Results: Contaminated Aquifer MO
(see table of naphthalene-degrading bacteria grown from site samples, and other results from this project)
- Using a combination of molecular techniques and field site sampling, we have microscopically probed the study site groundwater microbial community for nahAc, the best-described catabolic gene that initiates aerobic attack on naphthalene (the predominant site contaminant and carbon source for heterotrophic microorganisms). This assay microscopically detected nahAc transcripts in ~ 1% of the cells in well water (Bakermans and Madsen, 2002b).
- The geochemical parameters that define and are governed by in situ microbial activity have also been explored (Bakermans et al., 2002). By using on-site analyses of water characteristics (e.g., O2, NO3-, sulfate, alkalinity) as well as laboratory assays of field-fixed site samples (e.g., organic carbon, GC/MS assays of organic contaminants, CH4, and C13 values for DOC, methane and bicarbonate), we have documented aerobic and anaerobic carbon and electron flow at the site. GC/MS-based biodegradation assays of site contaminants [at ambient concentrations, incubated at the ambient temperatures (10ºC)] have documented primarily aerobic metabolism of naphthalene, 2-methylnaphthalene, benzothiophene, and indene (Bakermans et al., 2002).
- Harvesting of native cells along the on-site geochemical and contaminant gradient allowed extraction of DNA from well waters, preparation of 400 cloned 16S rRNA genes, RFLP screening, and assembly of 31 full sequences. Sixty percent of clones contained unique RFLP patterns—indicating relatively high diversity communities (Shannon Weaver indices from 3.53 to 3.69). Many of the full sequences (as high as 11% in one well) had no close relatives. Phylogenetic analysis using ARB software revealed distinctive compositions in subsurface microbial communities in each well. Contrasts in key community members were consistent with a gradient spanning aerobic conditions to contaminant-driven anaerobiosis across the site (Bakermans et al., 2002a).
- A critical feature of gene expression is the control exerted by the regulatory component of the naphthalene catabolic operon. We have thoroughly explored the presence and diversity of the nahR regulatory component of the naphthalene catabolic operon at our study site. NahR and the adjacent nahR-nahG intergenic region were amplified and sequenced from 7 site-derived naphthalene-degrading bacterial isolates, as well as 6 samples of sediment-derived DNA that produced 43 sequences. Our major conclusion was that the structure and function of nahR-nahG regulatory genes are remarkably conserved (Park et al., 2002a). This has significant implications for managing the biodegradation processes (for site clean-up) and our understanding of both on-site selective pressures and evolutionary events that assemble catabolic pathways.
- This MO study site is represented in a current compilation of studies on Horizontal Gene Transfer (HGT) (Madsen, 2002). In an effort to address a long-standing methodological limitation in environmental microbiology, (Padmanabhan et al. 2003) developed a field-based 13C-probing procedure that not only detects respiration of substrates added to field soils but also identifies the active populations in soil via their 13C-labeled 16S rDNA. (Wilson et al. 2003) used a library of naphthalene-degrading isolates from the study site to find evidence for phnAc-carrying mobile genetic element(s) in the study site (Park et al. 2003a) focused on the sequence of naphthalene catabolic plasmid pDTG1 native to site bacteria and used site-directed mutagenesis and reporter assays to assess controls on conjugal transfer. The two manuscripts by (Jeon et al. 2003a, b) report the characteristics of a new naphthalene-degrading bacterium Polaromonas naphthalenivorans and link its presence, traits, and unique nahAc-like sequence to its in situ role in naphthalene metabolism at the study site. Ongoing characterization of the naphthalene catabolic cluster of P. naphthalenivorans has revealed that the gene sequences and order resemble that of the nag operon of Ralstonia sp. U2. Thus, the (nag-type genetic elements (see Table 1, Section C.2.5) are present in the site. (Park et al. 2003b) showed that a functional nahR gene influences survival of naphthalene-degrading bacteria in soil probably by mitigating the accumulation of toxic metabolites. Finally, (Park and Madsen 2004) discovered a previously unexplored role of nahR in enhancing survival of bacteria in contaminated soil.
- See full list of publications related to this site.
Copyright on all images and material by Eugene L. Madsen, and William C. Ghiorse - 2006.