Grantee Research Project Results
2004 Progress Report: Rapid Determination of Microbial Pathways for Pollutant Degradation
EPA Grant Number: R831276C011Subproject: this is subproject number 011 , established and managed by the Center Director under grant CR831276
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: UT Center for Infrastructure Modeling and Management
Center Director: Hodges, Ben R.
Title: Rapid Determination of Microbial Pathways for Pollutant Degradation
Investigators: Kinney, Kerry A. , Kirisits, Mary Jo , Whitman, Christian
Institution: The University of Texas at Austin
EPA Project Officer: Aja, Hayley
Project Period: December 1, 2003 through November 30, 2004
Project Period Covered by this Report: December 1, 2003 through November 30, 2004
Project Amount: Refer to main center abstract for funding details.
RFA: Gulf Coast Hazardous Substance Research Center (Lamar University) (1996) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Targeted Research
Objective:
Molecular methods for investigating biological treatment processes are becoming increasingly popular at remediation sites across the country. Although most approaches involve detecting rDNA sequences to monitor the bacterial species present in a treatment system, directly monitoring the functional genes that actually catalyze pollutant degradation can be a better predictor of process performance. Our ability to employ such tools, however, depends on a priori knowledge of the sequences for the key functional genes involved in a particular biodegradation process. The current dearth of such gene sequence information for many environmentally relevant bacteria represents a fundamental barrier to the routine application of molecular biology tools at sites. The primary objective of this research project is therefore to develop a molecular tool, complementary DNA (cDNA) subtraction, for rapid and efficient determination of the metabolic pathways (and associated gene sequences) used by prokaryotic microorganisms to degrade organic pollutants. Because most microorganisms present in actual biological treatment systems are unsequenced, the potential for the cDNA technology to identify key genes in these poorly characterized bacteria represents a significant advancement.
Progress Summary:
In this project, we successfully developed Prokaryotic Suppression Subtractive Hybridization (SSH) Polymerase Chain Reaction (PCR) cDNA Subtraction. This molecular biology tool was developed previously for application to eukaryotes, and we have adapted it successfully for application to prokaryotes (e.g., bacteria), which mediate most biological treatment processes. This method isolates genes that are expressed (i.e., used) by a microorganism under specific conditions (e.g., growth on a specific pollutant), thereby identifying key genes without sequencing the microorganism’s entire genome.
The Prokaryotic SSH PCR cDNA Subtraction methodology can be summarized as follows. Bacteria are grown in the presence and absence of a pollutant; mRNA (expressed genes) is isolated from both cultures and used to synthesize cDNA (DNA copy of mRNA). This cDNA is then processed using two molecular screens to isolate the key genes involved in degradation of the specific pollutant (see Figure 1). Genes that are expressed in both cultures, which are not specifically required for the pollutant degradation, are “subtracted out” during the first hybridization screen. Then the cDNA pool undergoes a second type of screen, SSH PCR, which selectively amplifies the pollutant degradation genes. These two successive screens isolate key pollutant degradation genes from the total gene pool, and these genes then can be sequenced.
We developed the Prokaryotic SSH PCR cDNA Subtraction methodology using a well-studied model system to verify that the method was functioning as intended. The model system was Pseudomonas putida mt-2, a fully sequenced bacterium that was grown on toluene (model pollutant) or acetate (alternative simple carbon source).
Figure 1. Schematic of the Two Molecular Screens in SSH PCR cDNA Subtraction
cDNA from P. putida mt-2 grown on toluene or acetate was subjected to prokaryotic SSH PCR cDNA subtraction. A small sample of the gene fragments from the SSH PCR cDNA Subtraction-screened pool were sequenced. Ninety-five percent of these gene sequences were related to toluene degradation even though these genes represent less than 0.2 percent of the entire genome of approximately 5500 genes. The sequences represent 10 distinct genes within two key toluene biodegradation operons. Table 1 lists the key genes involved in toluene degradation and notes those that were isolated and sequenced using the Prokaryotic SSH PCR cDNA Subtraction method. Although only a small sample of the genes isolated by SSH PCR cDNA Subtraction were sequenced, we obtained sequences for 50 percent of the key genes involved in toluene degradation. These data demonstrate that the Prokaryotic SSH PCR cDNA Subtraction method was successful at isolating and sequencing key genes related to degradation of the pollutant of interest (toluene).
