Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a

Abstract

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.

FIG. 1

Distribution of radiolabel (added as [9-¹⁴C]phenanthrene) in the P. fluorescens LP6a-1 cell pellet and supernatant over time. The data points are means based on three independent experiments. The error bars (where visible) for supernatant and pellet data represent 1 standard deviation. The time when azide was added is indicated.

FIG. 2

Partitioning of phenanthrene between the cell pellet and the supernatant in LP6a-1 and wild-type strains. The error bars (where visible) represent 95% confidence intervals based on three to five independent measurements. Calculations for the wild-type strain are discussed in the text.

FIG. 3

Distribution of radiolabel (added as [9-¹⁴C]phenanthrene) in the P. fluorescens LP6a wild-type cell pellet and supernatant over time. The data points are means based on three independent experiments. The total is the sum of the data for a cell pellet and a supernatant. The error bars (where visible) for supernatant and pellet data represent 1 standard deviation. The time when azide was added is indicated.

FIG. 4

Relationship between PAH partition coefficients of LP6a-1, measured in the presence of azide (i.e., in the absence of active efflux), and the corresponding K_ow values (12). Partition coefficients were calculated by determining the ratio of pellet concentration to supernatant concentration (see text). The straight line represents the correlation calculated by Sikkema et al. (19).