Importantly, because the centrifugation assay is so rapid (~25 min duration), the observed effects must be due to existing efflux pumps and membrane fatty acid (FA) composition rather than being MK 8931 manufacturer influenced by induction of emhABC transcription or long-term membrane modifications through de novo synthesis of FA. Because incubation temperature affects FA composition and fluidity of membranes, which in turn can selleck kinase inhibitor affect protein-lipid interactions and integral membrane protein activity , we determined the effect of growth
temperature over a 25°C range on subsequent phenanthrene efflux activity. The cell-associated phenanthrene prior to azide addition was 1.34 ± 0.19 μmol/g, 1.93 ± 0.34 μmol/g and 2.30 ± 0.36 μmol/g in cLP6a cells grown at 10°C, 28°C and 35°C respectively, indicating
reduced efflux activity with increasing growth temperature. Consistent with previous work , cLP6a cells grown at 28°C exhibited active efflux of phenanthrene (Figure 2a): the steady state concentrations of phenanthrene associated with the cell pellet before (1.93 ± 0.34 μmol/g ) and after (5.28 ± 0.56 μmol/g ) azide addition were significantly different (P < 0.0001). Figure 2 Phenanthrene partitioning into P. fluorescens strains cLP6a and cLP6a-1. Partitioning of phenanthrene into the cell pellet of P. fluorescens strains, determined using a rapid efflux assay: (a) strain cLP6a grown at 10°C, 28°C or 35°C; (b) strain cLP6a-1 grown at 10°C, 28°C or 35°C. The vertical dashed line indicates selleck inhibitor the addition of azide (120 mM). Each data point is the mean of three independent experiments, and error bars, where visible, indicate the standard deviation. Efflux assays were also performed with the emhB disruption strain cLP6a-1 (Figure 2b) to determine the steady state concentration of phenanthrene in the absence of efflux in the cells. As expected, there was no evidence of phenanthrene efflux by mutant
cLP6a-1 Reverse transcriptase at 28°C and 35°C, as the steady state concentrations of cell-associated phenanthrene were unchanged before and after azide addition. Notably, the cell-associated phenanthrene prior to azide addition was significantly greater in cLP6a-1 cells grown at 28°C (6.60 ± 0.50 μmol/g) than in the parallel cLP6a cells (1.93 ± 0.34 μmol/g; P < 0.0001) (Figure 2). Thus, EmhABC is the sole efflux system responsible for phenanthrene efflux in cLP6a cells grown at 28°C and 35°C. The cell-associated phenanthrene concentration in cLP6a-1 cells grown at 35°C before azide addition (4.32 ± 0.19 μmol/g) was significantly lower (P < 0.0001) than in cells grown at 28°C (6.60 ± 0.50 μmol/g; Figure 2b), suggesting that phenanthrene partitioning into the cells was affected by changes in membrane FA composition induced by the incubation temperature.