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

Gayan K. A. Appuhamillage and T. G. Chasteen

Department of ChemistrySam Houston State University

1

Bioremediation of toxic oxyanions using genetically modified Escherichia coli strains

and study of their glutathione biosynthetic pathway

Objectives Introduction Methodology Results Discussion Conclusions Acknowledgement

2

To study the bioremediation potential of the genetically modified E. coli strains

on toxic oxyanions of Se (SeO32-, SeO4

2-) 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

3

Toxicity of oxyanions of Se and Te on humans

Oxyanions of Se (SeO32-, SeO4

2-)

• decrease body weight gains• liver cirrhosis• pancreatic enlargement• anemia• chronic hepatitis Oxyanion of Te (TeO3

2-)

• vomiting• renal pain• loss of consciousness• irregular breathing• cyanosis

4

Bioremediation

Toxic oxyanions(ex: SeO3

2-, SeO42-, TeO3

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=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

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.

Importance of Glutathione (GSH)

Acts as a reducing agent The thiol group of cysteine becomes oxidized while reducing reactive oxygen species (i.e. SeO3

2-, SeO42- , TeO3

2-) 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

7

E. coli strains used

8

gshBpCA24N plasmid

gshA

AG1

AG1/pCA24NgshA

AG1/pCA24NgshB

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

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

MethodologyMIC measurements:

11

Preparation of bacterial pre-cultures

Add a dye(after 24 h)

• Mixing with the toxicants (Na2SeO3, Na2SeO4, Na2Te2O3)

• Not mixing with the toxicants

OD600 measurements

(after 24 h)

Color observation (after 24 h)•Blue: dead cells•Pink: live cells

Specific growth rate (SGR) measurements:

12

OD600 measurements initially and at specific time intervals andSGR calculation by slope of log phaseIntracellular 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


• Mixing with the toxicants

• Not mixing with the toxicants


Filter and take out bacterial

cells

Break cell walls to take out proteins

13

MIC measurementsIPTG concentration/

mMMIC of Na2SeO3/ mM MIC of Na2SeO4/ mM MIC of Na2TeO3/ mM

0 31.25 250 0.0040.05 31.25 250 0.0040.1 31.25 250 0.0040.2 31.25 250 0.0040.4 31.25 250 0.0040.8 15.625 125 0.0021.0 15.625 125 0.002

IPTG concentration/ mM

MIC of Na2SeO3/ mM MIC of Na2SeO4/ mM MIC of Na2TeO3/ mM

0 125 1000 0.0040.05 125 1000 0.0040.1 62.5 1000 0.0160.2 62.5 1000 0.0160.4 62.5 1000 0.0080.8 62.5 500 0.0041.0 31.25 500 0.004


MIC of Na2SeO3/ mM MIC of Na2SeO4/ mM MIC of Na2TeO3/ mM

0 125 1000 0.0040.05 125 1000 0.0040.1 62.5 1000 0.0160.2 62.5 1000 0.0080.4 62.5 1000 0.0080.8 31.25 500 0.0041.0 31.25 500 0.004

For AG1

For AG1/pCA24NgshA

For AG1/pCA24NgshB

Specific growth rate measurements

14

Condition Specific growth rate/ min-1

control (no toxicant) 0.0093 ± 0.0002with Na2SeO3 (15.625 mM) 0.0077 ± 0.0002

with Na2SeO4 (125 mM) 0.0080 ± 0.0001with Na2TeO3 (0.002 mM) 0.0006 ± 0.0001

Condition Specific growth rate/ min-1

control (no toxicant) 0.0092 ± 0.0006with Na2SeO3 (62.5 mM) 0.0044 ± 0.0002with Na2SeO4 (500 mM) 0.0028 ± 0.0002

with Na2TeO3 (0.002 mM) 0.0011 ± 0.0001

Condition

Specific growth rate/ min-1

control (no toxicant) 0.0095 ± 0.0004

with Na2SeO3 (62.5 mM) 0.0047 ± 0.0001

with Na2SeO4 (500 mM) 0.0037 ± 0.0003with Na2TeO3 (0.002 mM) 0.0015 ± 0.0001

For AG1

For AG1/pCA24NgshA

For AG1/pCA24NgshB

Specific growth rate measurements (with IPTG)

15



of AG1


of AG1/pCA24N gshA


of AG1/pCA24N gshB

0 0.0093 ± 0.0002 0.0092 ± 0.0006 0.0095 ± 0.0004

0.05 0.0083 ± 0.0002

0.0092 ± 0.0006 0.0094 ± 0.0006

0.1 0.0085 ± 0.0002

0.0091 ± 0.0005 0.0093 ± 0.0003

0.2 0.0082 ± 0.0002

0.0092 ± 0.0004 0.0093 ± 0.0005

0.4 0.0084 ± 0.0001

0.0090 ± 0.0008 0.0093 ± 0.0003

0.8 0.0073 ± 0.0003

0.0076 ± 0.0003 0.0086 ± 0.0004

1.0 0.0072 ± 0.0002

0.0074 ± 0.0003 0.0080 ± 0.0005

Intracellular GSH measurements

16

Calibration curve for GSH level measurements

Calibration curve for protein content measurements

17


GSH content (µmol/ mg protein)

of AG1


of AG1/pCA24N gshA


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 ± 0.31 14.34 ± 0.49

0.1 10.51 ± 0.19

14.91 ± 0.19 14.44 ± 0.64

0.2 10.08 ± 0.34

14.35 ± 0.33 13.77 ± 0.32

0.4 9.20 ± 0.14

12.91 ± 0.62 12.43 ± 0.40

0.8 7.86 ± 0.28

11.01 ± 0.28 10.73 ± 0.18

1.0 7.04 ± 0.17

10.09 ± 0.38 9.82 ± 0.10

Intracellular GSH contents

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 Na2SeO4 and 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) 18

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

Toxicity of the tested oxyanions increases in the order of Na2SeO4< Na2SeO3< Na2TeO3

The toxicity of TeO32- is extremely large with respect to SeO3

2- and SeO4

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 Na2SeO4and Na2SeO3 and the concentration of IPTG should be controlled if it is used

20

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

22

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

MethodologyMIC measurements:

25

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. 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 OD600 ~ 0.1- 0.2

Mixing bacterial cultures (10 µL) with the toxicants •Controls: bacterial cultures and LB•Blank: LB

OD600 measurements

Color observation•Blue: dead cells•Pink: live cells

OD600 method

Resazurin

dye method

Specific growth rate (SGR) measurements:

26

Preparation of bacterial pre-cultures (18 h, 37 °C, in a shaker)OD600= 0.5

Dilution until OD600= 0.005

Incubation at 37 °C, in a shakeruntil OD600 ~ 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

OD600 measurements (initially and after 15 min intervals up to 15 h)

SGR determination by slope of the log phase of Ln OD600 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

27

28

Documents

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