The Use of Molecular and Biochemical Tools to Monitor Bioaugmented Microbial CommunitiesEPA Grant Number: U915831
Title: The Use of Molecular and Biochemical Tools to Monitor Bioaugmented Microbial Communities
Investigators: Larabee, Jeannine K.
Institution: Stanford University
EPA Project Officer: Broadway, Virginia
Project Period: June 1, 2000 through June 1, 2003
Project Amount: $102,000
RFA: STAR Graduate Fellowships (2000) RFA Text | Recipients Lists
Research Category: Fellowship - Microbiology , Academic Fellowships , Biology/Life Sciences
The objectives of this research project are to: (1) investigate and further characterize the expression of Psuedomonas stutzeri strain KC proteins involved in the production of pyridine-2,6-bis-thiocarboxylate (PDTC), a carbon tetrachloride-transforming factor; (2) investigate, further characterize, and partially purify secreted proteins that interact with PDTC and affect its carbon tetrachloride-transforming ability; and (3) develop techniques to monitor expression of these proteins in carbon tetrachloride-contaminated field sites.
Two methods have been used for protein expression analysis, Surface Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS) and 2-dimensional polyacrylamide gel electrophoresis, to characterize cytosolic and secreted protein expression under environmental conditions that lead to the production of PDTC. Mutants deficient in PDTC production also have been generated, and the protein expression profiles of these strains are being characterized as well. The eventual goal is to use these methods to detect PDTC and proteins that are expressed in the PDTC biosynthetic pathway in samples collected from carbon tetrachloride-contaminated field sites; these results will show that the "correct" organism is mediating carbon tetrachloride transformation by the "correct" metabolic pathway in the field (i.e., field results correlate with laboratory results). An assay is being developed, most likely using HPLC-MS, to measure the PDTC molecule itself. In the course of this work, evidence has been found to suggest that strain KC secretes a protein that interacts with PDTC and reduces its carbon tetrachloride transforming ability. The investigator currently is in the process of developing an assay using gas chromatography to study this effect and would like to confirm that the molecule responsible for reducing PDTC's carbon tetrachloride-transforming ability is a protein, further characterize the protein, and partially purify it. These results could have important implications for bioremediation in the field since we want to understand what happens to PDTC and its carbon tetrachloride-transforming ability after secretion from the cell.
This research is expected to result in a technique that can be used at carbon-tetrachloride contaminated sites to monitor expression of Pseudomonas stutzeri, which is involved in carbon tetrachloride transformation.