THE GROWTH AND STRESS RESPONSE CHARACTERIZATION
OF SYNECHOCOCCUs WH8 109 CYANOBACTERIA
MASSACHUSETTS INSTITUTE
OFTECHNOLOGY
BY ERIKA
M. ERICKSON
AUG 16 2010
S.B. BIOLOGICAL ENGINEERING, S.B. BIOLOGY
MASSACHUSETTS INSTITUTE OF TECHNOLOGY, 2008
S RARIES
ARCHIVES
SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL ENGINEERING
INTHE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ENGINEERING INBIOLOGICAL ENGINEERING
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
MAY2009
© 2009 Massachusetts Institute of Technology. All Rights Reserved
n
i'
Signature of Author
Erika Erickson
May 22, 2009
I
j--
Certified by.
Jonathan A. King
Professor of Biology
Thesis Supervisor
Certified by
i
As
, -
Eric J. Alm
istant Professor of Biological Engineering, and Civil and Environmental Engineering
A2
Thesis Supervisor
//
Accepted by.
wDarrell
J.Irvine
Associate Professor of Materials Science and Biological Engineering
Chairman, Master of Engineering in Biological Engineering Program
S
THE GROWTH AND STRESS RESPONSE CHARACTERIZATION OF
SYNECHOCOCCUs WH8109 CYANOBACTERIA
BY ERIKA M. ERICKSON
SUBMITTED TO THE DEPARTMENT OF BIOLOGICAL ENGINEERING ON MAY 22, 2009
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF ENGINEERING IN BIOLOGICAL ENGINEERING
AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY
ABSTRACT
Oceanic cyanobacteria are amongst the most populous species on the planet and
have been found in every ocean around the world. These photosynthetic organisms play a
major role in the global carbon cycle. They have adapted to a number of different
temperature, light, and nutrient niches. However, as important primary producers in the
oceans, these organisms play a vital role which may be threatened by global climate change
and pollution. As research on cyanobacterial species progresses, these organisms have
been found to show promise as potential sources of biofuel, renewable energy, and agents
for bioremediation. In order to utilize these organisms for future engineering applications
and basic scientific research, it is important to be able to grow the organism in a stable and
reproducible manner. This research characterizes the growth of Synechococcus WH8109 in
the laboratory. In the laboratory, cell culture densities of greater than 109 cells/mL with a
doubling time of approximately 24 hours were achieved when grown at 28'C with a 24 hour
light cycle in sea water and artificial salt water media.
Not only did cyanobacteria evolve long before their distant enteric cousins, but they
harness nearly all of their energy through photosynthesis. The photosystem is constantly
subjected to photo-oxidative damage and degradation. Interesting insight may be gained by
studying this complex repair process in the bacterial counterpart to plants, prior to applying
these concepts to higher order plant species. Chaperones have been implicated in this
repair process. In order to better characterize the stress response of WH8109, I have also
isolated the Synechococcus homologue of GroEL using anion exchange and gel filtration
chromatography and sucrose gradient centrifugation. The expression levels of this
chaperone were analyzed under normal and stress conditions and they have been shown to
respond to heat shock and infection.
Thesis Supervisor: Jonathan King
Title: Professor of Biology
Thesis Supervisor: Eric Alm
Title: Assistant Professor of Biological Engineering and Civil and Environmental
Engineering
Contents
A BST RACT ...................................................................................................................................................................
A2
Tables and Figures ...................................................................................................................................................
5
1.0
Introduction .................................................................................................................................................
6
The Photosynthetic A pparatus ..................................................................................................
6
1.1
1.1.1
Photosynthesis..............................................................................................................................6
1.1.2
Lim itations of Current Research ....................................................................................
1.2
8
Synechococcus......................................................................................................................................10
1.2.1
WH8109 and Its R elatives................................................................................................
10
1.2.2
Grow th Characteristics.....................................................................................................
11
1.2.3
Susceptibility to Phage Infection..................................................................................
12
1.3
Chaperones ...........................................................................................................................................
1.3.1
Roles and Functions.................................................................................................................15
1.3.2
Chaperonins................................................................................................................................17
1.3.3
GroELS...........................................................................................................................................18
1.4
2.0
15
Objectives and Motivation for Study .....................................................................................
19
Characterization of Stress Response..........................................................................................
21
2.1
Materials and Methods.....................................................................................................................21
2.1.1
Grow th Conditions ...................................................................................................................
21
2.1.2
Cell Count and Density Measurem ent.......................................................................
21
2.1.3
Infection........................................................................................................................................22
2.1.4
Heat Shock ...................................................................................................................................
2.1.5
Western Blot...............................................................................................................................24
2.2
Results.....................................................................................................................................................26
2.2.1
Grow th Curves ...........................................................................................................................
2.2.2
Infection Response...................................................................................................................29
2.2.3
Heat Shock Response ..............................................................................................................
2.3
24
26
30
Discussion..............................................................................................................................................32
2.3.1
Using Synechococcus W H8109 for Research ............................................................
32
2.3.2
Synechococcus WH8109 Growth Conditions and Characteristics ...................
33
2.3.3
Stress Response and GroEL .............................................................................................
33
3.0
Characterization of WH8109 GroELS Chaperonin Complex...........................................
3.1
Materials and Methods.....................................................................................................................37
3.1.1
Purification..................................................................................................................................37
3.1.2
B iochem ical Analysis...............................................................................................................41
3.1.3
Imaging .........................................................................................................................................
3.2
42
Results.....................................................................................................................................................42
3.2.1
Purification..................................................................................................................................42
3.2.2
Mass Spectrom etry ..................................................................................................................
3.3
37
50
D iscussion..............................................................................................................................................51
4.0
Sum m ary and Conclusion ....................................................................................................................
55
5.0
Future W ork ..............................................................................................................................................
58
6.0
References..................................................................................................................................................60
7.0
A ppendices.................................................................................................................................................65
Appendix A: Sea Water Media for Laboratory Growth of Synechococcus............................
65
A ppendix B: "W eigele Bubblers ................................................................................................................
66
Appendix C: Evaluation of Transcriptional Expression..............................................................
69
Appendix D: Mass Spectrometry Preliminary Results .................................................................
73
8.0
A cknow ledgm ents ..................................................................................................................................
77
Tables and Figures
7
Figure 1: Photosynthesis .......................................................................................................................................
9
Figure 2: Photosynthetic Electron Transport Chain ............................................................................
11
Figure 3: Sargasso Sea .........................................................................................................................................
Figure 4: Synechococcus Physiology...............................................................................................................13
14
Figure 5: Cyanophage ..........................................................................................................................................
Figure 6: Chaperone Networks........................................................................................................................16
Figure 7: Chaperonins..........................................................................................................................................18
19
Figure 8: GroELS Substrate Folding Cycle.............................................................................................
22
Figure 9: Synechococcus W H8109 Fluorescence ..................................................................................
24
Figure 10: Phage purification CsCl gradient...........................................................................................
26
Figure 11: Synechococcus W H8109 Growth Curves..........................................................................
Figure 12: Absorbance Spectra........................................................................................................................27
Figure 13: Cell Count vs. ODns.........................................................................................................................28
30
Figure 14: Syn 5 Infection of Synechococcus W H8109.......................................................................
31
Figure 15: Brief Heat Shock Characterization ....................................................................................
36
Figure 16: Prolonged Heat Shock Characterization .........................................................................
36
Figure 17: High Heat Shock Characterization........................................................................................
40
Figure 18: HPLC Chromatography Programs .......................................................................................
43
...............................................................................................
Gradient
Purification
19:
Sucrose
Figure
Figure 20: : Visualization of GroEL from Sucrose Gradient Purification...............44
45
Figure 21 Chromatography UV Absorbance...........................................................................................
Figure 22: Visualization of GroEL from Purification Trial #1.............................................................46
47
Figure 23: Purification Trial #1 Samples................................................................................................
48
Figure 24: Phycobiliprotein Separation..................................................................................................
Figure 25: Purification Trial #2.......................................................................................................................49
Figure 26: GroEL Samples from Purification Trial #2............................................................................50
51
Figure 27: MS Analysis ........................................................................................................................................
67
Figure 28: Synechococcus W H8109 Cultures .......................................................................................
68
...............................................................................................
Bubbler"
Blueprint
Figure 29: "W eigele
Table
Table
Table
Table
1: Chromatography Buffers ..................................................................................................................
2: Genes of Interest...................................................................................................................................70
3: PCR Primers ...........................................................................................................................................
4: Sequencing Primers ............................................................................................................................
41
71
72
1.0 Introduction
As global climate continues to change, it is crucial not only to gain
understanding of the species most susceptible to collapse, but also to find which
species may prove to be beneficial in attempts to alleviate an environmental crisis.
This thesis partly focuses on characterizing the growth conditions and stress
response of an abundant marine cyanobacteria species that plays a major role in the
global carbon cycle. Variations in ocean temperature and nutrient distribution
could heavily influence this organism; however it may also hold the key to
revolutionary advancement in alternative energy and bioremediation research. As
this country and others begin to focus more and more on alternative energy
initiatives, cyanobacterial and algal energy sources look extremely attractive and
certainly merit more investigation. To use this system, however, it must be feasible
to grow the organism in a laboratory under highly reproducible conditions.
Natural energy, such as photosynthesis, offers an intriguing yet challenging
avenue for alleviating the energy and resource crises the world may soon find itself
in. Not only is photosynthesis crucial for life on earth, but photosynthesis can
capture an incredible amount of energy. Manipulation of the photosystem in order
to harness this energy will prove most beneficial in future work on alternative
energy. To truly understand how the photosynthetic apparatus works, however,
one should also understand how it is put together.
