Progress 08/15/05 to 08/14/09
Outputs
OUTPUTS: 1. Trained and Mentored 2 Graduate students (Jincai Ma, Francis Jordan) and 1 full time researcher (David Ams) at Rutgers University. 2. Conducted selenium oxyanion reduction experiments using a soil bacterium Enterobacter cloacae SLD1a-1. Genetic experiments were also performed to identify the genes required for selenium oxyanion reduction. 3. Generated fundamental new knowledge which was disseminated via presentations at scientific meetings. These meetings include the American Society for Microbiology Meeting 2006, 2007 and 2008; the Goldschmidt Meeting 2005 and 2007, and the American Geophysical Union Meeting 2006, and the Soil Science Society of America Meeting 2006. 4. New genetic data were deposited in GenBank under the ascension numbers: DQ523830, EF633682, EU606203. PARTICIPANTS: Principal investigator: Nathan Yee Role: Designed the chemical kinetic and molecular genetic experiments; analyzed experimental data; prepared figures and tables for dissemination; co-wrote manuscripts for publication; mentored and trained graduate students. Collaborator: Donald Kobayashi Role: Co-designed molecular genetic experiments; analyzed experimental data; prepared figures and tables for dissemination; co-wrote manuscripts for publication; co-mentored and trained graduate students. Graduate Student: Jincai Ma Role: Performed the chemical kinetic and molecular genetic experiments; analyzed experimental data; prepared figures and tables for dissemination; co-wrote manuscripts for publication. Graduate Student: Francis Jordan Role: Developed analytical methods for detecting selenium compounds in soil samples; Maintained bacteria culture collection for laboratory experiments, conducted molecular genetic experiments. Full-time Researcher: David Ams Role: Isolated bacteria from soil samples; set-up analytical instruments in laboratory for chemical and genetic analysis TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Major Findings 1. The rates of microbial selenite [Se(IV), SeO32-] reduction is significantly more rapid than the reduction of selenate [Se(VI), SeO42-]. Kinetic experiments conducted as function of selenium oxyanion concentration, pH, and temperature showed that Se-reducing bacterium Enterobacter cloacae SLD1a-1 reduces selenite considerable faster than selenate. Both the rates of Se(VI) and Se(IV) reduction displayed strong temperature-dependence with Ea values of 121 kJ/mol and 71.2 kJ/mol respectively. These results suggest that higher activation energies are required to reduce Se(VI) than for the reduction of Se(IV). 2. The fnr gene is required for selenate reduction. Using directed mutagenesis, a knock-out mutation of the fnr (fumarate nitrate reduction regulator) gene was introduced into Enterobacter cloacae SLD1a-1. Mutation of the fnr gene in E. cloacae SLD1a-1 resulted in derivative strains that were deficient in selenate reduction activity. Complementation by the wild-type fnr sequence restored the ability of mutant strains to reduce Se(VI). Our findings suggest that Se(VI) reduction by E. cloacae is regulated by oxygen sensing transcription factors, and occurs under suboxic conditions. 3. The tatC gene is required for selenate reduction. A miniTn5 transposon mutant of E. cloacae SLD1a-1 was isolated that had lost the ability to reduce Se(VI), but was not affected in Se(IV) reduction activity. Nucleotide sequence analysis revealed the transposon was inserted within a tatC gene, which encodes for a central protein in the twin arginine translocation system. Complementation by the wild-type tatC sequence restored the ability of mutant strains to reduce Se(VI). The results suggest that Se(VI) reduction activity is dependent on enzyme export across the cytoplasmic membrane, and that reduction of Se(VI) and Se(IV) are catalyzed by different enzymatic systems. 4. The menD gene is required for selenate reduction. A mini-Tn5 transposon mutant of E. cloacae SLD1a-1, designated as 4E6, was isolated that had lost the ability to reduce Se(VI) to Se(0). Genetic analysis of mutant strain 4E6 showed that the transposon was inserted within a menD gene among a menFDHBCE gene cluster which encodes for proteins required for menaquinone biosynthesis. A group of E. coli K12 strains with single mutations in the menF, menD, menC and menE genes were tested for loss of selenate reduction activity. The results showed that E. coli K12 carrying a deletion of either the menD, menC or menE gene was unable to reduce selenate. Complementation using wild-type sequences of the E. cloacae SLD1a-1 menFDHBCE sequence successfully restored the selenate reduction activity in mutant strain 4E6, and E. coli K12 menD and menE mutants. The results of this work suggest that menaquinones are an important source of electrons for the selenate reductase, and are required for selenate reduction activity in Enterobacter cloacae SLD1a-1 and E. coli K12.
