Gayan K. A. Appuhamillage and T. G. Chasteen Department of Chemistry Sam Houston State University

1. Bioremediation of toxic oxyanions using genetically modified Escherichia coli strains and study of their glutathione biosynthetic pathway Gayan K. A. Appuhamillage and T. G. Chasteen Department of Chemistry Sam Houston State University

2. Objectives • Introduction • Methodology • Results • Discussion • Conclusions • Acknowledgement contents

3. To study the bioremediation potential of the genetically modified E. coli strains on toxic oxyanions of Se (SeO32-, SeO42-)and Te (TeO32-) • To study the effect of glutathione biosynthetic pathway in reducing the toxic oxyanions • To study the effect of isopropyl-β-D-1-thiogalactopyranoside (IPTG) towards the production of intracellular glutathione OBJECTIVES

4. Toxicity of oxyanions of Se and Te on humans • Oxyanions of Se (SeO32-, SeO42-) • decrease body weight gains • liver cirrhosis • pancreatic enlargement • anemia • chronic hepatitis • Oxyanion of Te (TeO32-) • vomiting • renal pain • loss of consciousness • irregular breathing • cyanosis Introduction

5. Bioremediation • Toxic oxyanions • (ex: SeO32-, SeO42-, TeO32-) Elemental forms (removable) (Se, Te) “We are bacteria” • The genesgshA and gshB play vital roles Ref: http://www.google.com/search?hl=en&site=imghp&tbm=isch&source=hp&biw=1280&bih=904&q=bacteria&oq=bacteria&gs_l=img.3..0l10.2656.5391.0.5662.10.7.1.2.2.0.114.583.6j1.7.0...0.0...1ac.1.6.img.d7WP0S3R9Fs

6. What are gshA and gshB ?? • Involve in the production of glutathione inside bacterial cells gshA gshB Ref: Kim, E. K., Cha, C. J., Cho, Y. J., Cho, Y. B., Roe, J. H. Synthesis ofγ-glutamylcysteine as a major low-molecular-weight thiol in lactic acid bacteria Leuconostoc spp. Biochem. Biophys. Res. Commun. 2008, 369, 1047-1051.

7. Importance of Glutathione (GSH) • Acts as a reducing agent • The thiol group of cysteine becomes oxidized while reducing reactive oxygen species (i.e. SeO32-, SeO42- , TeO32-) Reactions: 6 RSH + Na2SeO4 + 2 H+ Se + 3 RSSR + 4 H2O + 2 Na+ 4 RSH + Na2SeO3 + 2 H+ Se + 2 RSSR + 3 H2O + 2 Na+ 4 RSH + Na2TeO3+ 2 H+ Te + 2 RSSR + 3 H2O + 2 Na+ R: C10H16N3O6

8. E. coli strains used gshA gshB pCA24N plasmid AG1/pCA24NgshA AG1 AG1/pCA24NgshB

9. What is expected from IPTG? • IPTG increases the affinity of catabolic repression proteins (CRPs) to ribonucleic acid (RNA) polymerases • CRPs help attach RNA polymerases to promoter regions • Activated promoters can enhance production of more GshA or GshB enzymes • Production of more GSH is expected

10. Toxicity measurement methods • Minimum Inhibitory Concentration (MIC) • The lowestconcentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation • Specific Growth Rate • Increase in cell mass per unit time • Characteristic to a particular organism in a given medium at a given temperature • Can be calculated using the slope of the log phase in a bacterial growth curve (Ln Optical Density (OD)Vs. time)

11. Methodology MIC measurements: Preparation of bacterial pre-cultures • Mixing with the toxicants (Na2SeO3, Na2SeO4, Na2Te2O3) • Not mixing with the toxicants OD600 measurements (after 24 h) Add a dye (after 24 h) • Color observation (after 24 h) • Blue: dead cells • Pink: live cells

12. Specific growth rate (SGR) measurements: Bacterial pre-cultures • Mixing with the toxicants • Not mixing with the toxicants OD600 measurements initially and at specific time intervals and SGR calculation by slope of log phase Intracellular GSH measurements: Break cell walls to take out GSH Add a chemical reagent and measure absorbance at 412 nm Bacterial pre-cultures Filter and take out bacterial cells Intracellular protein contents: Break cell walls to take out proteins Bacterial pre-cultures Filter and take out bacterial cells Add a chemical reagent and measure absorbance at 595 nm * Above all were repeated with IPTG (0.05, 0.1, 0.2, 0.4, 0.8, 1.0 mM) added during pre-culture preparation

13. Results MIC measurements For AG1 For AG1/pCA24NgshA For AG1/pCA24NgshB

14. Specific growth rate measurements For AG1 For AG1/pCA24NgshA For AG1/pCA24NgshB

15. Specific growth rate measurements (with IPTG)

16. Intracellular GSH measurements Calibration curve for GSH level measurements Calibration curve for protein content measurements