I suspect this is largely
influenced by the group I chaperonin complex GroELS, which is found in many
bacteria and is homologous to the Hsp60/HsplO complexes in eukaryotes.
Therefore, this thesis also addresses the photosynthetic apparatus in cyanobacteria
species Synechococcus WH8109, and the characterization of one of its major protein
chaperones.
1.1
The Photosynthetic Apparatus
1.1.1
Photosynthesis
....
.
.....
.....
.........
.................................
...
..........
......
..................
....
....
....
......
...............
......
.. . .. ...
..
..
...
............
One of the major goals of plant science research is to increase efficiency of
photosynthetic systems. It has been shown that photosynthesis adjusts under stress
conditions such as high salt near marine environments, low water conditions in arid
environments,
or
under
increased
temperatures
(Schnackenberg,
1996).
Manipulation of food-producing plants in order to make hardier, larger yields under
varied environmental conditions has long been an aim of plant biologists facing the
challenge of feeding the Earth's ever-growing human population (NAS, 2000).
Climate change, spiking prices in transportation and food-processing, largely caused
by rising oil prices, and the financial lure of biodiesel crops over food-producing
crops has created the biggest stress in world food supply in years (Walt, 2008).
Monoculture crops and advancements in farming techniques have helped in some
ways with the global demand for food; however, these methods are essentially a
double-edged sword, threatening the genetic diversity and perhaps even the
nutrition of popular crops. Plant engineering has the potential to help assuage
world-wide hunger and malnutrition, if considered and implemented with care.
Photosynthesis is essential not only for maintaining food-sources, but also
for sustaining viable atmospheric conditions. It is well known that for every 6 moles
of carbon dioxide and 6 moles of water, 6 moles of molecular oxygen and 1 mole of
glucose are produced (See Figure 1). Oxygen production and carbon fixation support
oxygenic life on the planet. However, the efficiency of natural photosynthesis is
actually quite low. Less than 1% of the sun energy absorbed by photosynthetic
tissues is converted into biomass that is usable for fuel. The chemistry involved in
energ
caronlight
carbon
+water c
carbohydrates
idechlorophyll
'I
6COC+
Figure 1: Photosynthesis
6H20
light energy
chlorophyll
C6H120 6 +602
+ oxygen
..
..........
.
the light reactions, however, has closer to 50% energy conversion efficiency
(Korsfeldt, 2003). The focus of future research will be to mimic and improve this
process for large scale use. One application for increased photosynthetic activity is
the alleviation of some of the environmental carbon stresses caused by pollution
and industrialization, but breakthroughs in this line of research could also shift
society's dependence away from non-renewable fuel sources.
While the optimization of photosynthesis may seem quite straightforward,
the photosystem is very complicated.
Light reactions take place in the stacked
thylakoid membranes of photosynthetic organisms. In green plants, thylakoids are
found in the chloroplast, a pigment carrying organelle that is thought to have
evolved from engulfed cyanobacteria hundreds of millions of years ago (Bergsland,
1991). The light is harvested by antenna complexes (LHC proteins) associated with
the two photochemical reaction centers, photosystem I and photosystem II (PSI and
PSII, respectively). The electron transport chain also includes a cytochrome b6 /f
complex and an ATP synthase. Both PSI and PSII are composed of multiple protein
components used for binding and electron transfer. Well over 20 proteins make up
PSII alone (Hankamer, 1997). (See Figure 2) Manipulating such a complex protein
system is not a trivial task.
To make matters even more complicated, photosynthetic organisms are
constantly subjected to oxidative damage. The proteins in the photosystem reaction
center, chlorophyll, and the other light harvesting pigments are sensitive to light
damage. Oxidative stress and light damage result in a complex system of repair and
recovery under high light conditions, such that the large photosystem complexes are
frequently regenerated (Kim, 1993).
This damage and repair cycle presents a
significant challenge for the scientific application and imitation of PSI and PSII in
pursuit of an alternative energy source.
1.1.2
Limitations of Current Research
Cyanobacteria may be an attractive option for energy research, but there are
two major limitations impeding significant progress in this research.
First, the
........
........................
..........
M:
.1 1.
.............
.. ......
........
..............
.
1=
0-
.
......
......
- 11
"I'll"
genetic system of Synechococcus and many other cyanobacteria are not well
understood, as of yet. Second, while most of the components of the photosynthetic
apparatus are known, attempts to artificially achieve the PSII light conversion
reactions or to isolate and rebuild functioning PSI or PSII reaction centers in vitro
have met with only some success (Kanan, 2008; Hankamer, 1997; Dent, 2005).
In light of these limitations, Synechococcus WH8109 offers a few advantages
over other cyanobacteria.
First, Synechococcus is susceptible to cyanophage
infection, which bodes well for the development of genetic techniques in this
species. A common form of genetic manipulation in model bacterial species involves
phage-mediated gene transfer. While this has not yet been done in Synechococcus
WH8109, extensive characterization of Syn 5 and Syn 9, two of the cyanophage
capable of infecting Synechococcus WH8109, is currently underway (Pope, 2007;
Weigele, 2007). It seems reasonable to expect significant advances in the
understanding and use of Syn 5 in the future, potentially even for the genetic
Further advantages offered by
manipulation of Synechococcus WH8109.
Synechococcus WH8109 as a model organism include its ability to be grown to high
concentrations in the laboratory, and its small size. These traits have proven to be
particularly useful for some of the experiments described in the following pages.
Caibon fixn
reactions
JA14.
NADP-
PSII
Cyt bf
Figure 2: Photosynthetic Electron Transport Chain (Campbell, 2004)
9
PSI
ADP+Pi
ATP synthase
Finally, Synechococcus WH8109 an accessible source of a native essential
group I chaperonin. GroELS has been shown to frequently co-purify with thylakoid
membranes and RuBisCO proteins, strongly indicating its role in maintaining the
photosynthetic capacity of the cells (Lehel, 1992). It has been shown previously that
chloroplast and thylakoid development is defective in Arabidopsisthaliana mutants
lacking Clp complex components (Rudella, 2006). Cip protein complexes act as both
important chaperones and proteases, but deletion mutants are often viable (Mogk,
2001). Given what is known about damage in photosynthetic organisms and the
propensity for thylakoid-GroEL co-purification, GroEL may play a major role in
building and maintaining the photosynthetic apparatus of Synechococcus WH8109.
1.2
Synechococcus
1.2.1
WH8109 and Its Relatives
The simplest organisms that do complete oxygenic photosynthesis are the
single-celled phytoplankton, cyanobacteria.
Cyanobacteria are ubiquitous and
abundant in oceanic regimes all over the world. Picophytoplankton of the genus
Synechococcus are the predominant phycobilisome-containing cyanobacteria in the
world's aquatic environments (Scanlan, 2003). Synechococcus species are found in
most oceanic regimes around the world and boast a large range of viable
temperatures across the genus (Six, 2008). They also greatly contribute to global
marine carbon fixation, contributing between 32% and 89% of marine primary
production (Muhling, 2005). Synechococcus is a close relative of Prochlorococcus,a
genus on which a lot of characterization research has already been done (Partensky,
1999; Sullivan, 2003).
Synechococcus WH8109 grows predominantly in the Sargasso Sea, which is
located between the 40th and 70th meridians and the 25th and 35th parallels in the
middle of the North Atlantic Ocean (See Figure 3).
The culture grown in the
laboratory was isolated from coastal waters near the Bahamas, but the species has
been found both in open-ocean and coastal waters in concentrations of up to 105
..
. . .......
.
I
"I
"',
4' -
, -
..........
,
- -
z
...
....
........
,-
Figure 3: Sargasso Sea
cells/mL (Sullivan, 2003).
Prochlorococcus species from the same region grow
predominantly in the open ocean, and have been found to be far less abundant in the
more
variable
and potentially polluted
coastal waters
(Sullivan, 2003).
Synechococcus WH8109 has not been fully sequenced yet, but the project is in
progress
(Lindell, personal
communication, October 2008).
Nonetheless,
Synechococcus WH8109 is classified as an abundant clade II Synechococcus and
thought to be very similar to both Synechococcus CC9605, which was isolated off the
coast of California (Copeland, 2005), and Synechococcus WH8102, which is also from
the Sargasso Sea (Palenik, 2003), both of which have been sequenced: GenBank
Access ID's are CP000110.1, and BX548020.1, respectively.
1.2.2
Growth Characteristics
In the laboratory, Synechococcus WH8019 can be grown to concentrations in
excess of 1010 cells/mL in sea water media. Cultures in the exponential growth
phase are typically at concentrations between 107-108 cells/mL. Doubling time and
color depend on many factors, including seawater media, aeration rate, and light
conditions. WH8109 cells grow to an average of 0.8ptm diameter, which is slightly
larger than cyanobacteria of the genus Prochlorococcus,whose average diameter is
around 0.5pm, but small for the Synechococcus genus, which spans from 0.8pm2.0 pm (Waterbury, 1979). (See Figure 4)
Synechococcus have widely spaces thylakoid membranes (40-50nm) to allow
room for the phycobilisome complexes on the outside of the membranes. Other
marine cyanobacteria have been shown to have closely packed thylakoids and
different
light-harvesting-pigment
compositions
(Chisholm,
1988).
The
phycobilisomes are rod-shaped antenna complexes composed of phycoerythrins
(PE) that bind chromophores called phycobilins (See Figure 4). The phycobiliprotein
rods surround a central core of allophycocyanin (AP), which binds the blue
chromophore phycocyanobilin (PCB; Amax = 620nm).
