Elizabeth M. Marlowe

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

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.

Pathogen Detection in the Genomic Era

In the 21st century, one of the greatest challenges to public health and clinical microbiologists is the rapid detection and identification of emerging and reemerging pathogens. Complex factors such as genetic variation in the host and pathogen, environmental changes, population pressures, and global travel can all influence the emergence of infectious diseases. The SARS epidemic of 2003 highlighted the potential of an emerging pathogen to spread globally in a very short time frame (Peruski and Peruski, 2003). The diagnostics of such infectious diseases has been greatly affected in the past 20 years. No longer is cultivation and microscopy the only means of detecting infectious agents. With the introduction of molecular diagnostics, the ability to detect minute amounts of microbial nucleic acids in clinical specimens has revolutionized clinical microbiology. In particular, the utility of PCR allows the detection and quantitation of specific agents in a matter of hours. PCR sequencing of specific segments of nucleic acid allows for the determination of specific drug resistance that now aids in guiding viral therapies.