1. 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

2.  Objectives  Introduction  Methodology  Results  Discussion  Conclusions  Acknowledgement 2

3.  To study the bioremediation potential of the genetically modified E. coli strains on toxic oxyanions of Se (SeO 3 2-, SeO 4 2- ) and Te (TeO 3 2- )  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 3

4.  Toxicity of oxyanions of Se and Te on humans  Oxyanions of Se (SeO 3 2-, SeO 4 2- ) decrease body weight gains liver cirrhosis pancreatic enlargement anemia chronic hepatitis  Oxyanion of Te (TeO 3 2- ) vomiting renal pain loss of consciousness irregular breathing cyanosis 4

5.  Bioremediation Toxic oxyanions (ex: SeO 3 2-, SeO 4 2-, TeO 3 2- ) Elemental forms (removable) (Se, Te) 5  The genes gshA and gshB play vital roles Ref: http://www.google.com/search?hl=en&site=imghp&tbm=isch&source=hp&biw=1280&bih=904&q=bacteri a&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.d7W P0S3R9Fs

6.  What are gshA and gshB ??  Involve in the production of glutathione inside bacterial cells 6 gshA gshB Ref: Kim, E. K., Cha, C. J., Cho, Y. J., Cho, Y. B., Roe, J. H. Synthesis of γ-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. SeO 3 2-, SeO 4 2-, TeO 3 2- ) Reactions: 6 RSH + Na 2 SeO 4 + 2 H + Se + 3 RSSR + 4 H 2 O + 2 Na + 4 RSH + Na 2 SeO 3 + 2 H + Se + 2 RSSR + 3 H 2 O + 2 Na + 4 RSH + Na 2 TeO 3 + 2 H + Te + 2 RSSR + 3 H 2 O + 2 Na + R: C 10 H 16 N 3 O 6 7

8.  E. coli strains used 8 gshB pCA24N plasmid gshA AG1 AG1/pCA24NgshA AG1/pCA24Ngsh B

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 9

10.  Toxicity measurement methods  Minimum Inhibitory Concentration (MIC) The lowest concentration 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) 10

11. Methodology MIC measurements: 11 Preparation of bacterial pre- cultures Add a dye (after 24 h) Mixing with the toxicants (Na 2 SeO 3, Na 2 SeO 4, Na 2 Te 2 O 3 ) Not mixing with the toxicants OD 600 measurements (after 24 h) Color observation (after 24 h) Blue: dead cells Pink: live cells

12. Specific growth rate (SGR) measurements: 12 OD 600 measurements initially and at specific time intervals and SGR calculation by slope of log phase Intracellular GSH measurements: Bacterial pre-cultures Filter and take out bacterial cells Break cell walls to take out GSH Intracellular protein contents: 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 Add a chemical reagent and measure absorbance at 412 nm Bacterial pre-cultures Mixing with the toxicants Not mixing with the toxicants Bacterial pre-cultures Filter and take out bacterial cells Break cell walls to take out proteins

13. 13 MIC measurements IPTG concentration/ mMMIC of Na 2 SeO 3 / mMMIC of Na 2 SeO 4 / mMMIC of Na 2 TeO 3 / mM 0 31.252500.004 0.05 31.252500.004 0.1 31.252500.004 0.2 31.252500.004 0.4 31.252500.004 0.8 15.6251250.002 1.0 15.6251250.002 IPTG concentration/ mMMIC of Na 2 SeO 3 / mMMIC of Na 2 SeO 4 / mMMIC of Na 2 TeO 3 / mM 0 12510000.004 0.05 12510000.004 0.1 62.510000.016 0.2 62.510000.016 0.4 62.510000.008 0.8 62.55000.004 1.0 31.255000.004 IPTG concentration/ mMMIC of Na 2 SeO 3 / mMMIC of Na 2 SeO 4 / mMMIC of Na 2 TeO 3 / mM 0 12510000.004 0.05 12510000.004 0.1 62.510000.016 0.2 62.510000.008 0.4 62.510000.008 0.8 31.255000.004 1.0 31.255000.004 For AG1 For AG1/pCA2 4NgshA For AG1/pCA2 4NgshB

14. Specific growth rate measurements 14 Condition Specific growth rate/ min -1 control (no toxicant) 0.0093 ± 0.0002 with Na 2 SeO 3 (15.625 mM) 0.0077 ± 0.0002 with Na 2 SeO 4 (125 mM) 0.0080 ± 0.0001 with Na 2 TeO 3 (0.002 mM)0.0006 ± 0.0001 Condition Specific growth rate/ min -1 control (no toxicant) 0.0092 ± 0.0006 with Na 2 SeO 3 (62.5 mM) 0.0044 ± 0.0002 with Na 2 SeO 4 (500 mM) 0.0028 ± 0.0002 with Na 2 TeO 3 (0.002 mM)0.0011 ± 0.0001 Condition Specific growth rate/ min -1 control (no toxicant) 0.0095 ± 0.0004 with Na 2 SeO 3 (62.5 mM) 0.0047 ± 0.0001 with Na 2 SeO 4 (500 mM) 0.0037 ± 0.0003 with Na 2 TeO 3 (0.002 mM)0.0015 ± 0.0001 For AG1 For AG1/pCA2 4NgshA For AG1/pCA2 4NgshB