Synechococcus WH8109
phycobilisomes also bind the chromophores phycoerythrobilin and phycourobilin,
which are pink or red (PEB; Amax = 550nm) and orange (PUB; Amnax = 495nm)
chromophores respectively, in an approximately 1:1 ratio (Six, 2008). Due to its
pigment composition and light adaptability, Synechococcus WH8109 is categorized
as a pigment type 3c Synechococcus according to research by Six et al. (2008).
1.2.3
Susceptibility to Phage Infection
Synechococcus WH8109 is susceptible to infection by at least ten cyanophage,
including Syn 5 and Syn 9 (Sullivan, 2003). Synechococcus species are typically
susceptible to infection by Myoviridae, which is a morphological family of virus that
includes T4-like viruses and Syn 9. Syn 5 is in the Podoviridaevirus family, which
includes T7 and P22-like viruses.
Experimentation involving both phage have
revealed the structural components, protein sequences, genomic sequences (Pope,
2007; Weigele, 2007), and infection characteristics of each (Sullivan, 2003). (See
Figure 5)
..
............................
........
....
..........
......
..
........................
.....
.
.....
.
4a)
4b)
jj)
*Allophycocyanin
C-Phycocyanin
R-Phycocyanin 11
Phycoerythrin I
Phycoerythrin 11
Figure 4: Synechococcus Physiology
A - Electron micrograph of thin sections of Synechococcus isolated at 100m depth from 36*07.9'N,
64 018.1'W, 11 September 1987; scale bar 0.5pm (Chisholm, 1988). Note the widely spaced thylakoid
membranes (see red arrow) that occupy the majority of the intracellular space.
B - i) Electron micrograph of phycobilisome complexes isolated from Calothrixsp. PCC 7601 grown in
green light; mag. 250k (Glauser, 1992). ii) Model of the pigment type 3 Synechococcus phycobilisome
complex, adapted from Six et al. (2008)
......
..
...
. ............
..
Synechococcus WH8109's susceptibility to phage infection has major
Synechococcus and
consequences for the cyanobacteria's ecological role.
Prochlorococcus are two of the most abundant genera on the planet, and are
responsible for up to 90% of primary production in the open oceans (Muhling,
2005). Cyanophage infection plays a critical role in population control, nutrient
release and horizontal gene transfer for these species. Genetic evidence indicates
photosynthesis genes may be introduced to cyanobacteria species from phage
genomes (Sharon, 2007). How this affects expression of the endogenous genes and
phage genes is not yet fully understood, but cyanophage carrying photosynthetic
genes have become popular subjects of experimentation (unpublished data). Phage
and cyanobacteria sequence data is currently in the pipeline, which will facilitate
future experiments on modulation of expression and genetic manipulation of the
Synechococcus host via phage mediated gene insertion.
Figure 5: Cyanophage
A - Electron micrographs of Syn 5
stained with 2% uranyl acetate.
(Pope, 2007);
5b)
0
Q
4
B - Electron micrographs of Syn 9
stained with 1% uranyl acetate.
Taken by Peter Weigele, 2004.
1.3
Chaperones
Biology dogma is centralized around the theme of turning genetic
information into functional units, or proteins. Proteins have a huge diversity of
biological roles and abilities, from structural macromolecules to catalysts to
complex mechanical functions. However, proteins must achieve the proper fold
required for these functions, which is a complex thermodynamic process for the
polypeptide chain. In addition, the cytoplasm contains a high concentration of
protein which could lead to local situations of molecular crowding, sending a newly
folding polypeptide down the wrong folding pathway.
Both proper molecular
function of its proteins and efficient quality control is essential for cellular survival.
Some proteins, like ribonuclease are able to adopt their native structure
spontaneously while others require the assistance of a class of molecules called
chaperones (Anfinsen, 1973; Cheng, 1989). While many molecular chaperones are
referred to as heat shock proteins (HSPs), they are not all regulated in response to
heat shock and most play a maintenance role during normal cell growth in addition
to circumstances of stress and heat-mediated denaturation (Schlesinger, 1990).
Overall, the macromolecules called chaperones are structurally unrelated, but some
of the chaperones share similar characteristics, mechanisms, or structures (Mogk,
2001).
1.3.1
Roles and Functions
Macromolecules that aid in the folding of proteins differ slightly between
prokaryotes and eukaryotes.
The prokaryotes predominately utilize a single
chaperone network, the heat shock proteins, for the folding of newly synthesized
proteins and refolding of denatured or misfolded proteins. Included in this group
are the small heat shock proteins (sHSPs) and chaperonins. Bacteria also have a
chaperone called Trigger Factor (TF), which associates with the ribosomal exit
tunnel and interacts with newly synthesized polypeptide chains (Ludlam, 2004).
Eukaryotes, however, use two chaperone networks. The first includes the Clp-
.......
...
. ....................
...
.....
_ :...
....
...
.............
:::_
family of chaperones. Chaperones linked to protein synthesis (Clps) are linked to
the translational machinery of the cell and aid in co- and post-translational folding
of newly synthesized polypeptide chains and translocation of polypeptides (Reid,
2001). The second includes the heat shock proteins, which aid in folding and
refolding of proteins in response to stress or cytoplasmic crowding. (See Figure 6)
HSPs are named according to the approximate molecular weight of the
individual subunits of proteins observed to up regulate in response to heat stress.
Hsp90 is one of the most abundant proteins produced in cells, comprising between
1 and 2% of protein content under normal conditions and is found in bacteria and
A
B
Eukaryotes
Prokaryotes
TF
Ssb
CLIPS
Hsps
Hsps
Do novo
folding
Do novo
Denaturing
stress
folding
Denituring
straw
Figure 6: Chaperone Networks
A - Eukaryote chaperones include the heat shock proteins which function in folding and refolding of
nascent polypeptide chains and misfolded proteins under normal and stress conditions. Eukaryotes also
have Clp-family proteins, which associate with newly synthesized peptide chains.
B - Prokaryote chaperones include Trigger Factor (TF) and heat shock proteins. (Reissmann, 2007)
all branches of eukarya (Chen, 2006). Hsp70, also known as DnaK has been shown
to form a multi-chaperone complex with Hsp90 and DnaJ (Wegele, 2004). DnaJ is
the prokaryotic homologue of Hsp40 (Caplan, 1993).
The class of chaperones this
thesis will focus on are the Hsp60-like chaperonins and Hsp1O co-chaperonins.
1.3.2
Chaperonins
Chaperonins are ATP-dependent homo- and hetero-oligermeric complexes
that assemble into double-barreled structures.
The double barrel structure
provides two open cavities for sequestering misfolded or folding proteins and
isolating them from the crowded cytoplasm (Reissmann, 2007).
Group 1
chaperonins are found in bacteria and within eukaryotic organelles that originated
from an endosymbiont. The Group I chaperonins consist of two heptameric rings
composed of subunits of about 60kDa and use a co-chaperonin composed of a
heptameric lid complex (Kusmierczyk, 2003). This co-chaperonin is made of a set of
independent protein subunits that associates with the mouth of the barrel structure
to encapsulate the folding protein within one of the cavities (Fenton, 1996).
Included in this group is the GroELS complex from E. coli and its homologues that
are found in many bacterial species (See Figure 7).
Group II chaperonins are archaeal and eukaryotic in origin. While these
chaperonins still have the double-barrel structure, they have a built-in lid made
from the "floppy" apical domain of the subunits instead of a co-chaperone like GroES
(Reissmann, 2007).
The conformational changes involved in encapsulating the
protein target are different for the group II chaperonins and resemble a twisting
iris-like action as opposed to the cooperative binding of the GroES lid to GroEL
mechanism proposed for the group I chaperonins (Reissmann, 2007). (See Figure 7)
, --
-
- - - - ....
.......
........
. ....
.............................................................
........
-
-- -
I I - -,
,
,
GroEL-ES
GroEL
184A
GroES
142A
1
-
ATP
-140l
16 nm
side
tilted
top
Figure 7: Chaperonins
[top] Group I Chaperonin GroELS. (Frydman, 2001)
[bottom] Group H1Chaperonin Mm-Cpn. Three dimensional structure obtained by cryo-EM.
(Reissmann, 2007)
1.3.3
GroELS
The GroELS chaperonin complex has various functions in protein folding, DNA and
RNA synthesis, phage head assembly, and protein secretion (Georgopoulos, 1990;
Kusukawa, 1989; VanDyk, 1989). In E. coli, it is absolutely required for bacterial growth at
all temperatures (Fayet, 1989). It is speculated that the levels of GroELS could determine
the upper limit of temperatures cells can survive in (Lehel, 1992). The binding kinetics of
substrate, ATP, and the GroELS complex indicate a cooperative mechanism of binding and
release of the substrate in alternate cavities (Rye, 1997). Evidence of efficient folding of
.. ....
.................................
.................
.
.1. .
..........
...............
.....
...................
ADP
ATP
GroES
ATP
Li
CI
AP
ATP
Pi
ATP
I
T
Figure 8: GroELS Substrate Folding Cycle.
