role of glutathione reductase and glutathione-dependent formaldehyde dehydrogenase in ...

78 Pages·2015·1.39 MB·English

by  Michelle Abou Naoum

ABSTRACT
ROLE OF GLUTATHIONE REDUCTASE AND GLUTATHIONE-
DEPENDENT FORMALDEHYDE DEHYDROGENASE IN
SYNECHOCOCCUS PCC 7942
Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis,
generate reactive oxygen (ROS) and nitrogen (RNS) species as by-products of
photosynthesis and cellular metabolism. To defend against oxidative and
nitrosative stress caused by ROS and RNS, glutathione (GSH), a low molecular
weight thiol and an antioxidant, is employed by cyanobacteria and is involved in
maintenance of the intracellular reducing environment. Glutathione reductase
(Gor) regenerates GSH from glutathione disulfide (GSSG) maintaining a high
GSH:GSSG ratio. GSH-dependent formaldehyde dehydrogenase (Fdh) involved in
formaldehyde detoxification is able to metabolize S-nitrosoglutathione (GSNO).
To investigate the role of GSH dependent enzymes in protection against oxidative,
nitrosative and metal stress, gor:Tn and fdh:Tn mutants disrupted in gor and fdh
respectively were generated in Synechococcus sp. PCC 7942, a model organism
for photosynthetic studies and stress adaptation. Isolation of both mutants and their
complemented strains gorC:Tn and fdhC:Tn was successfully achieved. Compared
to wildtype, fdh:Tn mutant was susceptible to formaldehyde and nitrosative stress
while gor:Tn mutant grew similarly to wildtype when subjected to oxidative and
metal stress. Thiol levels were determined in wildtype, gor:Tn and gorC:Tn under
normal and stress conditions. GSH levels decreased under peroxide plumbagin,
,
Fe2+ and Cu2+ stress. The long-term goal of this research is to understand the role
of these enzymes in protecting photosynthetic organisms against stress.
Michelle Abou Naoum
August 2015

ROLE OF GLUTATHIONE REDUCTASE AND GLUTATHIONE-
DEPENDENT FORMALDEHYDE DEHYDROGENASE IN
SYNECHOCOCCUS PCC 7942

by
Michelle Abou Naoum

A thesis
submitted in partial
fulfillment of the requirements for the degree of
Master of Science in Biology
in the College of Science and Mathematics
California State University, Fresno
August 2015
APPROVED
For the Department of Biology:

We, the undersigned, certify that the thesis of the following student
meets the required standards of scholarship, format, and style of the
university and the student's graduate degree program for the
awarding of the master's degree.

Michelle Abou Naoum
Thesis Author

Mamta Rawat (Chair) Biology

Joseph Ross Biology

Karine Gousset Biology

For the University Graduate Committee:

Dean, Division of Graduate Studies
AUTHORIZATION FOR REPRODUCTION
OF MASTER’S THESIS

X I grant permission for the reproduction of this thesis in part or in
its entirety without further authorization from me, on the
condition that the person or agency requesting reproduction
absorbs the cost and provides proper acknowledgment of
authorship.

Permission to reproduce this thesis in part or in its entirety must
be obtained from me.

