An evaluation of bacterial gene expression during the biodegradation of organic contaminants

Abstract

The use of molecular techniques to examine remediation sites has primarily been used to track bacterial genetic potential and evaluate microbial diversity. Characterization of genetic potential, however, only indicates that the gene which encodes for a specific enzyme is present in a sample. It fails to give any information as to the expression of the degradative gene. Although specific microbial activity is a direct result of gene expression of the degradative gene, little is known about the expression of degradative genes in the environment. The objective of this study was to develop methodology to evaluate specific microbial gene expression to apply to understanding the biodegradation of organic contaminants. The model system used in this study was Pseudomona putida G7 (PpG7) which contains the NAH7 plasmid that encodes for the genes involved in the degradation of polyaromatic hydrocarbons (PAH) such as naphthalene. A qualitative RT-PCR assay was developed and used to evaluate gene expression as an index of biodegradation. Gene expression was correlated with substrate disappearance and degradation kinetics. This assay was used to evaluate the influence of two solubilizing agents, known to increase the biodegradation and bioavailablity of phenanthrene, to determine their influence on microbial gene expression. Gene expression patterns correlated with the presence of the solubilizing agents, indicating increased cellular activity due to increased bioavailability of the substrate. This assay was also used to evaluate gene expression and divergence of the nahAc genotype. Three Pseudomonas spp., isolated from a contaminated site, were compared to the well-studied PpG7 isolate. Although gene expression patterns correlated with degradation, the gene expression patterns of the three Pseudomonas sp. isolates were different from that of PpG7. These data suggest that gene expression is regulation-system dependent. Phylogenetic analysis of the nahAc genotype demonstrated that the Pseudomonas spp. were more closely related to another well studied naphthalene degrader, P. putida NCIB 9816, than to PpG7. These results demonstrated that gene expression can be used as an indicator of bioavailability and that divergence within a genotype is a factor in gene expression.