Cooperative ATP binding to the substrate bound cis-cavity of GroEL results in a slight conformational
change that induce GroES binding to the ds-cavity top. As ATP is hydrolyzed, a new substrate may bind
the trans-cavity, changing the conformation of the other cavity and releasing substrate, ADP, and GroES,
allowing for repeat of the cycle in the new cs-cavity. (Reissmann, 2007).
unfolded protein within the cis-cavity of the GroEL complex has been observed in
the presence of GroES and Mg-ATP (Weissmann, 1995). Binding of Mg-ATP and
substrate in the unoccupied cavity triggers the release of the GroES cap over the cisring and release of the partially or completely folded protein from within (Rye,
1997). See Figure 8 for the proposed model of substrate binding and release. The
enclosed cavity has a volume of approximately 175,000
A3 (Xu, 1997). This space
could therefore theoretically fit a globular protein of up to 142kDa within the cavity,
although the largest known substrate to be encapsulated by GroEL and GroES has
been 86kDA (Chen, 2006).
1.4
Objectives and Motivation for Study
The objective of the research presented here is to characterize the stress
response of an abundant photosynthetic oceanic cyanobacteria species important to
the global carbon cycle. To better understand the stress response of Synechococcus
WH8109, this research also focuses on the purification of one of its chaperonin
complexes that is homologous to the well-characterized E. coli chaperonin, GroELS.
Not only is this genus of cyanobacteria important in the natural world, but it
offers many advantages for study in the laboratory.
Photosynthesis is crucial for
life on this planet, yet the photosynthetic apparatus is constantly subjected to
oxidative and heat stress damage. As a photosynthetic oceanic cyanobacterium,
Synechococcus evolved long before enteric bacterial species.
To gain an
understanding of how some of the earliest organisms protected themselves against
damage as ubiquitous as photo-oxidative UV damage, research may uncover
interesting properties about oxidative stress in higher organisms.
It has been
shown that chaperones and small heat shock proteins co-purify with important
components of the photosynthetic apparatus and these chaperones offer protection
against UV damage (Balogi, 2008). One of the goals of modern research in multiple
biological fields is to understand how chaperones work, what their specific targets
include, and the implications this has on protein folding, disease, and development.
The motivation behind this research is to advance efforts to visualize
chaperones and other cellular machinery at work in vivo. The characterization of
the stress response of Synechococcus WH8109 and one of the important chaperones
in these cells is the first step to better understanding how these organisms respond
to stress conditions.
Once the experimental system is fully understood, these
organisms can be used for the visualization of chaperone localization and specificity
in vivo, even in higher organisms for the purpose of monitoring disease and
development.
Highly advanced imaging technology exists that could someday
accomplish this goal.
2.0
Characterization of Stress Response
2.1
Materials and Methods
2.1.1
Growth Conditions
Synechococcus WH8109, originally provided by John Waterbury and Matthew
Sullivan, was grown in the laboratory using a Percival Scientific (Boone, IA) Model
E-30B light incubator. Cultures were grown in artificial sea water (ASW) media and
SN media using water collected at the Northeastern University Marine Science
Center in Nahant, MA (See Appendix A). Cultures were grown under "cool white"
light supplied by a 40W bulb at an irradiance intensity of 50
[tEm-2 s-,
in a 24 hour
light cycle at 28'C. Cultures were aerated using the "Weigele" Bubbler System (See
Appendix C) in 500mL, 1L, or 2L polycarbonate bottles with polypropylene caps
(Nalgene). Cultures were started from frozen cell stock kept in 15% glycerol or 7%
DMSO at -80'C. Culture lines were continued via a 1% (v/v) inoculation of culture
in exponential growth phase into fresh SN Nahant or ASW media continuously for
up to 5 months.
2.1.2
Cell Count and Density Measurement
Cell count was done using a Zeiss HBO 50ac Epifluorescence Microscope with
mbq 52ac power supply (Thornwood, NY). Samples were loaded onto two glass
1/400 mm 2 Petroff-Hausser Counter slides (Hausser Scientific), and cell count was
calculated using an average of three squares from each slide. Antenna proteins
were excited using 450-490nm excitation wavelengths in an FITC (fluorescein
isothiocyanate)-type bandpass excitation filter, and viewed using a barrier filter that
allows emission beyond 515nm wavelength. The cyanobacteria emit yellow and
orange light in the visible spectrum (See Figure 9).
Cell density measurements were taken via two methods. Using the first
method, density measurements were made using an absorbance scan from
A3sonm -
....
..........
........
Figure 9: Synechococcus WH8109 Fluorescence
WH8109 visualized with Epifluorescence Microscope. Photo credit: Desislava Raytcheva.
Asoonm using the Varian Cary 50 Bio UV/Vis spectrophotometer (Walnut Creek, CA).
Density measurements were taken by a second method using an absorbance scan
from A3sonm - A75onm using a NanoDrop 2000 (Thermo Scientific). OD77s was
recorded from Varian scan results and OD750 from NanoDrop results.
2.1.3
Infection
2.1.3.1 Phage Purification and Concentration
Phage were concentrated and purified using a slightly modified PEG
precipitation procedure as described by Yamamoto et al. (1970). Synechococcus
WH8109 cultures were grown to a density between 1x10 8 and 1x10 9 cells/mL at
28'C in SN Nahant or ASW media in 50Em-2s-1 on a 24hr light cycle. Syn 5 stock
solution was added to an m.o.i. of 0.1. Infected cultures were grown in a separate
incubator from other cultures to prevent contamination by aerosolized phage. The
infected cultures were incubated until lysis, indicated by a change in color and
turbidity of the culture. The crude lysate was centrifuged at 7krpm at 4'C for 15min
to pellet debris.
Supernatant
was filtered through
a Whatman
0.45 im
polycarbonate filter to remove cellular and membrane debris. NaCl was then added
to a final concentration 0.5M and sample was stirred until all salt had dissolved.
PEG 8000 was added to a final concentration of 10% (w/v), mixed with stir bar, and
incubated for 2hrs at 4C. Phage were pelleted by centrifugation at 8krpm for
30min. Supernatant was poured off and the pellet was resuspended in 10mL SN
Nahant media/L crude lysate. SN Nahant media was supplemented with 50mM
Tris-HCl (pH 8.0) and 100mM MgCl2.
Phage suspension was loaded onto a 20% sucrose (w/v) CsCI gradient, p =
1.4 CsCl and p = 1.6 CsCl, where each of the gradient layers was made with SN
Nahant media. The gradients were ultracentrifuged at 28krpm for 4hrs at 4C in a
SW28 rotor. The opalescent phage band was visible at the interface between the p =
1.4 CsCl layer and the p = 1.6 CsCI layer (See Figure 10). The band was collected via
sterile syringe and dialyzed at 4C for 30min in 1M NaCl, 100mM MgCl2, 50mM TrisHCl, pH 8.0 solution. The dialyzed phage sample was then dialyzed again at 4*C in
100mM NaCl, 100mM MgCl2, 50mM Tris-HCl, pH 8.0 in a Slide-a-Lyzer dialysis
cassette (Pierce). Phage were concentrated using Amicon 50 kDa MWCO centrifugal
filtration units (Millipore) and stored at 4*C.
2.1.3.2 Infection of WH8109 Samples
Infection was performed on cultures grown to density of between 1x10 8 and
1x10 9 cells/mL grown at 28'C in SN Nahant or ASW media at an irradiance of 50
pEm-2 s1 on a 24hr light cycle. Syn 5 was added to culture at an m.o.i. of 5.0. 35mL
samples were collected before infection, and at the following time points: t = 0, 15,
30, 45, 60, and 90min. Time point samples were immediately centrifuged at 7krpm
for 10min at 4*C to pellet cells. Supernatant of the samples was discarded and the
pellet was resuspended in 350ptL Cyano-Lysis Buffer (25mM Tris, 300mM NaCl, pH
8.0).
Samples were frozen at -20'C for at least 2hrs before lysis. Samples were
thawed on ice and supplemented to have 10mM PMSF, 0.1% Triton-X100, 5mM
.
..
...
.
........
.....
.................
Figure 10: Phage purification
CsCI gradient.
Red arrow indicates opalescent
band of phage. Photo credit:
Jeannie Chew
MgCl2, 1U DNase/mL and 2mg/mL Lysozyme. Samples were lysed at 4*C using the
miniature SIM-Aminco FRENCH Pressure Cell at 14,000psi (SIM-Aminco Press
setting = 700 at medium ratio setting) for 3 passages.
2.1.4
Heat Shock
Cells grown at 28"C in SN Nahant or ASW media in bubblers at an irradiance
of 50pjEm-2s-1 on a 24hr light cycle were aliquoted into acid-washed 100mL flasks,
and placed in a VWR shaking water bath set to the appropriate shock temperature
under white-light fluorescent lamps. 35mL samples were centrifuged at 7krpm at
4C for 10min. Supernatant was discarded and the pellet was resuspended in 350 IL
Cyano-Lysis Buffer. Lysis conditions were identical to those described above.
2.1.5
Western Blot
Samples were prepared with 3xSDS loading dye and boiled for 5min, then
electrophoresed in duplicate through 12% SDS-PAGE gels run in parallel at 150V for
1.5hrs. One of the gels was stained using Krypton Protein Stain (Pierce Thermo
Scientific) for total protein imaging with a Typhoon 9400 scanner (GE Healthcare)
using fluorescent settings with green laser excitation at 532nm and 580 BP30
emission. The second gel was soaked in transfer buffer (20% methanol, 192mM
glycine, 25mM Tris) for 15min and then used in a wet-transfer western blot
protocol using the Bio-Rad Criterion blotter apparatus (Hercules, CA).
Proteins
were transferred at 15V overnight at 4*C onto Immobilon-FL PVDF membrane with
0.45im pores (Millipore). After electroblot transfer, the sandwiched gel was also
stained with Krypton Protein Stain and imaged as described above to confirm
successful and complete protein transfer.
