Furthermore, the Crizotinib lasB induction by 3-oxo-C9-HSL with the addition of 10 μM patulin decreased to 10% of the level in the absence of patulin (Figure 4a). The addition of 3-oxo-C10-HSL or 3-oxo-C12-HSL with patulin decreased the lasB expression levels to 50% and 60%, respectively

(Figure 4b and c). These data indicate that the order of LasR-binding affinity for 3-oxo-Cn-HSLs is: 3-oxo-C12-HSL > 3-oxo-C10-HSL > 3-oxo-C9-HSL. These results suggest that acyl-HSL entry into the cell is likely to be passive and acyl-HSLs were extruded by MexAB-OprM. As a result of the accumulation of these acyl-HSLs in the MexAB-OprM mutant, a non-natural response was induced. Figure 4 3-oxo-Cn-HSLs bind directly to LasR and the complexes are able to trigger lasB expression. Individual cultures of KG7403 (ΔlasI ΔrhlI PlasB-gfp) and KG7503 (ΔlasI ΔrhlI ΔmexB PlasB-gfp) were grown in LB medium with 5 μM 3-oxo-C9-HSL (a), 3-oxo-C10-HSL (b), or 3-oxo-C12-HSL (c) with 0, 10, 20, 50, or 100 μM patulin, respectively. Transcription of lasB was determined by measuring the fluorescence intensity (arbitrary units) depending on the amounts of green-fluorescence protein (GFP) derived from PlasB-gfp; emission at 490 nm and excitation at 510 nm. Open bars, KG7403; closed bars, KG7503. The data represent mean values

of three independent experiments. Error bars represent the standard errors of the means. Selection of a bacterial language by MexAB-OprM in bacterial Akt inhibitor selleck chemical communication As we have shown here, P. aeruginosa responds to several 3-oxo-Cn-HSLs in vitro. However, it was not known

whether this in vitro response to 3-oxo-Cn-HSLs was equivalent to a response to 3-oxo-Cn-HSLs in a natural environment. When grown in close proximity to the P. aeruginosa wild-type strain on LB plates, KG7004 (ΔlasIΔrhlI) carrying pMQG003 (lasB promoter-gfp) exhibited bright-green fluorescence, but the P. aeruginosa reporter strain near the QS-negative strain, KG7004 (ΔlasIΔrhlI), did not show GFP fluorescence (Figure 5). These results clearly demonstrated that physiological concentrations of AHLs derived from PAO1 were detectable as GFP fluorescence in KG7004 (ΔlasIΔrhlI) carrying pMQG003 (lasB promoter-gfp) (Figure 5). To examine the effect of MexAB-OprM on heterogeneous bacterial communication, P. aeruginosa was co-cultivated with C. violaceum P. chlororaphis P. agglomerans P. fluorescens or V. anguillarum (Figure 5 and Additional file 1: Figure S1). These bacteria are known to produce cognate acyl-HSLs [20–23]. It was shown that lasB expression by P. aeruginosaΔmexB was only strongly induced during co-cultivation with V. anguillarum (Figure 5 and Additional file 1: Figure S1). 3-oxo-C10-HSL production by V. anguillarum was confirmed by TLC assays using Chromobacterium violaceum VIR07, in agreement with a previous report ( Additional file 2: Figure S2) [22]. Figure 5 Role of MexAB-OprM in cross-talk between P. aeruginosa and V. anguilarum.