Signature of thesis author:
ACKNOWLEDGMENTS
First and foremost, I would like to express my sincere thanks to, Dr. Mamta
Rawat, my advisor and mentor, for providing me with the great opportunity to
work on a project in her laboratory with all the necessary capabilities to complete
the research. I am extremely thankful to Dr. Rawat for sharing her guidance and
expertise with me. Her continued support allowed me to accomplish goals and
tasks in an efficient and timely manner. Second, I take this opportunity to
acknowledge and express my gratitude to my committee members Dr. Karine
Gousset and Dr. Joseph Ross for their support, time and feedback. Third, I want to
thank Derek Vargas, Andrew Strankman, Jonathon Butin, Teresa Brooks, Dr.
Arishma Rajkarnikar Singh and all the laboratory members for their continued
help and support. At last, I am also grateful to my parents and husband for their
encouragement, support and tremendous help.
This work was supported by the National Science Foundation, and CSU-
Fresno College of Science and Mathematics Faculty Support Student Research
Award. The funds provided allowed for the timely completion of this project.
TABLE OF CONTENTS
Page
LIST OF TABLES ................................................................................................. vii
LIST OF FIGURES ............................................................................................... viii
LITERATURE REVIEW ......................................................................................... 1
Oxidative Stress: ROS and Free Radicals ......................................................... 3
Metal and Xenobiotic Stress ............................................................................. 6
Nitrosative Stress .............................................................................................. 9
Low Molecular Weight Thiols ........................................................................ 10
Glutathione ...................................................................................................... 11
Glutathione Biosynthesis ................................................................................ 12
Glutathione Reductase .................................................................................... 14
Ergothioneine .................................................................................................. 15
Formaldehyde Dehydrogenase/ S-nitrosoglutathione Reductase
(Fdh/GSNOR) ...................................................................................... 15
Short Chain Aldehydes ................................................................................... 18
Model Organism .............................................................................................. 20
Rationale ......................................................................................................... 21
Hypotheses ...................................................................................................... 22
METHODOLOGY ................................................................................................. 23
Culture Conditions .......................................................................................... 23
Mutagenesis ..................................................................................................... 23
Mutant Complementation ............................................................................... 26
Growth Curves ................................................................................................ 27
Thiol Labeling and HPLC ............................................................................... 29
Absorption Spectrum ...................................................................................... 31
vi vi
Page
RESULTS ............................................................................................................... 32
Isolation and Confirmation of Mutants ........................................................... 32
Isolation and Confirmation of Complemented Strains ................................... 34
Chlorophyll Extraction and Absorption Spectra ............................................. 34
Oxidative and Metal Stress in gor:Tn ............................................................. 39
Thiol Content in gor:Tn .................................................................................. 39
Nitrosative Stress in fdh:Tn ............................................................................. 44
Formaldehyde Stress in fdh:Tn ....................................................................... 44
DISCUSSION ......................................................................................................... 49
Conclusion ....................................................................................................... 54
Future Work .................................................................................................... 55
REFERENCES ....................................................................................................... 57
LIST OF TABLES
Page
Table 2.1. Strain Description ................................................................................. 24
Table 2.2 List of Primers: Oligonucleotides used to amplify DNA sequences.
Forward (F) and Reverse (R) primers are directed at 5’ and 3’ ORF of
the genes. .................................................................................................. 28
Table 3.1: Chlorophyll a levels in different strains. 10 mL of each wildtype,
gor:Tn, gorC:Tn, fdh:Tn and fdhC:Tn were grown to OD of 1.0
750
and treated with 1:1:1 mixture of pre-chilled acetone, methanol and
ethanol in order to extract pigments from the cells. After a 40 min
incubation at 4°C in dark, chlorophyll a levels were determined
through spectrophotometric analysis at 665nm. A two-tailed T-test
was used to determine whether difference between wildtype, mutant
and complemented strain was significant (significance p<0.01
represented by *) ...................................................................................... 37
Table 3.2: GSH levels in µmoles/g in untreated and treated samples. Results
represent mean and standard deviation. (Significance difference
between the treated and untreated strain is represented with * where
p<0.05 is considered significant) ............................................................. 43
LIST OF FIGURES
Page
Figure 1.1: Reactive oxygen species and reactive nitrogen species in the cell
as described by Bartosz 2009. ................................................................... 4
Figure 1.2: Low molecular weight thiols. A. Cysteine B. Thiocysteine C.
Cysteamine D. Lipoic acid E. Ergothioneine. F. Glutathione
(reduced form) G. Glutathione disulfide (oxidized form). ..................... 11
Figure 1.3: Glutathione Biosynthesis. GshA: ATP-dependent enzyme γ-
glutamylcysteine synthetase catalyzes addition of glutamic acid to
cysteine to form γ-glutamylcysteine. GshB: ATP-dependent
glutathione synthetase catalyzes addition of glycine to γ-
glutamylcysteine to form GSH. .............................................................. 13
Figure 1.4: Glutathione reductase reaction. Gor catalyzes the reduction of
GSSG to GSH with NADPH as the source of reducing equivalents. ..... 14
Figure 1.5: Oxidoreductase Activity of Fdh. A) Formaldehyde dehydrogenase
activity of Fdh. GSH reacts with formaldehyde to form
hydroxymethylglutathione which is a substrate for Fdh. S-formyl
glutathione is formed as a result. B) S-nitrosoglutathione reductase
activity of Fdh. GSH reacts with NO to form GSNO which is the
substrate for Fdh. .................................................................................... 17
Figure 1.6: Formaldehyde and short chain aldehydes. A) Formaldehyde B)
glyoxal C) Glycolaldehyde D) Methylglyoxal E) Glyceraldehyde. ....... 19
Figure 2.1: Knockout plasmids. Plasmid containing a disrupted fdh gene with
a Mu transposon insertion containing a chloramphenicol (Cm)
resistance cassette and ampicillin (Ap) and Kanamycin (kan)
resistance in the backbone of the vector (Top). Plasmid containing a
disrupted gor gene with Tn transposon insertion with kanamycin
resistance cassette and chloramphenicol resistance in the backbone
of the vector (Bottom). ............................................................................ 25
Figure 2.2: Map of pSyn_1/D-TOPO vector. Features of vector: TOPO
binding site 1 and binding site 2, rrnB transcriptional termination
region, NS1b and NS1a (neutral site 1b and 1a respectively), P
sc
(promoter), P (spectinomycin promoter), spectinomycin resistance
sp
gene, RBS (ribosome binding site) and GTGG overhang. (GeneArt®
Synechococcus TOPO® Engineering Kits: Life Technologies). ........... 28
Figure 3.1: Confirmation of gor:Tn mutant. pTn:gor, wildtype and gor:Tn
were amplified with gorF and gorR primers. .......................................... 33