The membrane was probed using an ECF anti-rabbit kit according to the
manufacturer's instructions (Amersham/GE Healthcare).
To briefly outline the
steps, first the membrane was washed in PBS-T (137nM NaCl, 2.7mM KCl, 4.3mM
Na2HPO4-7H20, 1.5mM KH2PO4 + 0.1% (v/v) Tween20, pH 7.5) with 5% (w/v)
Carnation powdered milk used for blocking agent, for 1hr at 25*C. The membrane
was then washed 3 times with PBS-T, where the first wash was for 15min, followed
by two additional washes for 5min, all at 25'C. The membrane was then probed for
1hr at 25*C with polyclonal anti-GroEL antibody developed in rabbit (Sigma) diluted
1:5000 in PBS-T with 0.25% blocking agent, and then washed again as described.
Finally, the membrane was probed with alkaline phosphatase conjugated anti-rabbit
2' antibody developed in goat (Sigma) diluted 1:10000 in PSB-T with 0.25%
blocking agent for 1hr at 25*C. The membrane was rinsed as described a final time
and then incubated at room temperature
for 10min with ECF substrate
(Amersham/GE Healthcare) and scanned using the Typhoon 9400 scanner using
fluorescent settings with green laser excitation at 532nm and 526 SP filter emission.
Gel bands and ECF fluorescence was quantified with ImageQuant TL software (GE
Healthcare).
Results
2.2
2.2.1
Growth Curves
Synechococcus WH8109 shows a robust ability to survive in the laboratory.
While it is more sensitive to environmental changes than E. coli, WH8109 can grow
in concentrations as high as approximately 1010 cells/mL in salt water media. To
prepare the media, we collected carboys of water from the Northeastern University
Marine Science Center in Nahant, MA. The water seems to need at least a month
after collection, particularly water collected during the winter months, before it will
consistently support cultures. To combat this inconsistency, we were also able to
grow WH8109 in artificial media (ASW). SN media was able to sustain growth more
quickly and to higher densities, while ASW sustained exponential growth over a
longer period of time (See Figure 11).
Cultures that originated from frozen stocks were continued by using
inoculation of 1 part existing culture in 100 parts fresh media, where existing
cultures were in the exponential growth phase (densities of approximately 108
cells/mL). Inoculation using cultures that had passed the exponential phase and
Growth of SynechococcusWH8109
1.6
1.4
-
1.2
Figure 11: Synechococcus WH8109 Growth Curves
l
Dashed line - SN Media Average Growth Curve
Solid line - ASW Media Average Growth Curve
0.8
0.6/
0.4
0.2
0
5
10
15
Age (Days)
20
25
30
were instead in the stationary growth phase had very low likelihood of survival
(data not shown).
One goal of characterizing the growth of the cultures was to determine if
culture density could be correlated to a single OD value in order to observe a simple
metric for growth assessment. The first step in developing this metric was to find
WH8109 has an absorbance signature
an absorbance wavelength to evaluate.
consisting of four peaks in the 400-800nm range. Two peaks at 440nm and 682nm
correspond to chlorophyll a absorbance. Normally, chlorophyll a shows peak
absorbance at 430nm and 660nm. The shifts observed in Synechococcus WH8109
correspond to interactions with secondary electron acceptors in close vicinity to the
chlorophyll a pigment (Bricaud, 1995). The specific shift in these values could be
due to the size of the cell (Fujiki, 2002) or the pigment packing and composition
As cultures aged, the peak at 682nm shifted
within the cell (Bricaud, 1995).
downward toward 660nm, indicating some separation of the chlorophyll a pigment
from the other antenna pigments or decreased intracellular pigment concentration.
This may indicate decomposition of the thylakoid membranes, lysis of a large
A
B
SynechococcusWI8109
Absorbance at 6 Days
0 .7 . . . . , . . . , , . . . . , . . . . , . . . . , , . , , , . , , , , , , . .1
.35
.
.
. .
PUB:PEB Ratio
with respect to Age
-
y=1.2278-0.00538x R= 0.51077
1.3
0.6
1.25
0.5
1.2
2
1.15
0.4
0.3
0
1.05
0.2
1 0.1
400
-
0.95
450
500
550
600
650
700
750
0
800
5
10
15
20
25
Age (Days)
Wavelength (nm)
Figure 12: Absorbance Spectra
A - Absorbance spectrum of 6 day old SN media culture.
B - PUB:PEB ratio of SN and ASW media cultures at different ages. Average Ration: 1.183; Std. Dev: 0.0595
27
30
.
.............................................................
...
1.44E+09
1.24E+09
1.04E+09
8.40E+08
0
6.40E+08
4.40E+08
2.40E+08
4.00E+07
0
2
4
6
8
12
10
14
0
2
Age (days)
4
6
8
10
14
12
Age (days)
5.50E+09
5.OOE+09
4.50E+09
4.OOE+09
3.50E+09
3.OOE+09
2.50E+09
2.OOE+09
1.50E+09
1.OOE+09
5.OOE+08
4
6
8
10
12
141
Age (days)
Figure 13: Cell Count vs. OD775
Comparison of cell count and oD775 of four independent cultures grown (distinguished by color) in
parallel in SN media at 28"C. Ratio of cell count to OD77S shows no direct correlation of the two values,
despite similar trends.
enough population of cells and subsequent release of free chlorophyll a into the
media, or that antenna complexes dissociated from the membrane as the
cyanobacteria aged. Which of the scenarios described is most accurate has not been
evaluated.
The other two peaks correspond to phycourobilin (PUB) and
phycoerythrobilin (PEB).
PUB showed a peak absorbance at 495nm and PEB
showed peak absorbance at 550nm (Six, 2008). The PUB:PEB ratio with respect to
age showed little variation in Synechococcus WH8109 (See Figure 12).
The
downward trend in PUB:PEB indicates a loss in the ratio of orange to pink or red
pigments. This supports the hypothesis that antenna complexes begin to dissociate,
losing PUB first.
In order to avoid the variability in peak data, the smooth and flat area after
the final chlorophyll a (See Figure 12) curve offered a place to attempt to correlate
population density to a quickly measurable quantity. However, analysis of the
absorbance density measurements showed no direct correlation between cell count
and OD7 7 5 or OD7 so. (See Figure 13). The implications of this are further addressed
in the discussion section, but the OD77s may be too variable due to effects from the
second chlorophyll a absorbance signature.
2.2.2
Infection Response
Cultures infected with 5 m.o.i. of Syn 5 began to show lysis after 60min and
were fully lysed within 3hrs of infection, as observed by color change. Analysis of
the GroEL response over the course of infection showed an increase in GroEL levels
until cells began to undergo lysis, with a peak at 30min. This is confirmed by the
relative concentrations of GroEL shown through western blot of both supernatant
and pellet samples at different time points. Progression of infection was monitored
by the formation of Syn 5 coat (gp39) and scaffolding (gp38) protein (Pope, 2007).
Cellular response to infection was monitored using OD775 to indicate changes in
culture density. (See Figure 14) Cultures have previously been observed to continue
growing until lysis begins, around 60min.
...
....
........................
....
......
D
0.148
0.146
0)
M.-5 CA
.
=3
-3L(
C
CZ
C;
V)(
0)
.-
A
4-
(
W
0
W
W-
0.144
.0
0.142
00
4mr4
01
o
A
0.138
0.136
0.134
0.132
66k
0.13
0
20
40
60
80
100
80
100
infection Time (min)
E
3
2.5
15
B
gp39 Protein
AP
C
0.5
-*
GroEL
0
20
F
A - SDS-PAGE of samples collected throughout an infection time course.
B - gp39 Protein from Syn5 band used for quantification of infection
level. gp39 encodes the coat protein of Syn 5 indicating cyanophage are
successfully multiplying.
C- ECF fluorescence from western blot of the gel in image A using
antibodies for GroEL.
60
.
-supematant
-Pellet
~
3
2.5
0.5
0
D - oD775 measurement of infected cells. Large square on y-axis
indicates oD775 of uninfected cultures. Large jump in OD at 15 minutes
probably due to experimental error.
40
Infection Time (min)
Figure 14: Syn 5 Infection of Synechococcus WH8109
20
40
60
80
Infection Time (m)
E - Quantification of gp39 Protein normalized against zero time-point
concentration.
F - GroEL concentration in supernatant and pellet normalized to
uninfected control sample concentration (indicated with the square
marker on the y-axis).
2.2.3
Heat Shock Response
Very little is known about what constitutes heat shock conditions for
Synechococcus WH8109.
For some freshwater Synechocystis species, heat shock
response has been observed at 42.5'C for cultures grown under normal conditions
at 30'C (Lehel, 1992). Small bodies of freshwater, however, may be subjected to
100
.
..
..
..
....
.........
higher temperature variation over shorter periods of time than the open ocean.
While Synechococcus is closely related to its freshwater cousins, oceans do not
change temperature as quickly, and the development of a heat shock response may
have occurred after the divergence of the Synechocystis progenitor from the
Synechococcus progenitor. In light of this, open ocean cyanobacteria may not be as
equipped to tolerate drastic changes in environmental temperature conditions. In
order to establish a heat shock protocol for Synechococcus WH8109, different times
and temperatures were analyzed. Initial tests of short exposure (30min) to 35*C
heat shock, where under normal conditions cells are grown at 28*C, showed a slight
increase in GroEL levels (See Figure 15).
