Grantee Research Project Results
1999 Progress Report: The Biochemistry and Molecular Biology of Microbial Selenate and Arsenate Transformation
EPA Grant Number: R826105Title: The Biochemistry and Molecular Biology of Microbial Selenate and Arsenate Transformation
Investigators: Stolz, John F.
Institution: Duquesne University , United States Geological Survey
Current Institution: Duquesne University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1997 through September 30, 2000
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $273,119
RFA: Exploratory Research - Environmental Biology (1997) RFA Text | Recipients Lists
Research Category: Biology/Life Sciences , Aquatic Ecosystems
Objective:
The mobilization of toxic elements and heavy metals in the environment can be strongly influenced by microbial activity. Sulfurospirillum barnesii has the ability to couple the oxidation of organic matter to the reduction of selenate and arsenate, compounds which have become significant environmental toxins. The hypothesis to be tested is that S. barnesii has separate pathways for arsenate and selenate reduction but that the selenate reductase is a less substrate-specific enzyme, capable of reducing a wide range of substrates. The objective of this proposal is to purify and characterize (Km, Vmax, substrate specificity, absorption spectrum, mid-point potential) the two reductases and associated cytochromes, and to develop biochemical and molecular probes based on the selenate reductase that can be used to detect S. barnesii in nature. The probes also will be used to determine the relatedness of selenate reductases from other bacterial species.Progress Summary:
N-terminal amino acid sequences for the three polypeptides of the arsenate reductase (AsR) have been obtained. The 31 and 22 kDa polypeptides are unique (i.e., do not match with the amino acid sequence of any known proteins) and thus are excellent candidates for biochemical and molecular probes. The 62 kDa polypeptide showed a high degree of similarity (85.7 percent) to hydrogenases. It has since been determined that the AsR has hydrogenase activity and the 62 kDa subunit crossreacts with the large subunit of the hydrogenase from Wolinella succinogenes. Oligonucleotide primers have been made based on the sequence data, and the gene has been cloned for the 31 kDa polypeptide and a portion of the 22 kDa polypeptide. Progress also has been made on other bacterial strains that respire oxyanions of selenate and arsenate. The AsR and the selenate reductase from Bacillus arsenicoselenatis have been localized to the cytoplasmic membrane. The AsR and the selenite reductase from Bacillus selenitireducens also are in the cytoplasmic membrane. All four enzymes are solubilized with CHAPS. A freshwater Bacillus from the Ohio River has been isolated that can respire both selenate and selenite. A selenate respirer isolated from the Dead Sea was found to be a member of the halobacteriales (gram positive, salt-loving, eubacteria).Future Activities:
It is anticipated that the AsR operon will be cloned and sequenced in the last year of the grant. Gene probes have been developed and are being tested for their specificity. Sediment samples from the Monongehela, Allegheny, and Ohio Rivers will be used for the environmental samples. Characterization of the Ohio River isolate will be completed. Characterization of the selenate reductase from S. barnesii also will be completed.Journal Articles on this Report : 4 Displayed | Download in RIS Format
| Other project views: | All 12 publications | 8 publications in selected types | All 5 journal articles |
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Blum JS, Bindi AB, Buzzelli J, Stolz JF, Oremland RS. Bacillus arsenicoselenatis, sp nov, and Bacillus selenitireducens, sp nov: two haloalkaliphiles from Mono Lake, California that respire oxyanions of selenium and arsenic. Archives of Microbiology 1998;171(1):19-30. |
R826105 (1999) R826105 (Final) |
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Oremland RS, Blum JS, Bindi AB, Dowdle PR, Herbel M, Stolz JF. Simultaneous reduction of nitrate and selenate by cell suspensions of Selenium-respiring bacteria. Applied Environmental Microbiology 1999;65(10):4385-4392. |
R826105 (1999) R826105 (2000) R826105 (Final) |
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Stolz JF, Oremland RS. Bacterial respiration of arsenic and selenium. FEMS Microbiology Reviews, Volume 23, Issue 5, October 1999, Pages 615-627. |
R826105 (1999) R826105 (2000) R826105 (Final) |
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Stolz JF, Ellis DJ, Blum JS, Ahmann D, Lovley DR, Oremland RS. Sulfurospirillum barnesii sp nov and Sulfurospirillum arsenophilum sp nov., new members of the Sulfurospirillum clade of the epsilon Proteobacteria. International Journal of Systematic Bacteriology 1999;49(Part 3):1177-1180. |
R826105 (1999) R826105 (Final) |
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Supplemental Keywords:
heavy metals, ecology, soil, water, enzymes, indicators, bioremediation., Scientific Discipline, Waste, Ecosystem Protection/Environmental Exposure & Risk, Environmental Microbiology, Fate & Transport, Biochemistry, Bioremediation, Ecological Risk Assessment, Molecular Biology/Genetics, microbiology, microbial selenate, fate and transport, aerobic degradation, biodegradation, arsenate compounds, oligonucleotide probes, biotechnology, reductases, sulfurospirillum barnesii, oligobacteria, heavy metalsRelevant Websites:
http://www.home.cc.duq.edu/~stolz/Research.html
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.