Progress 10/01/10 to 09/30/15
Outputs
Target Audience:Results from these studies are targeted to research scientists interested in herbicide degradation, enzymology, water reclamation, bioremediation, agricultural run-off issues, and to researchers interested in the evolution of bacterial genes and degradation pathways. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Results from these studies are useful in determining howpesticides in general, and specifically members of the neonicotinoids family of pesticides degrade in the environment and how the bacteria carry-out these degradation reactions. Results from these studies will allow researchers to determine how thesepesticides affect beneficial and harmful microbes in the environment and how this information can be used to bioremediate contaminated water supplies and soils. We also report here the development of strategies to biodegrade antibiotics in the environment. Antibiotic resistance among bacteria is becoming a major health problem, due in part to their indiscriminate use in the environment, their transport to sewage systems and the subsequent movement of antibiotic resistance genes into microbes associating with humans and animals.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kandil MM, Trigo C, Koskinen WC, Sadowsky MJ. 2015. Isolation and Characterization of a Novel Imidacloprid-Degrading Mycobacterium sp. Strain MK6 from an Egyptian Soil. J Agric Food Chem. 63:4721-4727.
- Type:
Other
Status:
Published
Year Published:
2015
Citation:
2. Burch TR, Sadowsky MJ, LaPara TM. 2015 Modeling the fate of antibiotic resistance genes and class 1 integrons during thermophilic anaerobic digestion of municipal wastewater solids. Appl. Microbiol. Biotechnol. PMID: 26481624.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Results from these studies are targeted to research scientists interested in herbicide degradation, enzymology, water reclamation, bioremediation, agricultural run-off issues, and to researchers interested in the evolution of bacterial genes and degradation pathways. Changes/Problems: While we initially think that resistance to mesitrione is likley due to a general adapation mechanisms involving oxidative stress repair systems, it may be that other mechanisms operate to allow resistance. If this is the case, we will determine the genetic basis for these other potential resistance mechanisms. What opportunities for training and professional development has the project provided? In this project both graduate students and postdocs are working with staff and a visiting scientist to understand the impact of selected herbicdes on soil microbial populations. How have the results been disseminated to communities of interest? Through refereed journal publications, verbal presentations and poster sessions at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? We will further examine mechanisms by which bacteria are resistant to mesitrione and how this alters competitiveness among soil microbes.
Impacts
What was accomplished under these goals?
The intensive use of agrochemicals has played an important role in increasing agricultural production in the US and elsewhere. One of the main impacts of agrochemical use has been changes in population structure of soil microorganisms. The aim of this study was to evaluate the influence of the herbicide mesotrione, which inhibits 4-hydroxyphenylpyruvate dioxygenase activity, on a model non-environmental microbe, E. coli. Mesotrione is a new herbicide developed for the selective pre- and post-emergence control of broad-leaved and grassy weeds in corn. This bacterium was chosen as its genome is well known. Results of our studies showed that this strain was able to tolerate high concentrations of the herbicide, and completely degraded mesotrione after 3 h of exposure. Growth rates in the presence of mesotrione were lower than in a control, prior to the period of degradation, showing the toxic effect on the bacterial cells. Considering that E. coli DH5-α had no previous contact with mesotrione, the defense system found in this strain can be considered as general and non-specific. This strategy may be a general adaptation mechanism by which bacterial strains in agricultural soils resist damage due to the presence of herbicides. We previously reported the cloning, sequencing, and expression of six genes involved in the atrazine biodegradation pathway of Pseudomonas sp. strain ADP, which is initiated by atzA, encoding atrazine chlorohydrolase. Here we explored the use of enhanced expression of a modified bacterial atrazine chlorohydrolase, p-AtzA, in transgenic grasses (tall fescue, perennial ryegrass, and switchgrass) and the legume alfalfa for the biodegradation of atrazine. Enhanced expression of p-AtzA was obtained by using combinations of the badnavirus promoter, the maize alcohol dehydrogenase first intron, and the maize ubiquitin promoter. For alfalfa, we used the first intron of the 5'-untranslated region tobacco alcohol dehydrogenase gene and the cassava vein mosaic virus promoter. Resistance of plants to atrazine in agar-based and hydroponic growth assays was correlated with in vivo levels of gene expression and atrazine degradation. The in planta expression of p-atzA enabled transgenic tall fescue to transform atrazine into hydroxyatrazine and other metabolites. Results of our studies highlight the potential use of transgenic plants for bioremediating atrazine in the environment. Results from these studies are useful in determining how herbicides in general, and specifically members of the benzoylcyclohexane-1,3-dione family of herbicides degrade in the environment and how the bacteria carry-out these degradation reactions. Results from these studies will allow researchers to determine how these herbicides affect beneficial and harmful microbes in the environment and how this information can be used to remediate contaminated water supplies and soils. We also report here the development of transgenic grasses that can be used to biodegrade atrazine in the environment. These grasses can be used for edge-of-field application to prevent atrazine run-off into waterways.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Olchanheski LR, Dourado MN, Beltrame FL, Zielinski AA, Demiate IM, Pileggi SA, Azevedo RA, Sadowsky MJ, Pileggi M. 2014. Mechanisms of tolerance and high degradation capacity of the herbicide mesotrione by Escherichia coli strain DH5-?. PLoS One. 2014 Jun 12;9(6):e99960. doi: 10.1371/journal.pone.0099960. eCollection 2014.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
2. Vail AW, Wang P, Uefuji H, Samac DA, Vance CP, Wackett LP, Sadowsky MJ. 2014. Biodegradation of atrazine by three transgenic grasses and alfalfa expressing a modified bacterial atrazine chlorohydrolase gene. Transgenic Res. 2014 Nov 29.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Results from these studies are targeted to research scientists interested in herbicide degradation, enzymology, water reclamation, agricultural run-off issues, and to researchers interested in the evolution of bacterial genes and degradation pathways. Changes/Problems: While we initially think that resistance to mesitrione is likley due to a general adapation mechanisms involving oxidative stress repair systems, it may be that other mechanisms operate to allow resistance. If this is the case, we will determine the genetic basis for these other potential resistance mechanisms. What opportunities for training and professional development has the project provided? In this project both graduate students and postdocs are working with staff and a visiting scientist to understand the impact of selected herbicdes on soil microbial populations. How have the results been disseminated to communities of interest? Through referreed journal publciations, verbal presentyations and poster sessions at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? We will further examine mechanisms by which bacteria are resistant to mesitrione and how this alters competitiveness among soil microbes.
