Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
2000-03-08
2004-08-10
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S091200, C435S471000, C435S463000, C435S170000, C435S029000, C435S008000, C435S004000, C435S139000, C536S023100
Reexamination Certificate
active
06773918
ABSTRACT:
BACKGROUND OF THE INVENTION
In the last three decades, there has been a significant increase in government regulations that hold industrial entities accountable for the chemical pollution that results from their manufacturing activities. In order to comply with environmentally sensitive regulations, businesses must be able to identify contamination and monitor its remediation processes. The cost and technical complexity of chromatographic methods currently in use may act to limit characterization of contaminated sites. One way to lower the cost of detection is to use biosensors derived from genetic systems of bacteria that have evolved to use organic contaminants as growth substrates.
Certain strains of soil bacteria have evolved the capacity to use toxic organic chemicals as food sources. Production of the required metabolic enzymes is, in some cases, controlled by a particular type of regulatory protein that detects the toxic chemical through a direct physical interaction. The protein-chemical complex binds to a cognate promoter sequence and activates expression of genes encoding the required metabolic enzymes. This type of regulatory protein can be utilized as a pollution detecting component in bacteria that have been engineered to signal the presence of environmental pollution.
The most basic whole cell bacterial biosensors are created by placing a reporter gene under control of an inducible promoter. Expression of the reporter gene provides a measurable signal when the appropriate transcription activator protein interacts with an effector chemical.
Phenol and various substituted phenols are used in the manufacture of dyes, photographic chemicals, pesticides, lumber preservatives, microbiocides and herbicides. Current methods for detecting phenol contaminants include gas chromatography and high-pressure liquid chromatography. These chromatographic methods require expensive equipment and highly trained technicians. In response to the U.S. Environmental Protection Agency having listed eleven phenols as priority pollutants, industries that use phenol and phenol derivatives require simple and inexpensive detection methods to identify spills, leaks, and other phenol contamination that result from their manufacturing and service activities.
The construction of bacterial biosensors is limited by the restricted availability of bacteria that are known to metabolize a chemical of interest and, in particular, by the absence of knowledge concerning the genetic systems that control bacterial response to the chemical. Fortunately, some of the bacterial genetic systems that support metabolism of polluting chemicals show significant genetic relatedness. Operons encoding genes required for metabolism of phenol, toluene, benzene, and xylene in some Pseudomonas and Acinetobacter species are headed by promoters recognized by sigma-54-associated RNA polymerase. Transcription directed by these promoters occurs when the system's regulatory protein detects the presence of the substrate for the catabolic enzymes. Proteins in this category include DmpR, XylR, MopR, PhhR, PhlR, and TbuT. These six proteins show significant similarity to one another at the amino acid level. Sequence information and domain swapping experiments indicate that the general arrangement of these regulatory proteins consists of discrete areas with three independent functions including chemical detection, polymerase activation, and DNA-binding.
XylR and DmpR are the most studied members of this group of transcription activators. The
Pseudomas putida
XylR has already served as the detection component for a number of biosensors based on its ability to activate transcription in response to xylene, toluene and benzene. DmpR, the product of the Pseudomonas CF600 dmpR gene, mediates expression of the dmp operon to allow growth on simple phenols. Transcription from Pdmp, the promoter heading the dmp operon, is activated when DmpR senses the presence of phenol, cresols, mono-chlorinated phenols, and some mono-methylated phenols (See, e.g., V. Shingler and T. Moore, “Sensing of aromatic compounds by the DmpR transcriptional activator of phenol-catabolizing Pseudomonas sp. strain CF600”, J. Bacteriol. 176:1555-1560 (1994)). Disubstituted phenols, such as 2,4-dichlorophenol or 2,4-dimethylphenol, are inferior inducers of dmp transcription.
Domain swapping experiments to form XylR-DmpR hybrids demonstrated that the sensor activity of these regulatory proteins is localized to the amino terminal region. By switching the first 234 amino acids of DmpR with those from XylR, Shingler and Moore, supra, created a chimeric protein that activated transcription from Pdmp in response to toluene and xylene, but not phenol or cresol. The results of the hybrid protein experiments indicated that transcription from Pdmp depends on a direct physical interaction between the sensor domain of DmpR and the inducing phenol.
The single regulatory protein, and the independent domain arrangement of DmpR and other proteins of this type make them particularly suitable candidates for genetic manipulation and suggests a way around the restrictions imposed by limited information about the genetics that control bacterial degradation of xenobiotics. Such altered proteins have the potential to extend the chemical target range of biosensors beyond that based on natural systems.
Therefore, it is an object of the present invention to alter the chemical sensing domain of the protein DmpR to respond to phenol and phenol derivatives which are poorly detected or undetected by the wild type protein.
Another object of the invention is to alter the chemical sensing domain of the protein DmpR to respond to phenol and phenol derivatives without disturbing its transcription activating functions.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
SUMMARY OF THE INVENTION
To achieve the foregoing and other objects, and in accordance with the purposes of the present invention, as embodied and broadly described herein, the method for enhancing bacterial response to organic molecules, where the bacteria have a regulatory protein with discrete functional domains for independent activities, one such domain being a sensor domain that detects the organic molecules through a direct physical interaction forming a protein-molecule complex which binds to a cognate promoter sequence and activates expression of genes encoding metabolic enzymes, includes modifying the sensor domain of the regulatory protein such that the response to the organic molecule is enhanced without altering the other domains.
Benefits and advantages of the present invention include the creation of a large variety of engineered proteins with abilities to detect toxic organic chemicals. Such engineered proteins will be useful in development of environmentally beneficial tools that both detect and degrade polluting chemicals.
REFERENCES:
patent: 5837458 (1998-11-01), Minshull et al.
Shingler et al. “An Aromatic effector specificity mutant of the transcriptional regulator DmpR overcomes the growth constraints of Pseudomonas sp. strain CF600 on para-substituted methylphenols”, (1994) J Bacteriol 176:7550-7.*
Willardson et al. “Development and testing of a bacterial biosensor for toluene-based environmental contaminants”, (1998) Appl Environ Microbiol 64:1006-1012.*
Schirmer et al. “Expression, inducer spectrum, domain structure, and function of MopR, the regulator of phenol degradation in Acinetobacter calcoaceticus NCIB8250”, (1997) J Bacteriol 179:1329-1336.*
Ng et al. “Aromatic effector activation of the NtrC-like transcriptional regulator PhhR limits the catabolic potential of the (methyl)phenol degradative pathway it controls”, (1
Kuske Cheryl R.
Terwilliger Thomas C.
Wise Arlene A.
Freund Samuel M.
Prouty Rebecca E.
Ramirez Delia
Sharples Kenneth K.
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