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

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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.