ACTIVITY-BASED PROTEIN PROFILING OF BACTERIAL MONOOXYGENASES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010922
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Plant & Microbial Biology

Non Technical Summary
Monooxygenases are enzymes that incorporate a single atom of oxygen from O2 and the reactions catalyzed by these enzymes play central roles in many of the major biogeochemical cycles such as the nitrogen and carbon cycles. Monooxygenases also often play key roles in natural and engineered process used for the remediation of environmental contamination by organic pollutants. Currently conventional molecular tools are often used to identify the microorganisms that are present in environmental samples and to assess the abundance of genes that encode important enzymes, including monooxygenases. However, for measurements of enzyme abundance, these approaches are indirect and quantify the potential for expression of these enzymes rather than measuring directly the amount of actual enzyme present. In this project the aim is to use activity-based protein profiling (ABPP) to identify and quantify bacterial monooxygenases. This technique relies on the initial in vivo inactivation of the target enzymes using diyne probes. After inactivation, the bacterial cells are recovered from the environmental matrix and the inactivated enzymes will be identified by proteomic approaches and quantified by fluorescence detection. The long-term aim will be to compare in situ bacterial activities with cell- or -protein-associated fluorescence to determine whether ABPP-based measurements can be used to accurately predict microbial activities in environmental samples. This approach could provide a rapid and effective method for predicting the environmental activities of monooxygenase-expressing bacteria and could find application in areas ranging from agriculture to waste water treatment through to bioremediation of pollutants.

Animal Health Component

15%

Research Effort Categories

Basic

65%

Applied

15%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	4099	1100	100%

Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

bacteria

activity-based protein profiling

environmental microbiology

Goals / Objectives
The overall aim of this project is to develop activity-based protein profiling as an approach for identifying and quantifying bacterial monooxygenase enzymes in pure cultures and environmental samples. The following are the specific objectives of the project.1) Determine the range of model bacterial monooxygenases that are susceptible to irreversible inactivation by diyne and similar inactivators.2) Develop appropriate methods for inactivating and recovering whole bacteria or bacterial enzymes from environmental samples.3) Develop methods for estimating rates of environmental processes based on activity-based profiling of bacterial monooxygenases.

Project Methods
The research will initially involve laboratory studies of pure cultures of bacteria that express well-characterized monoxygenase enzymes. The susceptibility of these enzymes to irreversible inactivation by diynes and other potential ABPP probes will be assessed using well-established kinetic approaches and physiological studies. After inactivation, the enzymes will be covalently conjugated with either fluors or affinity purification tags using copper-catalyzed alkyne/azide cycloaddition reactions. The labeled enzymes will then be identified using either in-gel or on-bead digestion followed by liquid chromatography/mass spectrometry. In subsequent studies using mixed microbial populations in environmental samples, the same approach outlined above will be used except that we will develop and optimize approaches for recovering either whole cells or total protein from the sample matrix prior to the analysis of protein-bound fluorescence. In the final part of the project rates of specific processes catalyzed by bacterial monooxygenases (e.g. methane or ammonia oxidation) will be determined in microcosm studies using samples taken from diverse environments. The identify and abundance of specific monooxygenase enzymes in these samples will then be determined using activity-based protein profiling approaches and levels of these enzymes will be compared with conventional molecular approaches such as qPCR and RT-qPCR that are widely used to establish gene abundance and gene transcription.This project will be used initially and primarily to train graduate students in modern and emerging protein-based approaches for characterizing microbial processes in the environment. As many monooxygenase enzymes are responsible for biodegrading organic pollutants it is also anticipated that efforts will be made to help environmental engineers and other relevant interested parties to use the techniques developed in this project and to interpret the results of these studies. The effectiveness of these efforts will be determined through several routes including the number of publications, the number of publication citations and the number of conference or workshop presentations made about the use the techniques and results of this project.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience for this project includes research scientists interested in determining the range and abundance of monooxygenase-expressing bacteria. In addition, this approach is of interest to environmental engineers who aim to understand the diversity of monooxygenase-expressing bacteria that may be involved in the biodegradation of environmental pollutants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training opportunities for 3 PhD and 1 MS-level graduate students How have the results been disseminated to communities of interest?The results of the project have been disseminated in two webinarsand two invited platform presentations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the last reporting period we have continued to explore the applicability of activity-based labeling of monooxygenase-expressing bacteria. However, rather than simply demonstrating that the underlying technique is widely applicable to diverse monooxygenases, we havestarted to examine whether this approach can be used in a quantitative fashion in three separate studies projects. The first of these studies continued to examine the toluene-2-monooxygenase (T2MO)found in Burkholderia vietnamiensis G4. One of the interesting aspects of this bacterium is that toluene-grown cells can oxidize the environmental pollutant, trichlorethylene (TCE). Phsyiological studies conducted by a collaborator at Oregon State University (Dr. Lewis Semprini) have demonstrated that TCE-degrading activity of this strain is very high in cells grown on benzyl alcohol and is also very high in cells grown on benzyl esters (e.g. benzyl acetate and benzyl butyrate) that can be hydrolyzed to release benzyl alcohol. In contrast, cells grown on acetate or butyrate alone have no detectable TCE-degrading activity. Our results have shown that the level of activity-based labeling of whole cell extracts analyzed by SDS-PAGE and flourescent imaging closely matches the measured in vivo TCE-degrading activity of cells grown on a wide variety of substrates. In the second study we have examined the sensitivity of two concurrently expressed monooxygenases to selective inactivation by treatments including illumination by near UV light and extremely low concentrations of acetylene (<1 Pa)are also accurately reflected in the overall level of protein labeling detected in SDS-PAGE analyses. Lastly, we have also used activity-based labeling to identify growth substrates that promote the expression of monooxygenases in Rhodococcus rhodochrous ATCC 21198. Our labeling results showthat cells grown on gaseous alkanes and exhibit high levels of short chain alkane monooxygenase activity are also strongly labeled using our approach. The sensitivity of our labeling approach has also enabled us to detect much lower levels of activity in alcohol grown cells. This discovery has in turn enable us to develop sklow release versions of these weakly inducing substrates that can then be used to maintain contaminant-degrading activity of cells for as long as a year.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Rasmussen MT, Saito A, Hyman MR, Semprini L. (2020) Co-encapsulation of slow release compounds and Rhodococcus rhodochrous ATCC 21198 in gellan gum beads to promote the long-term aerobic cometabolic transformation of 1,1,1-trichloroethane, cis-1,2-dichloroethene and 1,4-dioxane. Environmental Science: Processes & Impacts 22:771-791