Impacts
What was accomplished under these goals?
The intensive use of agrochemicals has played an important role in increasing agricultural production in the US and elsewhere. One of the main impacts of agrochemical use has been changes in population structure of soil microorganisms. The aim of this study was to evaluate the influence of the herbicide mesotrione, which inhibits 4-hydroxyphenylpyruvate dioxygenase activity, on a model non-environmental microbe, E. coli. Mesotrione is a new herbicide developed for the selective pre- and post-emergence control of broad-leaved and grassy weeds in corn. This bacterium was chosen as it genome is well known. Results of our studies showed that this strain was able to tolerate high concentrations of the herbicide, and completely degraded mesotrione after 3 h of exposure. Growth rates in the presence of mesotrione were lower than in a control, prior to the period of degradation, showing the toxic effect on the bacterial cells. Considering that E. coli DH5-α had no previous contact with mesotrione, the defense system found in this strain can be considered as general and non-specific. This strategy may be a general adaptation mechanism by which bacterial strains in agricultural soils resist damage due to the presence of herbicides. Results from these studies are useful in determining how herbicides in general, and specifically members of the benzoylcyclohexane-1,3-dione family of herbicides degrade in the environment and how the bacteria carry-out these degradation reactions. Results from these studies will allow researchers to determine how these herbicides effect beneficial and harmful microbes in the environment and how this information can be used to remediate contaminated water supplies and soils.
Publications
- Type:
Other
Status:
Submitted
Year Published:
2014
Citation:
Olchanheski, L.R., Dourado, M.N., Beltrame, F.L., Zielinski, A.A.F., Demiate, I.M., Pileggi, S.A.V., Azevedo, R.A., Sadowsky, M.J., and Pileggi, M. 2014. Mechanisms of tolerance and high capacity degradation of the herbicide mesotrione by the Escherichia coli DH5-?. Submitted.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Cyanuric acid is an intermediate in the bacterial metabolism of s-triazine ring compounds, including atrazine. The cyanuric acid produced via bacterial metabolism of atrazine is transformed to biuret via the action of cyanuric acid hydrolase. Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, are found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The current study employed 13C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze analogous reactions. Many enzymes that had been annotated incorrectly in genome projects have been reassigned by bioinformatics, gene cloning, and protein characterization studies. While the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, bioinformatics and the lack of bound metals argue against a connection to the amidohydrolase superfamily. Steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment. PARTICIPANTS: The participants in this project constitute a multidisciplinary team consisting of University faculty, postdocs, students and staff scientists, and employees of a not-for-profit research center (BTI). TARGET AUDIENCES: Results from these studies are targeted to research scientists interested in herbicide degradation, triazine herbicides, enzymology, water reclamation, and food adulteration, and to researchers interested the use of enzymes to detect adulterants. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results from these studies are useful in determining how s-triazine compounds degrade in the environment and how the bacteria carry-out these important set of reactions. Results from these studies will allow researchers to efficiently apply enzymes and microbes into the environment to bioremediate triazine compounds found in our water supplies and soils.
Publications
- Seffernick, J.L., Erickson, J.S., Cameron, S.M., Cho, S., Dodge, A.G., Richman, J.E., Sadowsky, M.J., and Wackett, L.P. 2012. Defining sequence space and reaction products within the cyanuric acid hydrolase (AtzD)/barbiturase protein family. J Bacteriol. 194:4579-4588.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The k(cat)/K(M) of 1.7 X 10(5) M(-1)s(-1) and the relatively low K(M) of 23 + 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway. We also isolated and characterized a new gene for the degradation of the food adulterant Melamine and will use this knowledge to better detect this compound in food and the environment. PARTICIPANTS: Participants: The participants in this project constitute a multidisciplinary team consisting of University faculty, postdocs, and staff scientists, and employees of a not-for-profit research center. TARGET AUDIENCES: Results from these studies are targeted to research scientists interested in herbicide degradation, triazine herbicides, enzymology, water reclamation, and food adulteration, and to researchers interested in the evolution of bacterial genes and degradation pathways and the use of enzymes to detect adulterants. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results from these studies are useful in determining how s-triazine compounds degrade in the environment and how the bacteria carry-out these important set of reactions. Results from these studies will allow researchers to efficiently apply enzymes and microbes into the environment to breakdown triazine compounds found in our water supplies and soils.
Publications
- Cameron, S. M., K. Durchschein, J. E. Richman, M. J. Sadowsky, and L. P. Wackett. 2011. A new family of biuret hydrolases functioning in s-triazine ring metabolism. ACS Cayalysis. 2011(1):1075-1082.
- Dodge, A. G., L. P. Wackett, and M. J. Sadowsky. 2012. Plasmid Localization and Organization of Melamine Degradation Genes in Rhodococcus sp. strain Mel. Appl. Environ. Microbial. In Press.
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