M
+
CI
+A
A
C
66kD;
Control
30min + 15min on ice
HS at 354C
30min
Supernatant
8 Pellet
GroEL
Figure 15: Brief Heat Shock Characterization
A - SDS-PAGE gel of heat shock condition lysates
B - Western blot of GroEL of gel above
C- Relative levels of GroEL as compared to Control sample supernatant and pellet concentrations,
respectively. Normalized to band darkness of Control samples.
More extensive testing under different temperature conditions revealed that
Synechococcus WH8109 is not tolerant of temperatures of 40'C and above. Even
after 30min of exposure, significant decrease in cell density was visible by change in
culture color (data not shown) and was evident in samples run on SDS-PAGE (See
Figure 17). Some culture density was also lost after 60min of exposure to 35'C, but
an increase in GroEL levels was evident after 30 and 60min of exposure to 35'C
shock. Longer exposure times (2-3hrs) showed elevated response proportional to
the time of stress exposure, with higher levels of GroEL for cells shocked at 35'C
than cells shocked at 32'C (See Figure 16).
2.3
Discussion
2.3.1
Using Synechococcus WH8109 for Research
Synechococcus WH8109 offers many advantages as a laboratory strain for
research. It grows reproducibly at high densities and a single frozen stock can grow
in media for multiple rounds of inoculation and growth. Cultures can be grown in
artificial media, allowing research to take place in any location where sea water may
not be available. The strain is susceptible to infection by at least ten different
cyanophage, which may prove beneficial in future genetic manipulation once the
system is better understood. As a photosynthetic organism, the principle findings
involving the photosystem, and its metabolism and maintenance may be applicable
to higher order organisms for energy research and agricultural applications. The
challenges of using Synechococcus WH8109 in the laboratory, however, include that
WH8109 is sensitive to environmental variations and achieving reproducible and
adequate growth requires careful attention and cleaning of all materials. Sequence
information has not yet been released or annotated, which prevents genetic
manipulation in vivo and prevents the construction of WH8109 vectors for
exogenous expression experiments.
Transformation of WH8109 has not been
attempted to the knowledge of this author.
2.3.2
Synechococcus WH8109 Growth Conditions and Characteristics
For the research described in this thesis, the growth conditions were
developed explicitly with the intent of producing high yields of cyanobacteria. The
prior research focused on the cyanophage that use Synechococcus WH8109 as a
host, and growth conditions were established in order to harvest high concentration
samples of phage.
Characterization of the growth tendencies of Synechococcus
WH8109 has informed some of the infection experiment protocols, however. The
cells are grown in a 24hr light cycle. The progress of infection is retarded when the
light source is removed (unpublished experiment). This has implications regarding
energy usage within the cells, but has not been studied well enough to make
conclusions. To better understand the metabolism and repair of these organisms,
growth using a night and day cycle may show differences in the stress response
tolerances. Inducing a night and day cycle will also slow the growth of the cells,
making it more difficult to achieve high enough cell densities for experimentation.
Despite attempts to develop an easily measured metric for observing culture
density, OD77s measurements did not correlate to cell count. The trends in increase
between OD77s and cell density were similar, so use of OD77s for some of the
measurements in this research does not necessarily negate the results, but should
be taken into consideration. In the future, a different wavelength may need to be
considered. Trends in growth characteristics based on OD77s roughly matched the
cell count, but not in a quantifiable way.
2.3.3
Stress Response and GroEL
Inducing heat stress and infection stress on the growth system showed
increase in expression of heat shock proteins. Quantification of the level of increase
of GroEL and GroES could be better monitored by radio-labeling experiments.
These experiments were not done for this set of data, but could be done in future
experiments to better understand Synechococcus WH8109's heat tolerance capacity.
Western blot analysis of the GroEL levels revealed a doublet band was
recognized by the anti-E. coli GroEL antibodies. This may have been due to nonspecific binding by the antibodies, however species of cyanobacteria most closely
related to Synechococcus WH8109 have two copies of a 60kDa chaperonin protein,
often called GroEL1 and GroEL2 in the protein annotation entries.
The slight
separation of these proteins on SDS-PAGE indicates some difference in sequence
length or electrophoresis migration character. It is impossible to tell at this point in
our understanding of Synechococcus WH8109 whether these two copies are
interchangeable in cellular function, or if they are specifically localized within the
cell.
Future experiments may help elucidate the difference in structure and
localization between the two copies of GroEL in WH8109, which will be discussed
more in the next chapter of this thesis.
Western blot analysis also shows a high concentration of GroEL pellets with
the insoluble fraction of the cell lysate. GroEL is known to be required for the
proper folding of RuBisCO and it has been shown to strongly associate with the
membrane-associated proteins of the thylakoid (Lorimer, 2001; Balogi, 2008; Lehel,
1992). Western blot using anti-Spinach RuBisCO antibodies showed that a high
concentration of RuBisCO remained in the insoluble fraction, though it was also
present to a significant extent in the soluble fraction as well (data not shown). Such
high concentrations of RuBisCO would necessitate an adequate amount of GroEL to
assist in folding.
Prior evidence indicates endogenous GroEL is hijacked for phage coat protein
folding (Lorimer, 1996; de Beus, 2000). Cyanophage infection of Synechococcus
WH8109 elicited a response from the cell that included higher levels of GroEL
around the 30min mark. While it has not been confirmed that GroEL associates with
Syn 5, the cyanophage that was used for the infections examined in this research,
infection did elicit an increase in GroEL levels. In other species, GroEL also spikes at
approximately 30min after being subjected to a stress condition (Lehel, 1992).
While it is unknown what GroEL is specifically doing in WH8109, there can be more
confidence in a claim stating that Synechococcus has a heat shock and stress
response in WH8109. This could be indicative of increased oxidative damage in the
cell, potentially caused by increased protein production as phage proteins are
expressed in addition to endogenous proteins. Increased translation within the cell
would require more energy, requiring higher levels of photosynthesis to provide the
energy, therefore resulting in more oxidative damage. If GroEL is assisting in the
folding of the phage capsid or protecting and repairing the endogenous photosystem
proteins in the cell or both has not been confirmed.
Further characterization of the stress response might include analysis of
transcriptional changes in the cells in response to changes in temperature, nutrient
levels, and infection in addition to the translational fluctuations that have been
documented herein. Attempts to perform this analysis are described in Appendix C.
The translational response documented above only evaluates the levels of protein
present. These experiments could be confirmed by radiolabeling and pulse chase
experiments to see how much protein is produced in response to the stress
condition. Chaperone production has been reported to change over the course of
hours (Lehel, 1992), however, and pulse chase is not as effective for such long lapses
in time.
Ln
I. L
rn
M
'd,
Cn
~
en
M -41
66k0
q
-
~0~0
~
Figure 17: High Heat Shock Characterization
A - SDS-PAGE of heat shock at 35 0Cand 40'C plus E. col shocked at 42'C for 30min.
B - Supernatant GroEL concentrations relative to Control sample and normalized to sample loading
concentration.
0
0
U
N~
.
LM
C-
On
U
M
o7
'
"
_7'~
A
66kDa
B
3
2.5
2
(Uu
c
.
g
Figure 16: Prolonged Heat Shock
Characterization
1.5
0 o
LU
~
A - SDS-PAGE of heat shock samples at 32 0Cand
35 0 C
0.5
Control
2hr, 32"C
3hr, 32*C
2hr, 35"C
Sample Condition
3hr, 35-C
B - GroEL concentration in supernatant relative
to control sample and normalized to sample
loading concentration.
3.0
Characterization of WH8109 GroELS Chaperonin Complex
3.1
Materials and Methods
3.1.1
Purification
3.1.1.1 Sucrose Gradient
Cell lysate samples were prepared using protocol described above. 300ptL of
each samples were layered on top of a sucrose step gradient in open top poly-clear
Beckman centrifuge tubes. Sucrose gradients were made from solutions in 50mM
Tris, 100mM NaCl, 100mM MgCl2, pH 7.5, with 5% to 20%, or 5% to 45% sucrose
mixed with settings SW50 tubes. Samples were ultracentrifuged in a Beckman
SW50 rotor for 3 hours at 40krpm at 4C. Twelve approximately 500[IL fractions
were collected from centrifuged samples and dialyzed into 50mM Tris, 100mM NaCl,
100mM MgCl2 solution, pH 7.5. Dialyzed samples were analyzed with SDS-PAGE gels
and Western blot to identify GroEL. Samples with the highest GroEL signals were
prepared for visualization with electron microscopy.
3.1.1.2 Ammonium Sulfate Precipitation
To test the ammonium sulfate precipitation response of GroEL, first
Synechococcus WH8109 crude lysate, prepared as described above with and without
0.1% Triton-X100, was centrifuged at 13krpm for 40min at 40 C to precipitate
membrane and insoluble proteins. To separate GroEL from the less soluble proteins
serial ammonium sulfate cuts were done from 10% to 66.7% in steps of
approximately 10% increase in ammonium sulfate concentration per cut. 100%
ammonium sulfate solution (3.9 M, pH 7.0) was added to the supernatant of crude
lysate sample to appropriate concentration, incubated on ice for 15min, and then
centrifuged at 13krpm for 15min at 4C. Resulting pellets were resuspended in
200ptL Buffer A + glycerol. Samples were prepared with 3xSDS loading dye and
boiled for 5min. Samples were electrophoresed through a 12% SDS-PAGE gel at
150V for 1.5hrs and analyzed through both Krypton Protein Stain visualization and
western blot using anti-GroEL antibodies and ECF as described.
