Conversely, antibiotics such as tetracycline and chloramphenicol that inhibit ribosomal function were shown to induce the expression of mexY, which encodes the MexY efflux pump in P. aeruginosa PAO1, but their effect on expression was concentration-dependent [8]. Induction of emhABC by tetracycline but not chloramphenicol (Figure 3) may likewise depend on concentration. Because single

sub-lethal concentrations of antibiotics were tested in this study we cannot make any conclusions about the effect of chloramphenicol on emhABC expression. Alternatively tetracycline may be a better substrate of the EmhABC efflux pump able to induce its expression compared to chloramphenicol and #JSH-23 randurls[1|1|,|CHEM1|]# phenanthrene. Dimethylformamide, the water-miscible solvent NCT-501 clinical trial used to add the PAHs, did not affect expression of emhABC genes in parallel control incubations (results not shown). Incubation temperature affects cLP6a membrane integrity Because the activity of EmhABC was low but the expression of emhABC was high in cLP6a cells grown at 35°C compared

to other incubation temperatures, we hypothesized that membrane integrity and (or) changes in membrane FA components might be responsible for these observations. To test the hypothesis, cell membrane integrity was determined using fluorescent dyes to determine the effect of incubation temperature on membrane permeability. Propidium iodide (PI) is a fluorescent reporter molecule that cannot cross intact cell membranes [23]. Therefore, cell fluorescence in the presence of PI only occurs if membrane integrity is compromised, allowing PI to penetrate and interact with intracellular DNA. Cetyltrimethylammonium bromide (CTAB) is a cationic surfactant that can permeabilize bacterial cell membranes and thus increase PI penetration. The fluorescence value of cells exposed to PI with CTAB treatment or without CTAB treatment represents, respectively, the total number of cells (with artificially induced membrane permeability) and the number of cells naturally exhibiting compromised membrane integrity [23]. A permeability index can be calculated as the percentage

of the net fluorescence value of PI-treated cells in the absence of CTAB relative to that in its presence. In Figure 4 the permeability index of cLP6a cells grown to stationary phase increased with next higher incubation temperature: cells grown at 10°C, 28°C or 35°C had permeability indices of approx. 9%, 12% and 20% respectively. This indicates that, as anticipated, cLP6a cells exhibit increasingly compromised membrane integrity when grown at 35°C, just below the maximum permissive growth temperature. Figure 4 The permeability index of P. fluorescens cLP6a. The permeability index of P. fluorescens cLP6a cells grown to stationary phase at 10°C, 28°C or 35°C. See text for definition of permeability index. Each bar represents the mean of three culture sub-samples.