An alternative physiological role for the EmhABC efflux pump in Pseudomonas fluorescens cLP6a

Abstract

Background: Efflux pumps belonging to the resistance-nodulation-division (RND) superfamily in bacteria are involved in antibiotic resistance and solvent tolerance but have an unknown physiological role. EmhABC, a RND-type efflux pump in Pseudomonas fluorescens strain cLP6a, extrudes hydrophobic antibiotics, dyes and polycyclic aromatic hydrocarbons including phenanthrene. The effects of physico-chemical factors such as temperature or antibiotics on the activity and expression of EmhABC were determined in order to deduce its physiological role(s) in strain cLP6a in comparison to the emhB disruptant strain, cLP6a-1.

Results: Efflux assays conducted with (14)C-phenanthrene showed that EmhABC activity is affected by incubation temperature. Increased phenanthrene efflux was measured in cLP6a cells grown at 10°C and decreased efflux was observed at 35°C compared with cells grown at the optimum temperature of 28°C. Membrane fatty acids in cLP6a cells were substantially altered by changes in growth temperature and in the presence of tetracycline. Changed membrane fatty acids and increased membrane permeability were associated with ~30-fold increased expression of emhABC in cLP6a cells grown at 35°C, and with increased extracellular free fatty acids. Growth of P. fluorescens cLP6a at supra-optimal temperature was enhanced by the presence of EmhABC compared to strain cLP6a-1.

Conclusions: Combined, these observations suggest that the EmhABC efflux pump may be involved in the management of membrane stress effects such as those due to unfavourable incubation temperatures. Efflux of fatty acids replaced as a result of membrane damage or phospholipid turnover may be the primary physiological role of the EmhABC efflux pump in P. fluorescens cLP6a.

Figure 1

Growth curves of P. fluorescens strains cLP6a and cLP6a-1. Growth of P. fluorescens strains cLP6a and cLP6a-1 at (a) 10°C, (b) 28°C or (c) 35°C determined as OD600 Each data point is the mean of three independent cultures, and error bars, where visible, indicate the standard deviation.

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 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.

Figure 3

Expression of emhABC efflux genes. Expression of emhABC in P. fluorescens strain cLP6a grown to stationary (Stat), logarithmic (Log) or Death phase at 28°C; grown to stationary phase at 10°C or 35°C; grown to stationary phase at 28°C in the presence of chloramphenicol (Chl) or tetracycline (Tet) at 1/4 MIC; or grown to stationary phase at 28°C in the presence of naphthalene (Nap) or phenanthrene (Phen) at 5 mmol l^-1, determined using RT-qPCR. The values shown are the fold-difference in expression of emhABC compared to expression levels in cells grown to stationary phase at 28°C (calibrator = 1). Each bar represents the mean of two independent experiments performed in duplicate. Error bars, where visible, indicate the average deviation.

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.

Figure 5

Free FA in cell free medium of P. fluorescens strains cLP6a and cLP6a-1 cultures. Free FA concentration in filtered medium from cLP6a and cLP6a-1 cultures grown to stationary phase at 10°C, 28°C or 35°C. Each bar represents the mean of two independent experiments, and error bars, where visible, indicate the average deviation.