Publications
- Yee N., Ma J., Kobayashi D.Y., 2008. Regulation and Transport of the Selenate Reductase in Enterobacter cloacae SLD1a-1. American Society for Microbiology, Abstract Q-044, Boston
- Yee N, Kobayashi DY, Ma J, 2005. The kinetics and mechanism of selenate reduction by Enterobacter cloacae. Geochimica et Cosmochimica Acta 69 (10): A670-A670 Suppl. S
- Cuiule C., Kobayashi D.Y., Yee N., 2007. Se(VI) Reduction by Enterobacter cloacae SLD1a-1 Requires Anaerobic Electron Carriers, American Society for Microbiology, Abstract Q-050, Toronto
- Yee N., Kobayashi D.Y., 2006. Genetic Identification of an Enzymatic Se(VI) Reduction Pathway, American Geophysical Union, Abstract B11A-0998, San Francisco
- Yee N., Dalia A., Kobayashi D.Y., Ma J, Boonfueng T., 2006. Heterologous expression of the fnr gene from Enterobacter cloacae SLD1-1a in Escherichia coli S17-1 activates selenate reductase activity and the ability to precipitate Se(0) Soil Science Society of America, Indianapolis, Abstract 178-17
- Yee N., Dalia A., Kobayashi D.Y., Ma J., 2006. The Genetics of Microbial Se(0) Biomineral Formation, American Society for Microbiology, Abstract Q-156, Orlando
- Kenward P, Yee N, Fowle D, 2005. Microbially controlled selenate reduction in nutrient limited systems. Geochimica et Cosmochimica Acta 69 (10): A455-A455 Suppl. S
- Ma J., Kobayashi D.Y., Yee N., 2008. The Role of Menaquinone in Se(VI) Reduction by Enterobacter cloacae SLD1a-1. American Society for Microbiology, Abstract Q-043, Boston
- Yee N., Ma J., Kobayashi D.Y., 2007. A Molecular Model for Microbial Se(VI) Reduction, Geochimica et Cosmochimica Acta 71 (15): A1145-A1145 Suppl. S
- Ma J., Kobayashi D.Y., Yee N., 2007. The kinetics of Se(VI) and Se(IV) reduction by Enterobacter cloacae, American Society for Microbiology, Abstract Q-049, Toronto
- Ma J., Kobayashi D.Y., and Yee N. (2008) Role of menaquinone biosynthesis genes in selenate reduction by Enterobacter cloacae SLD1a-1 and Escherichia coli K12, Environmental Micorbiology (in press)
- Yee N. and Kobayashi D.Y. (2008) Molecular genetics of selenate reduction by Enterobacter cloacae SLD1a-1, Advances in Applied Microbiology, 64, 107-123
- Ma J., Kobayashi D.Y., and Yee N. (2007) A chemical kinetic and molecular genetic study of selenium oxyanion reduction by Enterobacter cloacae SLD1a-1, Environmental Science & Technology 41, 7795-7801
- Yee N., Ma J., Dalia A., Boonfueng T., Kobayashi D.Y. (2007) Se(VI) reduction and the precipitation of Se(0) precipitation by the facultative bacterium Enterobacter cloacae SLD1a-1 is regulated by FNR, Applied and Environmental Microbiology, 73, 1914-1920
- Kenward P.A., Fowle D.A., Yee N. (2006) Microbial selenate sorption and reduction in nutrient limited systems, Environmental Science & Technology, 40, 3782-3786
|
Progress 08/15/06 to 08/14/07
Outputs
The focus of our USDA-NRI project is to understand how microorganisms transform the soluble selenium oxyanions selenate [Se(VI), SeO42-] and selenite [Se(IV), SeO32-] into insoluble elemental selenium [Se(0)]. We have completed the second year of this research project. Results have been generated by a graduate research assistant (Jincai Ma - Ph.D. student), a part-time postdoctoral researcher (Dr. David Ams), two undergraduate research assistants, and the principle investigator (Nathan Yee). Here are the main findings from the research we conducted in the past year: 1) Using directed mutagenesis, a knock-out mutation of the fnr gene was introduced into E. cloacae SLD1a-1. Mutation of the fnr gene in E. cloacae SLD1a-1 resulted in derivative strains that were deficient in selenate reduction activity and unable to precipitate elemental selenium. Complementation by the wild-type fnr sequence restored the ability of mutant strains to reduce Se(VI). Our findings suggest
that Se(VI) reduction and the precipitation of Se(0) by facultative anaerobes is regulated by oxygen sensing transcription factors, and occurs under suboxic conditions. Significance: This finding is scientifically important because it tells us that selenium oxyanion reduction by E. cloacae is a suboxic process, and occurs in environments where the oxygen concentration is low. 2) The rates of microbial selenium oxyanion reduction were measured as a function of initial selenium oxyanion concentration (0 to 1.0 mM) and temperature (10oC to 40oC), and transposon mutagenesis experiments were performed to identify gene(s) involved in the selenium oxyanion reduction pathway. The results indicate that Se(IV) reduction is significantly more rapid than the reduction of Se(VI). The kinetics of the reduction reactions were successfully quantified using the Michaelis-Menten kinetic equation. Both the rates of Se(VI) and Se(IV) reduction displayed strong temperature-dependence. X-ray absorption
near-edge spectra collected for the precipitates formed by Se(VI) and Se(IV) reduction confirmed the formation of Se(0). A miniTn5 transposon mutant of E. cloacae SLD1a-1 was isolated that had lost the ability to reduce Se(VI), but was not affected in Se(IV) reduction activity. Nucleotide sequence analysis revealed the transposon was inserted within a tatC gene, which encodes for a central protein in the twin arginine translocation system. Complementation by the wild-type tatC sequence restored the ability of mutant strains to reduce Se(VI). The results suggest that Se(VI) reduction activity is dependent on enzyme export across the cytoplasmic membrane, and that reduction of Se(VI) and Se(IV) are catalyzed by different enzymatic systems. Significance: These results are important because they show that E. cloacae employs different molecular mechanisms to reduce selenate [Se(VI), SeO42-] and selenite [Se(IV), SeO32-]. The results also demonstrate that the microbial reduction of selenate
is kinetically more favorable than the reduction of selenite. Non-technical Summary
Impacts
Understanding how microorganisms transform toxic selenium ions into non-toxic minerals will help improve water quality in selenium contaminated agricultural drainage ponds. In our research, we have identified essential genes in bacteria that perform this function. Using this knowledge, environmental managers will be able to design better strategies to clean-up toxic selenium ions in agricultural waters.
