MICROBIAL FORMALDEHYDE OXIDATION

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0181960
• Project Start Date: Oct 1, 1998
• Project End Date: Sep 30, 2005
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
BACTERIOLOGY

Non Technical Summary
(N/A)

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
206	4010	1100	100%

Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

microbial metabolism

bacteria

formaldehyde

energy production

toxins

bioremediation

photosynthesis

mutants

gene expression

signal transduction

carbon dioxide

methanol

metabolic pathways

bacterial genetics

glutathione

dehydrogenases

oxidation

transcription

gene regulation

rhodobacter sphaeroides

Goals / Objectives
Our long range goal is to define biological pathways for formaldehyde oxidation and sensing. In particular, we wish to identify how cells generate energy and one-carbon skeletons from formaldehyde oxidation. In addition, we will determine how cells sense formaldehyde and control expression of genes required for oxidation and assimilation of this metabolite. This project capitalizes on the existence of an inducible glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) in the facultative phototrophic bacterium Rhodobacter sphaeroides. In photosynthetic cells, GSH-FDH is required to use methylated compounds like methanol as a sole carbon source. Under respiratory conditions, GSH-FDH dissimilates the formaldehyde produced when methanol is co-metabolized with other carbon sources. Our experiments also take advantage of existing mutations that alter expression of the GSH-FDH structural gene (adhI). We have shown there is both negative and positive control over adhI exprssion. The sensor/histidine kinase-response regulator system (GfdRTS) represses adhI transcription while the redox-response PrrA protein activates promoter. A second class of trans-acting regulatory mutations (defined by alleles like spd-7) could alter function of an activator of adhI transcription (SpdA). Our short range goals are 1) to define the pathway for formaldehyde oxidation and assimilation and 2) to determine how cells sense and respond to formaldehyde. Our general approaches to these questions are presented below

Project Methods
1. Define the pathway for formaldehyde oxidation and assimilation. To accomplish this, we will take advantage of the inducible oxidation of formaldehyde that occurs when methanol is metabolized by R. sphaeroides. Specifically, we will characterize mutants defective in either respiratory methanol oxidation or photosynthetic utilization of methanol as a sole carbon source. Mutants defective in respiratory methanol metabolism will define proteins required to oxidize toxic formaldehyde. In contrast, mutants blocked in photosynthetic methanol utilization will identify enzymes that assimilate formaldehyde oxidation products into cellular material. By determining where individual lesions block formaldehyde metabolism, we will trace the flow of carbon and identify enzymes that remove this compound under respiratory conditions or assimilate it in photosynthetic cells. 2. Determine how cells sense and respond to formaldehyde. To answer this question, we will determine how adhI transcription is regulated. One set of experiments will dissect the GfdRS signal transduction chain. In particular, we will test the prediction that GfdS is a sensor/histidine kinase, ask if formaldehyde controls phosphorylation of this presumed sensor, monitor GfdR phosphorylation by GfdS, and test if phosphorylation of GfdR increases binding to a presumed adhI operator. To test if SpdA is a positive activator of adhI expression, we will characterize presumed gain-of-function mutations that increase transcription Ultimately, we seek to determine if GfdRS and SpdA respond to formaldehyde.

Progress 10/01/98 to 09/30/05

Outputs
This project analyzed how cells sense and generate energy from formaldehyde oxidation. Formaldehyde is a toxin that is produced naturally, chemically or by metabolism of a wide variety of methyl-containing compounds. Our goals are to identify how cells sense the presence of this toxic compound and determine how they generate energy and nutrients from the oxidation of formaldehyde. This research capitalizes on the role of the Rhodobacter sphaeroides glutathione dependent formaldehyde dehydrogenase (GSH FDH) in a formaldehyde oxidation pathway that is apparently found in a wide variety of microbes, plants and animals. Thus, our findings illustrate what is required for a large variety of cells to metabolize this toxic compound. A second major focus of our research is to determine how cells sense the presence of this toxic compound and control the expression of gene products required for its detoxification.

