Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals

Many gram-negative bacteria communicate by N-acyl homoserine lactone signal s called autoinducers (AIs). In Pseudomonas aeruginosa, cell-to-cell signal ing controls expression of extracellular virulence factors, the type II sec retion apparatus, a stationary-phase sigma factor (sigma(s)), and biofilm d ifferentiation. The fact that a similar signal, N-(3-oxohexanoyl) homoserin e lactone, freely diffuses through Vibrio fischeri and Escherichia coli cel ls has led to the assumption that all AIs are freely diffusible. In this wo rk, transport of the two P. aeruginosa AIs, N-(3-oxododecanoyl) homoserine lactone (3OC(12)-HSL) (formerly called PAI-1) and N-butyryl homoserine lact one (C-4-HSL) (formerly called PAI-2), was studied by using tritium-labeled signals. When [H-3] C-4-HSL was added to cell suspensions of P. aeruginosa , the cellular concentration reached a steady state in less than 30 s and w as nearly equal to the external concentration, as expected for a freely dif fusible compound. In contrast, [H-3]3OC(12)-HSL required about 5 min to rea ch a steady state, and the cellular concentration was 3 times higher than t he external level. Addition of inhibitors of the cytoplasmic membrane proto n gradient, such as azide, led to a strong increase in cellular accumulatio n of [3H]3OC(12)-HSL, suggesting the involvement of active efflux. A define d mutant lacking the mexA-mexB-oprM-encoded active efflux pump accumulated [H-3]3OC(12)-HSL to levels similar to those in the azide-treated wild-type cells. Efflux experiments confirmed these observations. Our results show th at in contrast to the case for C-4-HSL, P. aeruginosa cells are not freely permeable to 3OC(12)-HSL. Instead, the mexA-mexB-oprM-encoded efflux pump i s involved in active efflux of 3OC(12)-HSL. Apparently the length and/or de gree of substitution of the N-acyl side chain determines whether an AI is f reely diffusible or is subject to active efflux by P. aeruginosa.