17. Intracellular GSH contents

18. MIC results: • The resistance of the E. coli strains towards the toxicants is highest for Na2SeO4, followed by Na2SeO3 and Na2TeO3 (toxicity increases as; Na2SeO4< Na2SeO3<Na2TeO3 ) • The resistance to Na2SeO4and Na2SeO3 is higher in both AG1/pCA24NgshA and AG1/pCA24NgshB compared to AG1 • Increasing IPTG concentrations lower the resistance of the E. coli strains towards the toxicants • Specific growth rates: • Decrease in the presence of the toxicants with respect to the controls (reflect the relative toxicity of the toxicants) • Decrease when increasing IPTG concentrations (reflect a metabolic stress at higher IPTG levels) Discussion

19. Intracellular GSH contents: • Higher absorbance values for GSH and also higher intracellular GSH contents in both AG1/pCA24NgshA and AG1/pCA24NgshB compared to AG1 (shows the involvement of gshA and gshB genes for GSH synthesis) • Absorbance values for GSH in both AG1/pCA24NgshA and AG1/pCA24NgshB slightly increase with increasing IPTG concentrations up to a maximum and decrease again (shows that IPTG helps increase GSH production but to a limit) • Intracellular GSH contents (µmol/ mg protein) slightly decrease when increasing IPTG concentrations (intracellular protein contents slightly increase at higher IPTG levels) Discussion cont.

20. Toxicity of the tested oxyanions increases in the order of Na2SeO4< Na2SeO3< Na2TeO3 • The toxicity of TeO32- is extremely large with respect to SeO32- and SeO42- that it is hard to be controlled by intracellular GSH levels present in the strains • The presence of relatively higher GSH contents in both AG1/pCA24NgshA and AG1/pCA24NgshB than in AG1 confirms the involvement of gshAand gshB genes for GSH biosynthesis • IPTG can induce GSH production up to a certain limit but then the GSH production decreases due to metabolic stress at higher IPTG levels • Bioengineered E. coli strains AG1/pCA24NgshA and AG1/pCA24NgshB can be used successfully for the bioremediation of Na2SeO4and Na2SeO3 and the concentration of IPTG should be controlled if it is used CONCLUSIONS

21. Dr. T.G. Chasteen, Dr. D.C. Haines for excellent supervision and guidance • Dr. R. E. Norman the chair, and all the faculty members of the Department of Chemistry, Sam Houston State University • Robert A. Welch foundation for the excellent research support Acknowledgement

22. ThankYou

23. A Typical Bacterial Growth Curve Ref: http://www.google.com/search?hl=en&q=bacterial+growth+curves&bav=on.2,or.r_qf.&biw=1680&bih=878&wrapid=tlif136344729137810&um=1&ie=UTF-8&tbm=isch&source=og&sa=N&tab=wi&ei=Bo5EUcaOFfK14AODjoDwAw

24. Microwell with MIC

25. Methodology MIC measurements: Incubation at 37 °C, in a shaker until OD600 ~ 0.1- 0.2 Preparation of bacterial pre-cultures (18 h, 37 °C, in a shaker) OD600= 0.5 Dilution until OD600= 0.005 • Loading 96-microwell plates with toxicants (Na2SeO3, Na2SeO4, Te2O3) • Two-fold dilutions across the plate • final volume of each well =150 µL. OD600 measurements OD600 method • Mixing bacterial cultures (10 µL) • with the toxicants • Controls: bacterial cultures and LB • Blank: LB Incubation at 37 °C, in a shaker for 24 h Resazurin dye method Addition of resazurin sodium salt (10 µL, 6.75 mg/ mL) • Color observation • Blue: dead cells • Pink: live cells Further Incubation at 37 °C, in a shaker for 24 h

26. Specific growth rate (SGR) measurements: Dilution until OD600= 0.005 Incubation at 37 °C, in a shaker until OD600 ~ 0.1 Preparation of bacterial pre-cultures (18 h, 37 °C, in a shaker) OD600= 0.5 SGR determination by slope of the log phase of Ln OD600 Vs. time plots • Loading 96-microwell plates with bacterial cultures (150 µL)and the toxicants (50 µL, concentration= MIC/2) • Controls: bacterial cultures and LB • Blank: LB OD600 measurements (initially and after 15 min intervals up to 15 h) Intracellular GSH measurements: Centrifugation (10,000 rpm, 15 min) and pellet collection Pellet dissolution in Tris HCl (0.1 M, pH 8) and sonication (2 min) to break cell walls Centrifugation (10,000 rpm, 15 min) and collection of supernatant Pre-cultures (same as above) Addition of 5, 5’-dithiobis(2-nitrobenzoic acid), 50 µL into supernatants (725 µL) Incubation at 37 °C, 2 min and absorbance measurements at 412 nm Calculation of GSH levels using a calibration curve Intracellular protein contents: Calculation of protein contents using a calibration curve with bovine serum albumin (BSA) standards Addition of Bradford reagent (1 mL) to above supernatants (50 µL) Absorbance measurements at 595 nm after 2 min * Above all were repeated with IPTG (0.05, 0.1, 0.2, 0.4, 0.8, 1.0 mM) added during pre-culture preparation