Identification and Isolation of Bacterial Genes Essential for Arsenic Tolerance

1. Identification and Isolation of Bacterial Genes Essential for Arsenic Tolerance Jeffrey A. Parham Oklahoma State University

2. 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

3. Common forms of Organic Arsenic • Monomethyl arsenic acid (MMAA) • Dimethyl arsenic acid (DMAA) • Arseno-sugars

4. Common forms of Inorganic Arsenic • Arsenite (As III) as arsenious acid (H3AsO3) • Arsenate (As V) as H2AsO4- and HAsO42-

5. Potential Sources of Arsenic Contamination • Commercial and industrial chemicals used in • wood treating • computer and electronics • metal finishing • Residential chemicals • insecticides • weed killers

6. Potential Sources of Arsenic Contamination • Mining activities for • gold • copper • precious metals • Refining wastes • Natural deposits

7. Exposure Pathways • Ingestion of contaminated drinking water • Eating foods grown in arsenic contaminated soils • Inhalation of dusts, fumes, or mists • Dermal absorption

8. 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.

9. Early Symptoms of Arsenic Poisoning • Palpitations • Fatigue and weakness • Headaches and dizziness • Insomnia • Nightmares • Numbness in the extremities • Anemia

10. 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

11. Keratosis of the Hand and Foot

12. Gangrene Caused by Arsenic Poisoning

13. 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.

14. Bangladesh • Tens of millions of people are drinking arsenic contaminated water • > 8,500 people have been diagnosed with symptoms of arsenic poisoning

15. Bangladesh • More treatment facilities • Drill wells deeper than 150 meters

16. EPA Regulations • Currently 0.05 mg L-1 • Lowering to 0.005 mg L-1

17. Percentage of small public water-supply systems estimated to exceed targeted arsenic concentrations in their ground-water resource (ug/L).

18. 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.

19. Remediation Technologies • Precipatative processes • Adsorption processes • Ion exchange • Membrane filtration

20. Problems with Current Remediation Technologies • Frequent monitoring • Expensive • Impractical for large scale remediation • Require arsenite to be oxidized to arsenate to be effectively remediated

21. 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.

22. 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.

23. 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

24. 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

25. 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

26. 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.

27. 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.

28. 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

29. 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.

30. 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.