15. Specific growth rate measurements (with IPTG) 15 IPTG concentration/ mM Specific growth rate/ min -1 of AG1 Specific growth rate/ min -1 of AG1/pCA24N gshA Specific growth rate/ min -1 of AG1/pCA24N gshB 0 0.0093 ± 0.00020.0092 ± 0.00060.0095 ± 0.0004 0.05 0.0083 ± 0.0002 0.0092 ± 0.00060.0094 ± 0.0006 0.1 0.0085 ± 0.0002 0.0091 ± 0.00050.0093 ± 0.0003 0.2 0.0082 ± 0.0002 0.0092 ± 0.00040.0093 ± 0.0005 0.4 0.0084 ± 0.0001 0.0090 ± 0.00080.0093 ± 0.0003 0.8 0.0073 ± 0.0003 0.0076 ± 0.00030.0086 ± 0.0004 1.00.0072 ± 0.0002 0.0074 ± 0.00030.0080 ± 0.0005

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

17. 17 IPTG concentration/ mM GSH content (µmol/ mg protein) of AG1 GSH content (µmol/ mg protein) of AG1/pCA24N gshA GSH content (µmol/ mg protein) of AG1/pCA24N gshB 0 10.88 ± 0.27 15.16 ± 0.23 14.79 ± 0.22 0.05 10.59 ± 0.22 15.18 ± 0.3114.34 ± 0.49 0.1 10.51 ± 0.19 14.91 ± 0.1914.44 ± 0.64 0.2 10.08 ± 0.34 14.35 ± 0.3313.77 ± 0.32 0.4 9.20 ± 0.14 12.91 ± 0.6212.43 ± 0.40 0.8 7.86 ± 0.28 11.01 ± 0.2810.73 ± 0.18 1.07.04 ± 0.17 10.09 ± 0.389.82 ± 0.10 Intracellular GSH contents

18.  MIC results:  The resistance of the E. coli strains towards the toxicants is highest for Na 2 SeO 4, followed by Na 2 SeO 3 and Na 2 TeO 3 (toxicity increases as; Na 2 SeO 4 < Na 2 SeO 3 < Na 2 TeO 3 )  The resistance to Na 2 SeO 4 and Na 2 SeO 3 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) 18

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) 19

20.  Toxicity of the tested oxyanions increases in the order of Na 2 SeO 4 < Na 2 SeO 3 < Na 2 TeO 3  The toxicity of TeO 3 2- is extremely large with respect to SeO 3 2- and SeO 4 2- 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 gshA and 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 Na 2 SeO 4 and Na 2 SeO 3 and the concentration of IPTG should be controlled if it is used 20

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 21

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23. 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. 24 Microwell with MIC

25. Methodology MIC measurements: 25 Preparation of bacterial pre- cultures (18 h, 37 °C, in a shaker) OD 600 = 0.5 Dilution until OD 600 = 0.005 Loading 96-microwell plates with toxicants (Na 2 SeO 3, Na 2 SeO 4, Te 2 O 3 ) Two-fold dilutions across the plate final volume of each well =150 µL. Incubation at 37 °C, in a shaker for 24 h Addition of resazurin sodium salt (10 µL, 6.75 mg/ mL) Further Incubation at 37 °C, in a shaker for 24 h Incubation at 37 °C, in a shaker until OD 600 ~ 0.1- 0.2 Mixing bacterial cultures (10 µL) with the toxicants Controls: bacterial cultures and LB Blank: LB OD 600 measurements Color observation Blue: dead cells Pink: live cells OD 600 method Resazurin dye method

26. Specific growth rate (SGR) measurements: 26 Preparation of bacterial pre- cultures (18 h, 37 °C, in a shaker) OD 600 = 0.5 Dilution until OD 600 = 0.005 Incubation at 37 °C, in a shaker until OD 600 ~ 0.1 Loading 96-microwell plates with bacterial cultures (150 µL)and the toxicants (50 µL, concentration= MIC/2) Controls: bacterial cultures and LB Blank: LB OD 600 measurements (initially and after 15 min intervals up to 15 h) SGR determination by slope of the log phase of Ln OD 600 Vs. time plots Intracellular GSH measurements: Pre-cultures (same as above) 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 Intracellular protein contents: Addition of Bradford reagent (1 mL) to above supernatants (50 µL) Absorbance measurements at 595 nm after 2 min Calculation of protein contents using a calibration curve with bovine serum albumin (BSA) standards * Above all were repeated with IPTG (0.05, 0.1, 0.2, 0.4, 0.8, 1.0 mM) added during pre- culture preparation 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

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