A common physical factor affecting the composition and physiology of bacterial cells is incubation temperature [10, 11], which influences bacterial cell membrane fatty acid (FA) composition [11, 12]. Altered membrane FA composition is an adaptation mechanism used by bacteria to compensate for changes in membrane fluidity caused by physiological or biochemical stress. Fluidity of the membrane affects the interaction NVP-HSP990 of lipids and

proteins (including RND efflux pumps) anchored in the membrane and in turn permeation and transport of hydrophobic molecules across the membrane [11, 13, 14]. Changes in the resistance of cells grown at different AZD9291 temperatures to various environmental stresses have been reported [10]. However, increased resistance to antibiotics, environmental stresses or membrane-damaging agents has not previously been linked to the effect of growth temperature on increased activity of efflux pumps or expression of their genes. Pseudomonas fluorescens LP6a, an isolate from petroleum condensate-contaminated soil, utilizes PAHs such as naphthalene, phenanthrene and anthracene as sole carbon source [15, 16]. A RND-type efflux pump (EmhABC) in this strain that extrudes hydrophobic antibiotics and PAHs has been described previously [17–19], where EmhA

is the membrane fusion protein, EmhB is the RND protein www.selleckchem.com/products/nct-501.html and EmhC is the outer membrane protein [18]. The EmhABC efflux pump in P. fluorescens is a good model for investigating a physiological role for RND-type efflux pumps because it extrudes PAHs considered non-toxic to the cells as well as hydrophobic antibiotics [17] and its expression is not induced by Clomifene its PAH substrates [18]. PAH transport can be monitored in the absence of PAH metabolism [18] by using strain cLP6a,

a cured strain of P. fluorescens LP6a lacking the PAH catabolic plasmid pLP6a [16]. Comparing the properties of cLP6a with its emhB disruption mutant strain cLP6a-1 [18] allows inference of a physiological role for the RND efflux pump EmhABC based on the effect of growth temperature, antibiotics or PAHs on its activity and expression in relation to membrane FA changes. Methods Bacterial strains and growth conditions P. fluorescens cLP6a is a cured strain of the wild type P. fluorescens strain LP6a that lacks the catabolic plasmid pLP6a [16] and cannot metabolize PAHs. Strain cLP6a-1 is an emhB disruption mutant of the cured strain [18]. Strains were grown to stationary phase (unless otherwise indicated) in 100 ml of trypticase soy broth (TSB) (Difco) with gyratory shaking at 200 rpm at 10°C, 28°C (the optimal growth temperature; [15]) or 35°C. TSB inoculated with strain cLP6a-1 contained kanamycin (Sigma) at 25 μg ml-1 to maintain the gene disruption.