Microbial system for formaldehyde sensing and remediation

Details Microbial system for formaldehyde sensing and remediation Microbial system for formaldehyde sensing and remediation

1998-11-16

2001-06-05

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Process of utilizing an enzyme or micro-organism to destroy...

Destruction of hazardous or toxic waste

C435S252100, C435S252300

Reexamination Certificate

active

06242244

ABSTRACT:

BACKGROUND OF THE INVENTION
Increasing concern over environmental contaminants has made desirable systems for detecting and remediating such contaminants. Among the more important contaminants of industrial societies is formaldehyde. The health and environmental effects of formaldehyde have been well characterized, as has their distribution in soil and water. See, e.g. “Health and Environmental Effects Profile for Formaldehyde,” Report No. EPA/600/X-85/362, Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Office of Research and Development, US Environmental Protection Agency, Cincinnati, Ohio 45268 (NTIS document number PB88-174958) (October 1985) and “Exploratory Report Formaldehyde,” Report No. 710401018, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands (NTIS Report No. PB93-224483) (October 1992).
Evidence of formaldehyde carcinogenicity in rats and other epidemiological evidence have led to the classification of this compound as a probable human carcinogen. Formaldehyde is a common product of several industries (wood processing, paper production) that feed run-offs into aquatic ecosystems. Formaldehyde, which is present in approximately 2,000 entries of the Product Register Data Base, is also released from common cleaning agents, soaps, shampoos, paints, and lacquers. Little is known about how cells sense this toxin, metabolize it, or control the genes that are required for formaldehyde oxidation.
Existing chemical monitors for formaldehyde are time-consuming, exhibit variable sensitivity, and are prone to cross-reactivity with other aldehydes. It would be useful to utilize a biological system capable of specific response to, and detection of, formaldehyde. Moreover, a system capable of responding to the presence of formaldehyde could be useful as a bioremediation tool to reduce or eliminate formaldehyde as an environmental contaminant. However, to date, no biological formaldehyde-inducible detection or remediation system has been constructed.
Most organisms have the ability, using various metabolic pathways, to generate both energy and carbon skeletons by oxidizing a wide spectrum of substrates, including substrates that are themselves environmental toxins. Formaldehyde oxidation can be mediated by Class III alcohol dehydrogenase enzymes, also called glutathione-dependent formaldehyde-dehydrogenases or GSH-FDH, which are a well-studied class of the zinc-dependent alcohol dehydrogenase protein family that is known in both prokaryotes and eukaryotes.
GSH-FDH enzymes are believed to perform different functions depending upon the cell type. In some organisms, GSH-FDH serves a role in the catabolism of methylated compounds. For example, some methylotrophic microbes use GSH-FDH to generate carbon skeletons and NADH from the formaldehyde that is produced from methanol oxidation. In non-methylotrophic organisms, GSH-FDH rids the cells of toxic formaldehyde produced from the oxidation of methylated substrates such as choline, sarcosine, methionine, O-methylated amino acids, methanol, methyl halides, or several N-, O-, or S-methylated xenobiotics. In both roles, GSH-FDH enzymes generate reducing power, NADH, and a product, S-formylglutathione, that can be subsequently oxidized to generate one-carbon compounds such as formate or carbon dioxide.
In particular, S-hydroxy methyl glutathione (HMGSH), an adduct formed spontaneously by glutathione (GSH) and formaldehyde (HCHO) (reaction 1), is both the preferred in vitro substrate and the presumed physiologically relevant substrate in vivo for GSH-FDH enzymes (reaction 2).
(1) HCHO+GSH→HMGSH (spontaneous)
(2) HMGSH+AND
+
→S-formylglutathione+NADH+H
+
Unlike other classes of alcohol dehydrogenase enzymes, members of the GSH-FDH family do not exhibit appreciable activity with short aliphatic alcohol substrates such as ethanol. Instead, GSH-FDH enzymes catalyze the AND-dependent oxidation of long chain hydroxylated fatty acids (i.e., 12-hydroxydodecanoic acid) or long chain alcohols.
In the photosynthetic purple bacterium
Rhodobacter sphaeroides
, a glutathione-dependent formaldehyde dehydrogenase protein (AdhI) is encoded by adhI in an operon that also includes cycI which encodes an isoform of the cytochrome c
2
family of electron transport proteins. The AdhI protein encoded by adhI has the characteristic substrate preference of a glutathione-dependent formaldehyde dehydrogenase. Ferguson plot analysis, using zymograms, suggests that the functional form of AdhI is a homodimer of approximately 40 kDa subunits, analogous to other such enzymes. The complete nucleotide sequence of
R. sphaeroides
adhI has not heretofore been disclosed.
Expression of the adhI-cycI operon is thought to be regulated since the abundance of isocytochrome c
2
was increased by a trans-acting regulatory mutation. Rott, et al., “Genetic Evidence for the Role of Isocytochrome c
2
in Photosynthetic Growth of
Rhodobacter sphaeroides
Spd Mutants,”
J. Bacteriol
. 175:358-66 (1993).
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized in that a protein-encoding genetic sequence that is operably linked to a formaldehyde-inducible transcriptional promoter sequence and a cis-acting operator in a genetic construct can be specifically transcribed in a bacterial host cell that contains a formaldehyde-specific regulatory system disclosed herein, when the cell is exposed to formaldehyde. The genetic construct is useful in an assay for detecting formaldehyde.
The invention is further summarized in that a formaldehyde dehydrogenase gene that is operably linked to the formaldehyde-inducible transcriptional promoter/operator sequence in a genetic construct can be specifically transcribed in a bacterial host cell that contains the formaldehyde-specific regulatory system when the cell is exposed to formaldehyde, thereby producing a formaldehyde dehydrogenase enzyme. The construct is useful in a bioremediation assay capable of metabolizing formaldehyde into single-carbon skeletons.
The invention is further summarized in that a bacterial cell can respond in a biosensing assay or bioremediation assay if it contains a genetic construct that includes a protein-encoding genetic sequence operably linked to the formaldehyde-inducible transcriptional promoter sequence linked to a cis-acting operator, a trans-acting regulator of the transcriptional promoter and a formaldehyde-specific sensor.
When linked to the cis-acting operator sequence described herein, the transcriptional promoter can be induced in the presence of formaldehyde to transcribe the operably linked protein-encoding sequence in the presence of a two-component regulatory system (also referred to as a histidine kinase-response regulator) wherein one or more components sense the presence of formaldehyde (hereinafter referred to as a “sensor”) and another component, the so-called “response regulator”) regulates transcription of the protein-encoding sequence. The sensor and the regulator molecule can be provided as proteins or can be encoded on a genetic construct when placed under the control of a suitable promoter. The gene or genes encoding the sensor and/or regulator can be provided on the same construct as the formaldehyde-inducible promoter operably linked to the coding sequence, or can be provided on separate constructs. If provided on the same construct, the gene or genes should be under the transcriptional control of an unregulated or constitutive promoter.
The present invention is also summarized in that a method for detecting the presence of formaldehyde includes the steps of (1) adding to a sample to be tested a bacterial cell that contains (a) a genetic construct that includes a genetic sequence encoding a detectable protein operably linked to the disclosed promoter/operator sequence, and further contains (b) a trans-acting regulator of the formaldehyde-specific transcriptional promoter as well as (c) a formaldehyde-specific sensor, and then (2) monitoring for the presence of t

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