The EmhABC efflux pump decreases the efficiency of phenanthrene biodegradation by Pseudomonas fluorescens strain LP6a

Abstract

Pseudomonas fluorescens strain LP6a, designated here as strain WEN (wild-type PAH catabolism, efflux positive), utilizes the polycyclic aromatic hydrocarbon phenanthrene as a carbon source but also extrudes it into the extracellular medium using the efflux pump EmhABC. Because phenanthrene is considered a nontoxic carbon source for P. fluorescens WEP, its energy-dependent efflux seems counter-productive. We hypothesized that the efflux of phenanthrene would decrease the efficiency of its biodegradation. Indeed, an emhB disruptant strain, wild-type PAH catabolism, efflux negative (WEN), biodegraded 44% more phenanthrene than its parent strain WEP during a 6-day incubation. To determine whether efflux affected the degree of oxidation of phenanthrene, we quantified the conversion of ¹⁴C-phenanthrene to radiolabeled polar metabolites and ¹⁴CO₂. The emhB ⁻ WEN strain produced approximately twice as much ¹⁴CO₂ and radiolabeled water-soluble metabolites as the WEP strain. In contrast, the mineralization of ¹⁴C-glucose, which is not a known EmhB efflux substrate, was equivalent in both strains. An early open-ring metabolite of phenanthrene, trans-4-(1-hydroxynaphth-2-yl)-2-oxo-3-butenoic acid, also was found to be a substrate of the EmhABC pump and accumulated in the supernatant of WEP but not WEN cultures. The analogous open-ring metabolite of dibenzothiophene, a heterocyclic analog of phenanthrene, was extruded by EmhABC plus a putative alternative efflux pump, whereas the end product 3-hydroxy-2-formylbenzothiophene was not actively extruded from either WEP or WEN cells. These results indicate that the active efflux of phenanthrene and its early metabolite(s) decreases the efficiency of phenanthrene degradation by the WEP strain. This activity has implications for the bioremediation and biocatalytic transformation of polycyclic aromatic hydrocarbons and heterocycles.