In addition, we have now shown that temperature affects expression
and activity of the EmhABC RND efflux pump (measured by using RT-qPCR, phenanthrene efflux and antibiotic MIC assays). The FA content of cLP6a followed the expected trends at 10°C and at 35°C, shifting Sepantronium cost towards unsaturation and saturation respectively [11, 32]. The FA content of the membrane affected the partitioning of phenanthrene into the membrane, since cLP6a-1 cells grown at 35°C contained lower fractions of phenanthrene in the absence of active efflux compared to those grown at 28°C. This observation is consistent with the rationale that saturated FA pack closely, hindering partitioning of hydrophobic molecules like PAHs into the lipid bilayer [11] whereas angular cis-unsaturated FA pack more loosely, facilitating partitioning. The observed changes in FA with temperature are also consistent with results from the membrane integrity assay in which the permeability index increased with temperature. Growth temperature also affected EmhABC activity in cLP6a, possibly indirectly through membrane perturbation including the modulation of FA. cLP6a cells having high unsaturated FA
content (i.e., 72% in cells grown at 10°C) and greater membrane integrity VX-770 molecular weight had higher efflux activity than cells with lower proportions of unsaturated FA (i.e., 14% at 28°C or 4% at 35°C) and increased permeability. This observation suggests that increased unsaturated FA content may allow efficient or stable association of the three protein components of RND efflux pumps, which spans two membranes and the periplasm. The enhanced phenanthrene efflux observed in cLP6a at 10°C is consistent with the additive effect of EmhABC with a postulated alternate efflux pump that is active
Bay 11-7085 at 10°C. The presence of an alternate pump in P. fluorescens is not unexpected, as multiple efflux pumps have been identified in other Pseudomonas species [2, 7] and additional efflux pumps were invoked by Hearn et al. [18] to explain anthracene and fluoranthene efflux in P. fluorescens strain cLP6a. The Angiogenesis inhibitor induction of emhABC genes was observed in cLP6a cells exhibiting major changes in membrane FA composition due to sub-optimal growth conditions, namely at 10°C, 35°C and in the presence of tetracycline. Expression was also increased in logarithmic phase cells, which undergo rapid synthesis and turnover of FA, and in death phase cells that experience membrane deterioration. The relationship between induction of emhABC genes and membrane FA modulation indicates that the EmhABC efflux pump may be involved in the extrusion of replaced membrane FA as a result of membrane turnover. This possibility is further supported by the higher concentration of free FA in the medium of cLP6a cultures grown at 35°C concomitant with high membrane permeability and over-expression of emhABC genes.