identification and isolation of bacterial genes essential for arsenic tolerance jeffrey a. parham...
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Identification and Isolation of Bacterial Genes Essential for
Arsenic Tolerance
Jeffrey A. Parham
Oklahoma State University
Arsenic may be found in air, water, and soil as
• Sulfide minerals
• Complex sulfides of metal cations
• Adsorbed on mineral colloids
• Bound to organic matter
• Bound with Al, Fe, Ca, or Mg
Common forms of Organic Arsenic
• Monomethyl arsenic acid (MMAA)
• Dimethyl arsenic acid (DMAA)
• Arseno-sugars
Common forms of Inorganic Arsenic
• Arsenite (As III) as arsenious acid (H3AsO3)
• Arsenate (As V) as H2AsO4- and HAsO4
2-
Potential Sources of Arsenic Contamination
• Commercial and industrial chemicals used in – wood treating– computer and electronics– metal finishing
• Residential chemicals– insecticides – weed killers
Potential Sources of Arsenic Contamination
• Mining activities for– gold– copper– precious metals
• Refining wastes
• Natural deposits
Exposure Pathways
• Ingestion of contaminated drinking water
• Eating foods grown in arsenic contaminated soils
• Inhalation of dusts, fumes, or mists
• Dermal absorption
Arsenic in the Human Body
• The major portion of absorbed arsenic is excreted through the urine (about 50 %)
• A small portion of As can be stored by the body in metabolically dead tissues, such as skin, hair, feces, and nails, thereby slowly eliminating arsenic.
Early Symptoms of Arsenic Poisoning
• Palpitations
• Fatigue and weakness
• Headaches and dizziness
• Insomnia
• Nightmares
• Numbness in the extremities
• Anemia
Symptoms Resulting from Prolonged Exposure to As
• Melanosis-keratosis
• Leucomelanosis
• Edema
• Conjunctival congestion
• Squamous and basal cell carcinomas
• Bowen’s disease
• Carcinoma of the lungs, uterus, bladder, and genitourinary tract
Keratosis of the Hand and Foot
Gangrene Caused by Arsenic Poisoning
Bangladesh
• Surface water was contaminated with microorganisms so wells were drilled to pump water from an aquifer
• Aquifer contaminated with arsenic that had been eroded from hard rocks by the Ganges River system and deposited in alluvial sediments.
Bangladesh
• Tens of millions of people are drinking arsenic contaminated water
• > 8,500 people have been diagnosed with symptoms of arsenic poisoning
Bangladesh
• More treatment facilities
• Drill wells deeper than 150 meters
EPA Regulations
• Currently 0.05 mg L-1
• Lowering to 0.005 mg L-1
Percentage of small public water-supply systems estimated to exceed targeted arsenic concentrations in their ground-water resource (ug/L).
Arsenic concentrations exceeding 10 µg/L in 10 percent of samples
Arsenic concentrations exceeding 5 µg/L in 10 percent of samples
Arsenic concentrations exceeding 3 µg/L in 10 percent of samples
Counties with fewer than 10 percent of samples exceeding 3 µg/L.
Insufficient data.
Remediation Technologies
• Precipatative processes
• Adsorption processes
• Ion exchange
• Membrane filtration
Problems with Current Remediation Technologies
• Frequent monitoring
• Expensive
• Impractical for large scale remediation
• Require arsenite to be oxidized to arsenate to be effectively remediated
Phytostabalization
• Phytostabalization is an emerging technology for treating As contamination.
• Phytostabalization does not detoxify As, it simply prevents the transport of As off the site.
• A method must be developed to us in conjunction with phytostabilization to detoxify the arsenic.
Creation of a Transgenic Phytostabalizer
• It may be possible to modify the genome of a phytostabalizer, such as poplar trees, to detoxify As(III) by oxidizing it to As(V) or creating an organoarsenical compound.
• Before the transgenic plant can be created, a gene capable of evoking the transformation of As must first be identified.
Objectives
• Isolation and identification of arsenic tolerant strains of bacteria
• Construction of the genomic library from an isolated strain and identification of genes essential for arsenic tolerance
• Determination of the physiological processes for arsenic tolerance of the isolated strains
Isolation of Arsenic Tolerant Bacteria
• Bacteria will be extracted from soil samples using De Leij et al’s (1993) method
• Bacteria extracts will be spread on TSA plates spiked with Sodium m-Arsenite
• Isolates will be plated and pure cultures will be obtained for identification and creation of the genomic library
Identification of Arsenic Tolerant Bacteria
• Isolates will be identified by– Morphology– Gram-Staining– Genetics
• Genetic identification will be done using 16s rDNA and a universal bacterial primer. Isolated DNA will be cloned to a plasmid, sequenced, and compared to known organisms using a BLAST search
Construction of the Genomic Library
• Total DNA will be extracted from a tolerant isolate.
• This DNA will be digested using restriction enzymes.
• Fragments will be cloned to a plasmid, such as pUC 18, in an E. coli strain lacking As tolerance to create the genomic library.
Identification of Genes Essential for Arsenic Tolerance
• The transformed E. coli will be plated on TSA plates containing arsenic to confer As tolerance.
• DNA from Tolerant isolates will be sequenced to identify the new gene responsible for As tolerance.
Determination of Physiological Processes for As Tolerance
• As tolerant species will be cultured in serum vials sealed with rubber septum using TSB spiked with As(III) as the growth medium.
• Cultures will be incubated for a predetermined amount of time at 30oC
Determination of Physiological Processes for As Tolerance
• Following incubation, As species present will be measured in the cell bodies, growth medium, and air in the vials.
• A GC will be used to analyze gas samples
• HPLC Mass Spec will be used to measure organoarsenic compounds
• Hydride generation, a liquid-N trap, and atomic absorption spectrometry will be used to determine inorganic species.
Conclusion
• Once this work is complete, a gene responsible for As tolerance will be available for use in creating a transgenic plant that can detoxify arsenic and lessen the threat of human harm.
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