Grantee Research Project Results
2003 Progress Report: Metal Biosensors: Development and Environmental Testing
EPA Grant Number: R830907Title: Metal Biosensors: Development and Environmental Testing
Investigators: Anderson, Anne J. , Miller, Charles D. , McLean, Joan E
Institution: Utah State University
EPA Project Officer: Hahn, Intaek
Project Period: May 1, 2003 through April 30, 2006 (Extended to April 30, 2007)
Project Period Covered by this Report: May 1, 2003 through April 30, 2004
Project Amount: $336,000
RFA: Environmental Futures Research in Nanoscale Science Engineering and Technology (2002) RFA Text | Recipients Lists
Research Category: Nanotechnology , Safer Chemicals
Objective:
The objective of this research project is to develop and test biosensors and DNA arrays that will detect copper (Cu) and cadmium (Cd) specifically and indicate the bioavailability of these metals to a bacterium.
Progress Summary:
The project’s goals concern nanoscale detection of the bioavailability of toxic metals using Cu and Cd as models and a bacterium, Pseudomonas putida KT2440, as the detector. The following studies are underway: (1) development of a suite of sensitive-promoter fusion biosensors that react to low levels of metals with differing degrees of specificity; (2) construction of a cell plate array consisting of a library of these metal-sensitive promoter-fusion biosensors; and (3) generation of a gene array to detect changes in gene expression indicating that the a sensor cell is under metal stress.
Two Pseudomonas putida strains, KT2440 and Corvallis, respond to the toxic metal Cu with cell death being initiated at about 10 mg/L. Two-dimensional (2-D) gel electrophoresis demonstrates changes in the levels of several distinct peptides after exposure to Cu and Fe, showing degrees of specificity in response to these different metals. Using MALDI-TOF analysis, two of the peptides that changed in response to 10 mg/L Cu were shown to correspond to a flagellin, which decreased threefold, and the lipoamide dehydrogenase component of 2-oxoglutarate dehydrogenase (OGDH), which increased threefold. Decreased flagellin production has been correlated by other researchers with water stress in the cell for P. putida. The increase in OGDH suggests that carbon flow through the tricarboxylic acid cycle may be enhanced by Cu exposure, as anticipated from activated metabolism to overcome stress. Members of a library in P. putida KT2440 containing promoter fusions with a luxAB reporter cassette displayed different degrees of change in lux expression upon exposure to Fe. Expression was increased from a gene encoding guanosine triphosphate (GTP). Its product, guanosine 3',5'-bispyrophosphate (ppGpp), accumulates in bacteria in response to either amino acid depletion or energy source starvation. Thus, this is a novel finding for a metal stress response. Our findings confirm that the pseudomonads can be developed into accurate and sensitive biosensors for toxic metals.
Future Activities:
In Years 2 and 3 of this research project, we will focus on Goal 2 (construction of a cell plate array consisting of a library of metal-sensitive promoter-fusion biosensors) and Goal 3 (a gene array to detect changes in gene expression indicating that a sensor cell is under metal stress).
Goal 2
The luxAB: insertional library in KT2440 will be screened for members that show differential sensitivity to Cu and Cd to assemble the plate array.
Goal 3
Approach A: Detection of Genes Associated With Metal Stress By Light Emission From Fusion of the Promoter of Responsive Genes to the LuxAB Detector System. The site of insertion for promoters that show differential regulation upon exposure to Cu and Cd will be pursued in Year 2 and will be continued to Year 3.
Approach B: RNA Profiling. PCR identification of genes predicted to change in response to toxic metals will be initiated in Year 2.
Approach C: Detection of Proteins Changed In Level After Exposure to Toxic Metals By 2-D Gel Electrophoresis Followed By Gene Identification Through Reverse Genetics. The identification of peptides that are shown by 2-D electrophoresis to increase in accumulation in response to Cu and Cd will be continued in Year 2. Identification of the corresponding genes by reverse genomics will be pursued.
By Year 3 of this research project, we anticipate the generation of a gene array that will permit cell responses to metals to be analyzed. This array will be tested with authentic metal solutions and then with environmental samples.
Journal Articles:
No journal articles submitted with this report: View all 8 publications for this projectSupplemental Keywords:
watersheds, groundwater, mine spoils, risk assessment, proteomics, genomics, DNA chip arrays, microbiology, bioavailability, bioremediation, Pseudomonas putida, restoration, runoff, bacterial sensors, bioengineering, biosensors, cadmium and cadmium compounds, Cd, chromium and chromium compounds, Cr, environmental measurement, field detection, field monitoring, heavy metals, metal biosensors, nanoengineering, nanosensors,, RFA, Scientific Discipline, Toxics, Water, POLLUTANTS/TOXICS, Ecosystem Protection/Environmental Exposure & Risk, Sustainable Industry/Business, Environmental Chemistry, Chemicals, Monitoring/Modeling, New/Innovative technologies, 33/50, Engineering, Chemistry, & Physics, Environmental Engineering, environmental monitoring, nanosensors, cadmium & cadmium compounds, chromium & chromium compounds, bioengineering, environmental measurement, nanotechnology, field monitoring, bacterial sensors, field detection, biosensors, cadmium, heavy metalsProgress and Final Reports:
Original AbstractThe 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.