Application of a reverse transcription-PCR assay to monitor regulation of the catabolic nahAc gene during phenanthrene degradation.

Abstract

Biodegradation of polycyclic aromatic hydrocarbons (PAH), such as phenanthrene, in environmental samples is often limited by low bioavailability which results from a combination of low aqueous solubility and/or high sorption. The purpose of this study was to investigate the influence of agents that increase PAH bioavailability on expression of the PAH catabolic gene nahAc. Phenanthrene was used as a model PAH and Pseudomonasputida PpG7, which contains the NAH7 plasmid that encodes the genes responsible for naphthalene and phenanthrene degradation, was used as a model degrader. PAH bioavailability was altered by the addition of two biosurfactants, rhamnolipid and hydroxypropyl-beta-cyclodextrin (HPCD). Gene expression was determined by extraction of bacterial mRNA followed by RT-PCR amplification of two transcripts; nahAc, a naphthalene dioxygenase gene, and rpoD, a housekeeping gene. Results indicate that the lag period preceding nahAc gene induction decreased from 312 to 48 h in the presence of biosurfactants. Expression of the nahAc gene, as measured by RT-PCR, in the presence of surfactants was bimodal on a temporal basis, indicating that induction stopped briefly during biodegradation. Cessation of induction could have resulted from the up-regulation of alternate pathways or the accumulation of toxic intermediates. In contrast, expression of the rpoD gene was maintained throughout the duration of each experiment. This research demonstrates that the use of a gene expression assay to monitor the impact of substrate bioavailability on substrate utilization provides unique information concerning the biodegradation process that cannot be obtained from more traditional biodegradation assays such as cell growth or substrate disappearance. Gene expression assays also have the potential for use in assessing the impact of other environmental factors on biodegradation.