Impacts
From this work, processes and strains have been patented by WARF. These tools use bacteria to sense formaldehyde in the environment and efficiently remove this toxic compound from industrial or natural sites that routinely contain this to this chemical.

Publications

• No publications reported this period

Progress 01/01/04 to 12/31/04

Outputs
This project analyzed how cells sense and generate energy from formaldehyde oxidation. Formaldehyde is a toxin that is produced naturally, chemically or by metabolism of a wide variety of methyl-containing compounds. Our goals are to identify how cells sense the presence of this toxic compound and determine how they generate energy and nutrients from the oxidation of formaldehyde. This research capitalizes on the role of the Rhodobacter sphaeroides glutathione dependent formaldehyde dehydrogenase (GSH FDH) in a formaldehyde oxidation pathway that is apparently found in a wide variety of microbes, plants and animals. Thus, our findings illustrate what is required for a large variety of cells to metabolize this toxic compound. A second major focus of our research is to determine how cells sense the presence of this toxic compound and control the expression of gene products required for its detoxification.

Impacts
From this work, processes and strains have been patented by WARF. These tools use bacteria to sense formaldehyde in the environment and efficiently remove this toxic compound from industrial or natural sites that routinely contain this to this chemical.

Publications

• Barber, R. D. and T. J. Donohue. 1998. Function of a glutathione-dependent formaldehyde dehydrogenase in Rhodobacter sphaeroides formaldehyde oxidation and assimilation. Biochemistry 37:530-537.
• Barber, R. D. and T. J. Donohue. 1998. Pathways for transcriptional activation of a glutathione-dependent formaldehyde dehydrogenase gene. J. Mol. Biol. 280:775-784.
• Hickman, J., Barber, R. D., Skaar, E. and T. J. Donohue. 2002. A link between the membrane-bound pyridine nucleotide transhydrogenase and glutathione-dependent processes in Rhodobacter sphaeroides. J. Bacteriol. 184:400-409.
• Hickman, J., Witthuhn, V. C., Dominguez, M., and T. J. Donohue. 2004. Positive and negative transcriptional regulators of bacterial glutathione-dependent formaldehyde dehydrogenase gene expression. J. Bacteriol. 186:7914-7925.

Progress 01/01/03 to 12/31/03

Outputs
This project analyzed how cells sense and generate energy from formaldehyde oxidation. Formaldehyde is a toxin that is produced naturally, chemically or by metabolism of a wide variety of methyl-containing compounds. Our goals are to identify how cells sense the presence of this toxic compound and determine how they generate energy and nutrients from the oxidation of formaldehyde. This research capitalizes on the role of the Rhodobacter sphaeroides glutathione dependent formaldehyde dehydrogenase (GSH FDH) in a formaldehyde oxidation pathway that is apparently found in a wide variety of microbes, plants and animals. Thus, our findings illustrate what is required for a large variety of cells to metabolize this toxic compound. A second major focus of our research is to determine how cells sense the presence of this toxic compound and control the expression of gene products required for its detoxification.

Impacts
From this work, processes and strains have been patented by WARF. These tools use bacteria to sense formaldehyde in the environment and efficiently remove this toxic compound from industrial or natural sites that routinely contain this to this chemical.

Publications

• No publications reported this period

Progress 01/01/02 to 12/31/02

Outputs
This project analyzes how cells generate energy from the oxidation of formaldehyde. Formaldehyde is a toxin, potent mutagen and possible carcinogen that is produced naturally, chemically or by metabolism of a wide variety of methyl-containing compounds. Our goals are to identify how cells sense the presence of this toxic compound and determine how they generate energy and nutrients from the oxidation of formaldehyde. This research capitalizes on the role of the Rhodobacter sphaeroides glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) in a formaldehyde oxidation pathway that is apparently found in a wide variety of microbes, plants and animals. Thus, our findings illustrate what is required for a large variety of cells to metabolize this toxic compound. A second major focus of our research is to determine how cells sense the presence of this toxic compound and control the expression of gene products required for its detoxification.

