Application of a Reverse Transcription-PCRassay to monitor regulation of the catabolicnahAc gene during phenanthrene degradation

Abstract

Biodegradation of polycyclic aromatic hydrocarbons (PAH), such as phenanthrene, in environmental samples is often limited by low bioavailability whichresults from a combination of low aqueous solubility and/or high sorption. The purpose of thisstudy was to investigate the influence of agents that increase PAH bioavailability on expressionof the PAH catabolic gene nahAc. Phenanthrene was used as a model PAH andPseudomonas putida PpG7, which contains the NAH7 plasmid that encodes the genesresponsible for naphthalene and phenanthrene degradation, was used as a model degrader.PAH bioavailability was altered by the addition of two biosurfactants, rhamnolipid andhydroxypropyl-β-cyclodextrin (HPCD). Gene expression was determined by extraction of bacterialmRNA followed by RT-PCR amplification of two transcripts; nahAc, a naphthalenedioxygenase gene, and rpoD, a housekeeping gene. Results indicate that the lag period precedingnahAc 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 wasbimodal 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 theaccumulation of toxic intermediates. In contrast, expression of the rpoD gene wasmaintained throughout the duration of each experiment. This research demonstrates thatthe use of a gene expression assay to monitor the impact of substrate bioavailability on substrateutilization provides unique information concerning the biodegradation process that cannot beobtained 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 otherenvironmental factors on biodegradation.