To avoid dilution of the sample, a second ammonium sulfate precipitation
was done using dry ammonium sulfate added directly to the sample. Dry weights of
ammonium sulfate were added to a final concentration of 20%, 40% and 60%
ammonium sulfate concentration, mixed at 4C for 15min, and then centrifuged at
13krpm for 15min at 4C. The resulting pellets were resuspended in equal volume
to the supernatant of Buffer A + 10% (v/v) glycerol.
Samples were dialyzed
overnight at 4*C in Buffer A + glycerol in Novagen D-Tube Dialyzer Mini 6-8kDa
MWCO tubes.
Dialyzed samples analyzed with SDS-PAGE electrophoresis and
Western blot as described above. The primary antibody used for Western blotting
was anti-GroEL polyclonal antibody developed in rabbit (Sigma), and the secondary
antibody used was anti-rabbit alkaline phosphatase conjugated whole antibody
developed in goat (Sigma). Gel bands and ECF fluorescence was quantified using
ImageQuant TL software (GE Healthcare).
3.1.1.3 Chromatography
3.1.1.3.1
Anion Exchange
Anion exchange (AIEX) chromatography was performed using the HiPrep Q
FF 16/10 Sepharose Column (GE Healthcare) with 20mL column volume (CV) on an
AKTAPurifier FPLC platform (GE Healthcare) using Unicorn 4.1 software.
The
column was stored at 4C in EtOH Buffer and equilibrated for 5 CV with Buffer A or
Buffer A Plus (See Table 1). Lysate was loaded onto column using a 50mL glass
SuperLoop (GE Healthcare) and eluted into fractions using gradients from 0% to
100% Buffer B or Buffer B Plus. For detailed information about elution protocols,
see Figure 18. Samples were analyzed using electrophoresis through 12% SDS-PAGE
and Western blotting as described. Fractions were dialyzed in 3.5KDa MWCO Slidea-Lyzer dialysis cassettes in 4L Buffer A Plus for 4 hours at 4C.
To prepare lysates, Synechococcus cultures were centrifuged at 7krpm at 4*C
for 10min. Pellet was resuspended in Buffer A at 100x concentration. Suspension
was frozen at -80*C until use. Cells were thawed in a 20'C water bath and then
returned to ice. 1 tablet per 1OmL culture of Complete-Mini@ EDTA-free Protease
Inhibitor tablets (Roche) was dissolved in 0.5mL Resuspension Buffer (25mM Tris,
300mM NaCl, 50mM MgCl2, pH 8.0) and added to thawed cells along with 10 L/mL
lysate O.5mg/mL DNase mixed in 10ptL/mL lysate DNase Buffer (100mM Tris-HCl,
500mM MgCl2, 13mM CaCl2, pH 8.0).
1% Triton-X100 was added to a final
concentration of 0.1%. Cells were lysed at 4C using the Thermo FRENCH Pressure
Cell (Thermo Scientific) at 14,000psi (SIM-Aminco Press setting = 1100 at high ratio
setting) for 3 passages. 90mM PMSF was added to lysate to a final concentration of
9mM.
Lysate was centrifuged at 13krpm for 35min at 4C.
Supernatant was
collected and 7.5% PEI was added to a final concentration of 0.12%, incubated on
ice for 10min, and then centrifuged again at 13krpm for 15min at 4C. Supernatant
was collected and filtered using a 0.2im PALL syringe filter. Lysate was loaded onto
AIEX column by injection of 45-50mL of filtered lysate and washed with Buffer A or
Buffer A Plus.
3.1.1.3.2
Size Exclusion
Size exclusion chromatography was performed using the Superose 6 HR
10/30 Gel Filtration Column (GE Healthcare) with 23.56mL column volume (CV) on
an AKTAPurifier FPLC platform (GE Healthcare) using Unicorn 4.1 software. The
column was equilibrated with 2 CV Buffer Cor Buffer C Plus. 500jL of concentrated,
0.2ptm filtered samples were loaded onto SEC column using 1.5mL injection.
Samples were eluted in Buffer C or Buffer C Plus for 1 CV in 0.5mL fractions.
Fractions were analyzed using electrophoresis through 12% SDS-PAGE and Western
blotting as described. SEC fractions were pooled and used for transmission electron
microscopy (TEM) imaging.
AIEX Program 2
AlEX Program 1
0
5
10
15
20
25
0
30
2
9
6
4
10
Column Volumes (20mL)
Column Volumes (20mL)
U'
SEC Program
AlEX Program 3
100 |
0
4
8
12
Column Volumes (20mL)
16
0
0.5
1
1.5
2
Column Volumes (23.56mL)
Figure 18: HPLC Chromatography Programs
Anion exchange (AIEX) and size exclusion chromatography (SEC) strategies for purification of Synechococcus
WH8109 GroEL and GroES using HPLC. Blue dot indicates injection of sample. Buffer B and Buffer Ccould also be
Buffer B Plus and Buffer CPlus, respectively.
Table 1: chromatography Buffers. All filtered with 0.2pm filter and kept at 4*C
Buffer
Ingredients
Buffer A
Buffer A Plus
j 50mM Tris, 1mM EDTA, 1mM DTT, 5mM MgCI2, 1mM ATP, 10% (v/vj Glycerol, pH 7.6
1
I100mM NaCl
I
Buffer B
Buffer B Plus
Buffer C
Buffer C Plus
High Salt
Low Salt
9
EtOH
J
V
30% Isopropanol in MilliQ H20
Iso
ddH20
3.1.2
Biochemical Analysis
3.1.2.1 Mass Spectrometry
Purification
samples
were
prepared
with
3xSDS
loading dye
and
electrophoresed through 12% SDS-PAGE gel at 150V for 1.5hrs. Duplicate gels were
made. One gel was stained with Coomassie and stored in 2% acetic acid overnight.
Gel bands were extracted and submitted to the Koch Institute Proteomics Core
Facility (Cambridge, MA) for protein identification via Ion Trap LC mass
spectrometry using a linear trap quadropole (LTQ) Ion Trap spectrometer (Thermo
Electron Corporation, FL) connected to an Agilent Model 1100 Nanoflow highpressure liquid chromatography (HPLC) system. The second gel was stained with
Krypton Protein Stain and visualized as described above.
3.1.3
Imaging
3.1.3.1 Transmission Electron Microscopy (TEM)
Purification samples were visualized with negative stain electron microscopy
and prepared with 1% (w/v) uranyl acetate stain on Formvar and carbon coated
400 mesh copper grids. Images were taken using a JEOL Model 1200 transmission
electron microscope at 60kV.
3.1.3.2 Proposed Imaging
Once samples have reached a purity of approximately 75-85% total protein
content, cryo-electron microscopy (cryo-EM) can be performed. Cryo-EM work had
not commenced at the time of submission.
3.2
Results
3.2.1
Purification
3.2.1.1 Sucrose Gradient
Sucrose gradients from 5% to 20% sucrose resulted in the best separation of
GroEL from lysate supernatant samples. SDS-PAGE and anti-GroEL western blot
analysis showed GroEL separates in the first four fractions of the 5-20% gradient,
and within the first three fractions of the 5-45% gradient. Figure 19 shows the
separation of GroEL from cells grown under normal conditions on a 5-20% gradient.
Attempts to compare the concentration of GroEL from samples grown under HS and
infection conditions proved inconclusive (data not shown). ECF signal from antiGroEL antibodies from a western blot appeared diffusively throughout all 12
fractions of each sucrose gradient. At the time these experiments were done, the
lysis procedure used sonication as opposed to French press for rupture of the
membrane. It is possible sonication disrupted the multimeric GroEL complex and
fragmented the protein into many different sizes of peptide. The fragments would
G-o
-J
ureru at 4kp
*
-
Fiur 19:
cos
thehaeZa
at 4
-aWI
Grdet
difr
c.
6 2
I
IRA
I
uiicto
nseimntain
coficet andCoul haeapardiWl
B
Figure 19: Sucrose Gradient Purification
A - SDS-PAGE of supernatant and pellet samples from cell lysate grown at normal conditions and
ultracentrifuged for Zhrs at 4Okrpm at V0C.
B - Anti-GroEL western blot of gel above.
then have had different sedimentation coefficients and could have appeared in all
sucrose gradient fractions.
Samples collected from the GroEL containing fractions were dialyzed and
stained for EM imaging. Sucrose gradient samples were poorly clarified and images
of these grids revealed extensive contamination. Filtering the samples through a
5OkDa MWCO filter and making new grids facilitated the imaging of GroEL
complexes via EM, though samples were still heavily contaminated (See Figure 20).
Figure 20: Visualization of GroEL from
a
b
c
Sucrose Gradient Purification
100 nm
a - diameter = 0.0125pm
HV=6 0.0kV
b - diameter = 0.0135pm
c - diameter = 0.0157pm
Dire ct Mag: 250000 x
King Lab - MIT
Electron micrographs, samples stained with
1%uranylacetate.
3.2.1.2 Ammonium Sulfate Precipitation
Synechococcus WH8109 GroEL from the soluble fraction of cell lysate,
partially lost solubility with each step in ammonium sulfate concentration above
10% saturation. By 60% ammonium sulfate saturation, there was approximately an
equal concentration of GroEL still in the supernatant as the concentration that
precipitated in that step, which showed a significant loss. Addition of Triton-X100
to the lysis buffer facilitated in pelleting phycibiliproteins at around 40-50%
ammonium sulfate, whereas without Triton-X100, the phycobiliproteins clumped
together but could not be precipitated efficiently even by 60% saturation of
ammonium sulfate. Addition of Triton-X100 had no effect on percentage of GroEL
that precipitated with the insoluble fraction of the cell pellet (data not shown).