Table 1. Genes Related to Toluene Degradation That Were Sequenced Using Prokaryotic SSH PCR cDNA Subtraction
*X indicates that the gene was isolated and sequenced
by Prokaryotic SSH PCR cDNA Subtraction.
Prokaryotic SSH PCR cDNA Subtraction shows promise as a focused gene sequencing tool for obtaining gene sequences that are key to pollutant degradation in bacteria. Even though the P. putida mt-2 genome contains a total of approximately 5500 genes, most of which are not directly related to pollutant degradation, the Prokaryotic SSH PCR cDNA Subtraction method successfully identified 10 genes that are involved in toluene degradation. These gene sequences were identified without sequencing the entire P. putida mt-2 genome, which is a costly and time consuming endeavor. Prokaryotic SSH PCR cDNA Subtraction can be applied quickly and inexpensively to any cultureable bacteria of interest allowing one to efficiently obtain gene sequences relevant to a particular biological treatment process. Access to functional gene sequences enables researchers to harness the power of the sophisticated molecular biology tools becoming available for investigating and monitoring biological treatment processes.
Future Activities:
Now that we have demonstrated the utility of the Prokaryotic SSH PCR cDNA Subtraction technique for a model organism (P. putida) degrading a model pollutant (toluene), we plan to extend the work to unsequenced bacteria degrading perchlorate, a pollutant of relevance at many hazardous waste sites.
Journal Articles:
No journal articles submitted with this report: View all 4 publications for this subprojectSupplemental Keywords:
biomarker, cDNA subtraction, functional gene, waste, ecological risk assessment, environmental engineering, hazardous waste, advanced treatment technologies, bioremediation, contaminated waste sites, groundwater contamination, petroleum contaminants, hydrocarbon,, RFA, Scientific Discipline, INTERNATIONAL COOPERATION, Waste, TREATMENT/CONTROL, Remediation, Environmental Chemistry, Treatment Technologies, Technology, Hazardous Waste, Hazardous, hazardous waste treatment, microbial degradation, BTEX, biotechnology, cDNA subtraction tool, bioremediationRelevant Websites:
http://dept.lamar.edu/gchsrc/ Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
CR831276 UT Center for Infrastructure Modeling and Management Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R831276C001 DNAPL Source Control by Reductive Dechlorination with Fe(II)
R831276C002 Arsenic Removal and Stabilization with Synthesized Pyrite
R831276C003 A Large-Scale Experimental Investigation of the Impact of Ethanol on Groundwater Contamination
R831276C004 Visible-Light-Responsive Titania Modified with Aerogel/Ferroelectric Optical Materials for VOC Oxidation
R831276C005 Development of a Microwave-Induced On-Site Regeneration Technology for Advancing the Control of Mercury and VOC Emissions Employing Activated Carbon
R831276C006 Pollution Prevention through Functionality Tracking and Property Integration
R831276C007 Compact Nephelometer System for On-Line Monitoring of Particulate Matter Emissions
R831276C008 Effect of Pitting Corrosion Promoters on the Treatment of Waters Contaminated with a Nitroaromatic Compounds Using Integrated Reductive/Oxidative Processes
R831276C009 Linear Polymer Chain and Bioengineered Chelators for Metals Remediation
R831276C010 Treatment of Perchlorate Contaminated Water Using a Combined Biotic/Abiotic Process
R831276C011 Rapid Determination of Microbial Pathways for Pollutant Degradation
R831276C012 Simulations of the Emission, Transport, Chemistry and Deposition of Atmospheric Mercury in the Upper Gulf Coast Region
R831276C013 Reduction of Environmental Impact and Improvement of Intrinsic Security in Unsteady-state
R831276C014 Integrated Chemical Complex and Cogeneration Analysis System: Greenhouse Gas Management and Pollution Prevention Solutions
R831276C015 Improved Combustion Catalysts for NOx Emission Reduction
R831276C016 A Large-Scale Experimental Investigation of the Impact of Ethanol on Groundwater Contamination
R831276C017 Minimization of Hazardous Ion-Exchange Brine Waste by Biological Treatment of Perchlorate and Nitrate to Allow Brine Recycle
R831276C018 Integrated Chemical Complex and Cogeneration Analysis System: Greenhouse Gas Management and Pollution Prevention Solutions
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
Main Center: CR831276
64 publications for this center
18 journal articles for this center