Superfund Research Program
A Cellular Biosensor to Detect Chlorocatechols
Catechols, including chlorocatechols, are introduced into the environment by both industrial and natural sources. Because these compounds are resistant to many standard wastewater treatment processes, they may be discharged in the effluents of paper, rubber, dyes, plastics, pharmaceuticals, and cosmetics manufacturing facilities and accumulate in lake and river sediments. More importantly, catechols are produced naturally in the environment as the byproduct of microbial degradation of chlorinated aromatic compounds including polychlorinated biphenyls (PCBs). The catechol byproducts are more toxic than the parent compounds. Catechol and chlorocatechols have been characterized as strong irritants to eyes, skin, and respiratory tract and have been proven to cause DNA damage.
Scientists at the University of Kentucky (UK) have developed a cellular biosensor system to identify and quantify 3-chlorocatechol and 4-chlorocatechol, toxic breakdown products of PCBs. A biosensor is a biological monitor that recognizes a chemical or physical change and produces a measurable signal in response to the environmental change. Cellular biosensors can be designed for high sensitivity and selectivity, but the greatest advantage of cellular systems is they are capable of measuring the bioavailability of the compound being studied. Cellular biosensor systems operate as follows:
- The compound being studied passes through the cell membrane.
- The compound binds to a regulatory protein.
- The regulatory protein activates a promoter.
- The promoter activates the reporter gene.
- Translation of the reporter gene produces a reporter protein.
- Upon addition of an external substrate, the reporter protein produces a measurable signal.
To detect 3-chlorocatechol and 4-chlorocatechol, the UK researchers applied genetic engineering techniques to design and develop a cellular biosensor system using the bacteria Pseudomonas putida. These bacteria can use chlorocatechols as a carbon source, biodegrading chlorocatechols via a degradative pathway regulated by the ClcR protein. The UK researchers introduced a plasmid that contains the lacZ gene into P. putida. The lacZ gene is also controlled by the ClcR protein and encodes for b-galactosidase.
The P. putida chlorocatechol cellular biosensor developed by the UK scientists operates as follows:
- In the absence of chlorocatechol, the regulatory protein ClcR binds to the operator/promoter (O/P) region of the plasmid and prevents gene transcription.
- When bioavailable chlorocatechol is present, it passes through the cell membrane.
- Chlorocatechol binds to ClcR, releasing it from the O/P region.
- This allows transcription and translation of the lacZ gene to produce b-galactosidase.
- A chemiluminescent substrate is added to the cellular biosensor system and reacts with the b-galactosidase, producing a measurable chemiluminescent signal.
The UK scientists have verified that the level of production of b-galactosidase by the bacteria is related to the dose of chlorocatechols. They have optimized procedures used in several phases of the biosensor technique, producing an extremely sensitive system that can detect 3-chlorocatechol and 4-chlorocatechol at concentrations as low as 8 x 10-10 and 2 x 10-9 M, respectively.
The researchers verified that the system is extremely specific. They exposed the biosensor system to a series of organic compounds (catechols, chlorophenols, biphenyl) that are structurally related to chlorocatechols and might interfere with the biosensor system. No appreciable levels of b-galactosidase were produced. The UK scientists have also demonstrated that the system is capable of distinguishing 3-chlorocatechol from 4-chlorocatechol.
By developing this sensitive, selective system, the UK scientists have provided hazardous waste site remediators with a valuable tool for directly identifying and quantifying chlorocatechol compounds in the complex mixtures commonly found at Superfund sites. This cellular biosensor system may be useful for several practical applications:
- Assessment of environmental contamination ¾ the system can be used to detect low concentrations of chlorocatechols in soil, sediment, water, and biological samples.
- Evaluation of remediation efforts ¾ it is important to ensure that remediation systems designed to cleanup PCBs do not leave the site contaminated with chlorocatechols.
- Exposure studies ¾ chlorocatechols have been found in the urine of individuals exposed to chlorobenzene and therefore can serve as biomarkers of exposure.
This biosensor system is a tailor-made probe to directly monitor the level of chlorocatechols in soil, sediment, and water samples at Superfund sites. Because it does not require expensive equipment or extensive pretreatment of environmental samples, the UK biosensor system is simpler and more economical than standard methods. The UK scientists are working to develop additional biosensor systems. By using different reporter proteins that emit fluorescence or bioluminescence at different wavelengths, they are developing array sensing systems for a variety of environmental pollutants. Analysis of the color of the light generated by the bacteria will allow for identification of the pollutant(s) present in a particular environment. This would provide tools for in situ monitoring of multiple contaminants at Superfund sites.
For More Information Contact:
To learn more about this research, please refer to the following sources:
- Daunert S, Barrett G, Feliciano JS, Shetty RS, Shrestha S, Smith-Spencer W. 2000. Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev 100(7):2705-2738. PMID:11749302
- Guan X, Ramanathan S, Garris JP, Shetty RS, Ensor CM, Bachas LG, Daunert S. 2000. Chlorocatechol detection based on a clc operon/reporter gene system. Anal Chem 72(11):2423-2427. PMID:10857616
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