Publications
- Ma J., Kobayashi D.Y., and Yee N. (2007) A chemical kinetic and molecular genetic study of selenium oxyanion reduction by Enterobacter cloacae SLD1a-1, Environmental Science & Technology (in press)
- Yee N., Ma J., Dalia A., Boonfueng T., Kobayashi D.Y. (2007) Se(VI) reduction and the precipitation of Se(0) precipitation by the facultative bacterium Enterobacter cloacae SLD1a-1 is regulated by FNR, Applied and Environmental Microbiology, 73, 1914-1920
- Kenward P.A., Fowle D.A., Yee N. (2006) Microbial selenate sorption and reduction in nutrient limited systems, Environmental Science & Technology, 40, 3782-3786
|
Progress 08/15/05 to 08/14/06
Outputs
The focus of our USDA-NRI project is to understand how soil microorganisms transform soluble selenate oxyanions into insoluble elemental selenium. When have completed the first year of this research project. Results have been generated by a graduate research assistant (Jincai Ma, Ph.D. student), two undergraduate research assistants, and the principle investigator (Nathan Yee). Here are the main research findings: 1) In sorption experiments with the metal-reducing bacterium Shewanella putrefaciens, we observed that selenate sorption is strongly dependent on solution ionic strength, suggesting the formation of outer-sphere complexes with the cell wall functional groups. XANES spectroscopic analysis of the cells after the sorption experiments revealed the presence of elemental selenium, indicating that S. putrefaciens has can reduce Se(VI) to Se(0). Interestingly, this reduction reaction occurs in the absence of external electron donors. Significance: These findings are
scientifically important because they elucidate the mechanisms of selenium oxyanion sorption onto microbial cell walls. 2) In kinetics studies with the Se-reducing bacterium Enterobacter cloacae, we developed a rate law that quantitatively describes the rates of microbial selenium oxyanion reduction. We measured the rate of microbial selenate and selenite reduction as a function of initial Se concentration, pH, and temperature. Using the Michaelis-Menten equation, we successfully modeled the reduction kinetics. We found that the rates of selenate and selenite reduction are described by different kinetic parameters, suggesting that reduction of Se(IV) and Se(VI) is catalyzed by different enzymatic pathways. Significance: These results are important because they allow us to predict how fast soil microorganisms transform soluble selenium oxyanions to insoluble elemental selenium. 3) Using a direct cloning technique, we identify a genetic pathway that confers selenate reductase activity
in the Se-reducing bacterium Enterobacter cloacae. We show that heterologous expression of the global anaerobic regulatory gene fnr from E. cloacae in the non Se-reducing strain E. coli S17-1 activates selenate reductase activity and the ability to precipitate Se(0) particles. SEM and XAFS spectroscopic analysis shows that E. cloacae and the cosmid clone produce elemental selenium particles with identical morphologies and short range atomic order. Significance: These findings are scientifically important because they reveal that Se(0) biomineral formation by facultative anaerobes is activated by oxygen sensing transcription factors, and occurs under suboxic conditions.
Impacts
Understanding how microorganisms transform toxic selenium ions into non-toxic minerals will help improve water quality in selenium contaminated agricultural drainage ponds. By measuring the rates of microbial selenium transformation, and identifying the genes that perform this function, researchers will be able to predict how fast microbes can remove toxic selenium ions from agricultural waters.
Publications
- Yee N., Dalia A. Kobayashi D.Y., Ma J., Boonfueng T. 2006. Heterologous expression of the fnr gene from Enterobacter cloacae SLD1-1a in Escherichia coli S17-1 activates selenate reductase activity and the ability to precipitate Se(0) biominerals, Applied and Environmental Microbiology (submitted)
- Ma. J, Kobayashi D.Y, Yee N. 2006. The kinetics of Se(VI) and Se(VI) reduction by Enterobacter cloacae (in prep)
- Kenward P.A., Fowle D.A., Yee N. 2006. Microbial selenate sorption and reduction in nutrient limited systems, Environmental Science and Technology, 40, 3782-3786
|