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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
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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 HAsO42-
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
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Palpitations
Fatigue and weakness
Headaches and dizziness
Insomnia
Nightmares
Numbness in the extremities
Anemia
Symptoms Resulting from
Prolonged Exposure to As
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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
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Precipatative processes
Adsorption processes
Ion exchange
Membrane filtration
Problems with Current
Remediation Technologies
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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.