anaerobically in LB medium supplemented with 100 mM NO₃⁻. Symbols: open diamonds, PAO1; open triangles, ΔrhlI mutant; solid triangles, ΔrhlI mutant with 10 μM C₄-HSL; open circles, ΔlasI mutant; solid circles, ΔlasI mutant with 1 μM 3-oxo-C₁₂-HSL. Three independent experiments were conducted, and representative data are shown.

Effects of AHL signals on denitrification. (A) Effects of AHL signals on NO₃⁻ reduction and N₂ production. The amounts of NO₃⁻ reduced and N₂ produced over 12 h were measured in PAO1 and in ΔrhlI and ΔlasI mutants. Solid bars, NO₃⁻ reduced; open bars, N₂ produced. C₄-HSL and 3-oxo-C₁₂-HSL were added to the cultures at final concentrations of 10 μM and 1 μM, respectively. Data are means ± standard deviations from three independent experiments. (B) Effects of AHL signals on NO₃⁻-respiring activity. NADH-dependent NAR activity was measured in cells grown anaerobically for 12 h. C₄-HSL at 10 μM and 3-oxo-C₁₂-HSL at 1 μM were added to the cultures of ΔrhlI and ΔlasI mutants, respectively. The experiment was performed in triplicate.

Effects of RhlR and LasR transcriptional regulators on denitrification regulation. The amounts of NO₃⁻ reduced and N₂ produced over 12 h were measured. Solid bars, NO₃⁻ reduced; open bars, N₂ produced. C₄-HSL and 3-oxo-C₁₂-HSL were added to the cultures at final concentrations of 10 μM and 1 μM, respectively. rhlR or lasR was inserted into a pUCP24 plasmid (pUCP-rhlR or pUCP-lasR) and transformed into a ΔrhlI ΔrhlR or ΔlasI ΔlasR double mutant for complementation. pUCP24 (29) was transformed into a ΔrhlI ΔrhlR or ΔlasI ΔlasR mutant as a control. Data are means ± standard deviations from three independent experiments.

Denitrification regulation by rhl and las quorum-sensing systems. (A) Effect of 10 μM C₄-HSL and 1 μM 3-oxo-C₁₂-HSL on a ΔrhlI ΔlasI double mutant. The amounts of NO₃⁻ reduced and N₂ produced over 12 h were measured. Solid bars, NO₃⁻ reduced; open bars, N₂ produced. Data are means ± standard deviations from three independent experiments. (B) Effects of 10 μM C₄-HSL and 1 μM 3-oxo-C₁₂-HSL on the transcription of denitrifying genes in a ΔrhlI ΔlasI mutant. C23O activity was measured in cells cultured for 6 h (late-exponential phase). Data are means ± standard deviations from three independent experiments.