Progress 10/01/18 to 09/30/19

Outputs
Target Audience: The target audience for this project includes research scientists interested in determining the range and abundance of monooxygenase-expressing bacteria. In addition, this approach is of interest to environmental engineers who are interested in understanding the diversity of monooxygenase-expressing bacteria that may be involved in the biodegradation of environmental pollutants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provioded training opportunities for 3 PhD and 1 MS-level graduate students How have the results been disseminated to communities of interest? The results of the project have been disseminated in a webinar and two invited platform presentations,. Several student posters have also been presented at national conferences. What do you plan to do during the next reporting period to accomplish the goals? Our main aims in the next reporting period will be to complete our studies of the toluene-oxidizing bacterium, Burkholderia vietnamiensis G4 and an alkane-oxidizing bacterium, Mycobacterium vaccae JOB5. This research will emphasize the development of selective probes for specific monooxygenases, with particular emphasis on the copper-containing monooxygenase found in several alkane oxidizing strains. A longer term aim is to harness our current technique, and a simplified one step labelling process, to examine bacteria using fluorescence-activated cell soryting.

Impacts
What was accomplished under these goals? We have determined that the basic approach used in our studies can be applied to many model bacteria that are known to express monooxygenase enzymes, these include representative ammonia, methane, gaseous alkane, gaseous alkene, toluene, and phenol-metabolizing bacteria. We have also developed the use of Nycodenz-density gradient centrifugation as a mechanism for extracting intact bacteria from soil samples. In the case of alkane-oxidizing bacteria, we have also established that there is a strong relationship between the levels of monooxygenase-associated protein fluorescence (as determined by SDS-PAGE) and monooxygenase activity. A similar relationship has also been established for model toluene-oxidizing ?bacteria and whole cell fluorescence, as determined by flow cytometry.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Chu M-Y, J, Bennett PJ, Dolan ME, Hyman MR, Peacock AD, Bodour A, Andersen RH, Mackay DM, Goltz MN (2018) Concurrent in situ cometabolic biodegradation of 1,4-dioxane and chlorinated aliphatics using groundwater recirculation. Groundwater Monitoring and Remediation 38: 53-64

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience for this project includes research scientists interested in determining the range and abundance of monooxygenase-expressing bacteria. In addition, this approach is of interest to environmental engineers who are interested in understanding the diversity of monooxygenase-expressing bacteria that may be involved in the biodegradation of environmental pollutants. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provioded training opportunities for 3 PhD and 1 MS-level graduate students How have the results been disseminated to communities of interest?The results of the project have been disseminated in two separate webinars and several student posters presented at n ational conferences. What do you plan to do during the next reporting period to accomplish the goals?Our main aims in the next reporting period will be to publsih two paers that make extensive use of this technique. These papers will focus on studies on a toluene-oxidizing bacterium, Burkholderia vietnamiensis G4 and an alkane-oxidizing bacterium, Mycobacterium vaccae JOB5. Our research will also focus on moving this technique away from pure culture studies through to examining monooxygenase-expressing bacteria in environmental samples. A longer term aim is to harness our current technique, and a simplified one step labelling process, to examine bacteria using fluorescence-activated cell soryting.

Impacts
What was accomplished under these goals? We have determined that the basic approach used in our studies can be applied to many model bacteria that are known to express monooxygenase enzymes, these include representative ammonia, methane, gaseous alkane, gaseous alkene, toluene, and phenol-metabolizing bacteria. We have also developed the use of Nycodenz-density gradient centrifugation as a mechanism for extracting intact bacteria from soil samples. In the case of alkane-oxidizing bacteria, we have also established that there is a strong relationship between the levels of monooxygenase-associated protein fluorescence (as determined by SDS-PAGE) and monooxygenase activity. A similar relationship has also been established for model toluene-oxidizing bacteria and whole cell fluoresecnce, as determined by flow cytometry.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience: The target audiences for the research in this project include researchers involved in fundamental studies of microbiological processes as well as environmental engineers and state regulators interested in methods for remediating contaminated sites Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has providing training opportunities for graduate students enrolled in at least two different graduate programs. How have the results been disseminated to communities of interest?See publication list What do you plan to do during the next reporting period to accomplish the goals?Our main efforts in the next reporting period will be to determine how to effectively isolate intact bacteria from soil samples as well as from ground water containing sediment.

Impacts
What was accomplished under these goals? We completed a study of the activity-based protein profiling (ABPP) of ammonia monooxygenase in the canonical ammonia-oxidizing bacterium, Nitrosomonas europaea. We also extended similar approaches to a wide range of other bacteraiial monooxygenases including several soluble diiron monooxygenases (SDIMOs).

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Activity-based protein profiling of ammonia monooxygenase in Nitrosomonas europaea. Bennett, K, Sadler, NC, Wright AT, Yeager C, and Hyman M