Impacts
From this work, processes have been developed and patented in which bacteria could be used to sense formaldehyde in the environment and efficiently remove this toxic compound from industrial or natural sites that routinely contain this chemical.

Publications

• Hickman, J., Barber, R. D., Skaar, E. and T. J. Donohue. 2002 A link between the membrane-bound pyridine nucleotide transhyrdogenase and glutathione-dependent processes in Rhodobacter sphaeroides. J. Bacteriol. 184:400-409.

Progress 01/01/01 to 12/31/01

Outputs
Our research seeks to determine how cells generate energy from the oxidation of formaldehyde. Formaldehyde is a toxin, potent mutagen and possible carcinogen that is produced naturally, chemically or by metabolism of a wide variety of methyl-containing compounds. Our immediate goals are to identify how cells sense the presence of this toxic compound and determine how they generate energy and nutrients from the oxidation of formaldehyde. This research capitalizes on the known roles of the Rhodobacter sphaeroides glutathione﷓dependent formaldehyde dehydrogenase (GSH﷓FDH) in formaldehyde oxidation under respiratory and photosynthetic growth conditions. This enzyme is part of a formaldehyde oxidation pathway that is apparently found in a wide variety of microbes, plants and animals, so our findings will illustrate what is required for a large variety of cells to metabolize this toxic compound. A second major focus of our research is to determine how cells sense the presence of this toxic compound and control the expression of gene products required for its detoxification. From this work, we expect to develop novel ways in which bacteria could be used to sense formaldehyde in the environment and efficiently remove this toxic compound from industrial or natural sites that routinely contain this to this chemical

Impacts
This project studies the process and control of formaldehyde metabolism by a glutathione-dependent pathway in bacteria. This pathway is present in other microbes, plants and animals so our findings are relavant to many cells. In addition, the findings of our studies on the metabolism of this toxic compound have led to several patents with importance in the agricultural, industrial and medical settings

Publications

• Hickman, J., Barber, R. D., Skaar, E. and T. J. Donohue. 2002 A link between the membrane-bound pyridine nucleotide transhyrdogenase and glutathione-dependent processes in Rhodobacter sphaeroides. J. Bacteriol. 184:400-409.

Progress 01/01/00 to 12/31/00

Outputs
Formaldehyde is a potent mutagen and toxin that is produced naturally, chemically or biologically, by the oxidation of a wide variety of methyl-containing compounds. Our goal is to dissect pathways used for sensing formaldehyde and generating energy or carbon skeletons from its oxidation. This research capitalizes on the known roles of the Rhodobacter sphaeroides glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) in formaldehyde oxidation under respiratory and photosynthetic growth conditions. It also takes advantage of existing metabolic signals and mutations that alter expression of the GSH-FDH structural gene (adhI). Our short term goals are to: 1. Determine the role of genes within a potential "formaldehyde metabolism" gene cluster. We know that the region downstream of adhI encodes proteins with significant amino acid similarity to gene products that are believed to participate in formaldehyde metabolism by other cells. To determine how these gene products function in formaldehyde metabolism, we will test how loss of individual genes alters the formation of formaldehyde or the generation of energy its oxidation. 2. Identify additional gene products involved in formaldehyde oxidation. To identify other functions that are required for formaldehyde oxidation, we will analyze mutants that are killed by the addition of a metabolic source of formaldehyde like methanol under respiratory growth conditions. By identifying the lesions in methanol sensitive strains, determining if they result in formaldehyde accumulation, and testing if these genes are co-regulated with adhI, we will identify new functions and regulatory strategies that are required for sensing formaldehyde and generating energy from its oxidation. 3. Determine how cells respond to formaldehyde. Our data predicts that an intermediate in formaldehyde metabolism and the reducing power formed by formaldehyde oxidation each control the activity of proteins that positively and negatively regulate adhI expression. In one set of experiments, we will dissect what is required for GfdRTS to reduce adhI expression and test our model that an intermediate in formaldehyde oxidation relieves repression by this signal transduction pathway. To determine if PrrA directly activates adhI expression, presumably in response to the reducing power generated by formaldehyde oxidation, we will test if this protein binds to and stimulates transcription from this promoter in vitro. To test if SpdA is a second activator of adhI expression, we will characterize presumed gain-of-function mutations (spdA*) that increase function of this promoter.

Impacts
This project seeks to determine how cell sense, respond to and remove a toxic and potentially carcinogeinc compound like formaldehyde. This research is of basic interest since all cells can generate or encounter this toxic compound in their environment. Formaldehyde is also a major product of industrial society, either itself or from biodegradation of compounds that generate formaldehyde. Thus we are also actively prusuing the licensing of our findings in order to use bacteria as a formaldehyde bioremediation agent.

Publications

• Hickman, J., R. D. Barber, Skaar, E. and T. J. Donohue. 2001. A role for the membrane-bound pyridine nucleotide transhyrdogenase in biological formaldehyde oxidation. (In preparation).
• Witthuhn, V. C., and T. J. Donohue. 2001. GfdRTS controls transcription of Rhodobacter sphaeroides gene products involved in formaldehyde oxidation. (In preparation).

Progress 01/01/99 to 12/31/99

Outputs
Formaldehyde is a potent mutagen and toxin that is produced naturally, chemically or biologically, by the oxidation of a wide variety of methyl-containing compounds. Our goal is to dissect pathways used for sensing formaldehyde and generating energy or carbon skeletons from its oxidation. This research capitalizes on the known roles of the Rhodobacter sphaeroides glutathione-dependent formaldehyde dehydrogenase (GSH-FDH) in formaldehyde oxidation under respiratory and photosynthetic growth conditions. It also takes advantage of existing metabolic signals and mutations that alter expression of the GSH-FDH structural gene (adhI). Our short term goals are to: 1. Determine the role of genes within a potential "formaldehyde metabolism" gene cluster. We know that the region downstream of adhI encodes proteins with significant amino acid similarity to gene products that are believed to participate in formaldehyde metabolism by other cells. To determine how these gene products function in formaldehyde metabolism, we will test how loss of individual genes alters the formation of formaldehyde or the generation of energy its oxidation. 2. Identify additional gene products involved in formaldehyde oxidation. To identify other functions that are required for formaldehyde oxidation, we will analyze mutants that are killed by the addition of a metabolic source of formaldehyde like methanol under respiratory growth conditions. By identifying the lesions in methanol sensitive strains, determining if they result in formaldehyde accumulation, and testing if these genes are co-regulated with adhI, we will identify new functions and regulatory strategies that are required for sensing formaldehyde and generating energy from its oxidation. 3. Determine how cells respond to formaldehyde. Our data predicts that an intermediate in formaldehyde metabolism and the reducing power formed by formaldehyde oxidation each control the activity of proteins that positively and negatively regulate adhI expression. In one set of experiments, we will dissect what is required for GfdRTS to reduce adhI expression and test our model that an intermediate in formaldehyde oxidation relieves repression by this signal transduction pathway. To determine if PrrA directly activates adhI expression, presumably in response to the reducing power generated by formaldehyde oxidation, we will test if this protein binds to and stimulates transcription from this promoter in vitro. To test if SpdA is a second activator of adhI expression, we will characterize presumed gain-of-function mutations (spdA*) that increase function of this promoter.

Impacts
This research seeks to determine the process by which cells sense and metabolize toxic one-carbon compunds like formaldehyde. The pathway being analyzed in this bacterium is present in all cell types so our information will be relevant to other bacteria, plants and animals. We are also using this work to develop formaldehyde bioremediation systems and biosensors which have many basic and applied uses in the private sector.

Publications

• Barber, R. D. and T. J. Donohue. 1998. Function of a glutathione-dependent formaldehyde dehydrogenase in Rhodobacter sphaeroides formaldehyde oxidation and assimilation. Biochemistry 37:530-537.
• Barber, R. D. and T. J. Donohue. 1998 Pathways for transcriptional activation of a glutathione-dependent formaldehyde dehydrogenase gene. J. Mol. Biol. 280:775-784.