3.2.1.3 Purification Trial #1
The first purification trial first used anion exchange chromatography of
approximately 50mL of cell lysate and used AIEX Program 1 (See Figure 18). This
trial used Buffer A and Buffer B. The flow through fractions were collected, dialyzed
in Buffer A, and passed through another round of anion exchange using the same
AIEX program. SDS-PAGE and western blot analysis of the flow through and elution
fractions from each column showed GroEL eluted at approximately 15% Buffer B,
which roughly corresponded to 150mM NaCl concentration. The lysate loaded onto
the column already had a high salt concentration from residual sea water media in
the pelleted cells. The flow through fractions achieved the same conductance levels
Anion Exchange 1
1600.00
1400.00
1200.00
E
1000.00
C
M 800.00
600.00
>
400.00
200.00
0.00
130.00
180.00
230.00
280.00
Elution Volume (mL)
Anion Exchange 2
-
-
3500.00
3000.00
E
2500.00
2000.00
1500.00
1000.00
s00.00
0.00
0.00
200.00
100.00
400.00
300.00
Elution Volume (mL)
Figure 21 Chromatography UV Absorbance
-i-Excludo
2000.00
1800.00
absorbance from purification trial #1
anion exchange columns and first size
exclusion column. Peaks correspond to
pigments eluting from the
160.00Elution
-
31600.00
1400.00
1200.00
C
M1000.00
(U
100.00phcyobiliprotein
800.00
4
600.00
D
400.00
200.00
0.00
0.00
10.00
20.00
30.00
40.00
50.00
Elution Volume (mL)
as the elution gradient at approximately 15% Buffer B. Samples from these two
anion exchange columns were pooled to maintain similar protein composition, and
concentrated to a volume of 500tL using a 50kDa MWCO filter.
500[tL of
concentrated samples were passed through a Superose 6 gel filtration column and
eluted using Buffer C. GroEL eluted between 12-14mL when eluted at a flow rate of
0.3mL/min. In calibration tests, Thyroglobulin, MW = 669kDa, eluted from the
Superose 6 column at 12.61mL when eluted at .5mL/min (data provided by Ligia
Acosta-Sampson). The estimated size of the Synechococcus WH8109 GroEL complex
is around 840kDa. If the calibration of the column still applies for lower flow rates,
the species of GroEL eluted from the column is smaller than expected.
These
samples were evaluated with SDS-PAGE (See Figure 23 for assessment of purity) and
visualized using TEM.
Electron micrographs of the samples showed highly
concentrated and predominately pure samples of ring structures
with 7
distinguishable subunits (See Figure 22). Whether the proteins imaged by TEM are
double barreled structures or single barreled structures was not apparent.
100 nm
100 nm
HV=60.OkV
Direct Mag: 150000x
HV=60.OkV
Direct Maq: 200000x
100 nm
HV=60.OkV
Direct Maq: 250000x
Figure 22: Visualization of GroEL from Purification Trial #1
Electron micrographs at magnification indicated below image of select samples from purification trial #1.
Molecules in the barrel characteristic barrel orientation indicated by red arrow.
:................. iww.
..
-
__ -
z ._
;.............
::
.............
.........................
4
Figure 23: Purification Trial #1 Samples
SDS-PAGE and corresponding samples from first purification. Order is the same for gel lanes and sample
tubes. Take note of the orange pigmentation in most samples tubes, as well as the large 16kDa bands in
the gel from the phycobiliproteins. The red arrow indicates GroEL band.
Figure 21 shows the elution absorbance from both anion exchange columns
run and one of the four size exclusion columns run for the first purification trial.
The high peaks in absorbance are indicative of the pigments that also eluted from
the columns. Synechococcus type 3c species have 3 or 4 chromophores: PUB, PEB,
AP, and two forms of phycocyanin. The associated pigments are orange, red, blue,
and violet, respectively.
These pigment-associated proteins eluted at slightly
different salt concentrations on the anion exchange columns (See Figure 24) and
gradated even more during size exclusion chromatography. In the first purification
trial, the pigment-associated proteins persisted through all chromatography steps to
Figure Z4: Phycobiliprotein Separation
A,B - Anion exchange column,. Red arrow indicates visible segregation of pigments in the column.
C- SEC column exhibiting pigment segregation, as indicated by red arrow.
co-purify with GroEL in the elution fractions that were pooled for TEM visualization
(See Figure 23).
The predominant contaminating pigment was the orange PEB
phycobiliprotein.
3.2.1.4 Purification Trial #2
For the second trial, approximately 50mL of cell lysate supernatant was
loaded onto an initial anion exchange column and purified using AlEX program 2.
The 5% Buffer B Plus wash step ensured a high enough salt concentration to allow
all of the GroEL to be eluted in the flow through fractions. The fluorescent orange
pigment bound firmly to the column and eluted in the waste fractions at 100%
Buffer B Plus (See image B in Figure 25). The flow through fractions were dialyzed in
Buffer A Plus and then used for passage through another anion exchange column,
where this time AlEX Program 3 was used. Steps in Buffer B concentration provided
the appropriate salt concentration to elute GroEL in only two fractions. These
.......................................
...........
.
..
...........
..............................
.........
.
fractions were concentrated from 9mL each to approximately 800gL each and used
for size exclusion chromatography.
AIEX program 3 allowed for greater segregation of pigment-associated
proteins than had been seen in the other anion exchange column eluants (See Figure
25). The two GroEL-containing fractions from the second anion exchange step were
predominately purple and somewhat pink (See image E of Figure 25). The blue and
orange pigments were mostly removed through the anion exchange steps, which
Figure 25: Purification Trial #2
A - Flow through fraction of first anion exchange column, which were pooled, dialyzed and loaded onto second
anion exchange column.
B - Waste fractions from first anion exchange column.
C - Second anion exchange column.
D,E,F - Separation of phycobiliproteins. The leftmost fraction in figure E contained the majority of the GroEL from
the second anion exchange column and was concentrated and run through the size exclusion column.
significantly reduced the pigment absorbance signal in the SEC FPLC results. SDSPAGE and western blot analysis showed that GroEL eluted predominately with
purple and red pigments after the SEC step for this purification trial (See Figure 26).
Western blot analysis also showed that GroEL eluted later than expected in the SEC
columns from trial #2, as well.
During the four SEC columns in the second
purification trial, the FPLC exhibited signs of a clogged hose, which resulted in
periodic pressure spikes and frequently paused during the SEC program.
To
alleviate stalling problems, the flow rate was further reduced to 0.2mL/min.
3.2.2
Mass Spectrometry
Final samples from the first purification trial and size exclusion fractions
from the second purification trial were submitted for MS analysis. Gel bands were
selected to indicate contaminants, confirm presence of GroEL, and track proteins
that co-purify with GroEL to aid in future sample clarification strategies (See Figure
27). Mass spectrometry results for protein identification were not available at the
time of the original submission. Partial results can be found in Appendix D.
Figure 26: GroEL Samples from Purification Trial #2
Fractions containing GroEL eluted from size exclusion column of leftmost fraction shown above in image E of
Figure 25. The purple phycobiliprotein, phycocyanin-R, predominately co-purified with GroEL in this trial.
.
. . .......
..
Lf~
N-
n
I
I
I
Ln
N
NN
N
L(
00i~
0N
Ln
................
q
r-41
NS
-~
-~
'-i
N
r
N
0N
-~
6i
-
N
0
75kDa
50kDa
-
Figure 27: MS Analysis
[top] SDS-PAGE of purification trial #1 GroEL
isolate samples. Bands submitted for protein
identification by mass spectrometry are
circled. AIEXI refers to samples from the
initial anion exchange column, and AIEX2
refers to samples obtained from the anion
exchange step done on the AIEX1 flow
through samples.
-:
,LDE
ONCo
75kDa 5OkDa,
3.3
[bottom] SDS-PAGE of purification trial #2
SEC fractions. Bands submitted for protein
identification by mass spectrometry are
circled. Fraction numbers indicated above
lanes, where F29 is at 14.5mL elution.
Discussion
GroEL has been purified from a number of bacterial species, most notably E.
coli. E. coli and Synechococcus have many similarities, including, that both are gram
negative bacteria and have a diameter of approximately 1tm. One notable
difference that is important for the motivation behind this research, however, is that
cyanobacteria are believed to very closely resemble their ancient predecessors, who
evolved long before enteric bacteria. The purification of Synechococcus WH8109
GroEL has not been attempted before, to the knowledge of this author.
Furthermore, purification of cyanobacterial Hsp60 chaperonins has only been
documented in one paper. Attempts to purify the GroEL-related protein from
Synechocystis PCC 6803 met with limited success (Lehel, 1992).
experienced
difficulties
with
dissociation
of the
GroEL
The authors
complex
during
chromatography and co-purification with other multimeric high molecular weight
proteins, including the phycobilisome complex and RuBisCO in the carboxysome.
Similar challenges were met during the purification trials attempted in the research
presented in this thesis. The highly expressed 16-2OkDa phycobilisome proteins
appear to have a similar pl to GroEL and therefore eluted in the same anion
exchange chromatography fractions. Lehel et al. instead chose to select conditions
that favored the dissociation of the GroEL complex, which allowed them to obtain
high purity samples of the dissociated chaperonin subunits.
For the research presented in this thesis, the goal of purification was
predominantly
for
cyro-EM
Contamination by another
visualization
of
the
native
GroEL
protein.
protein complex may not affect the final 3D
reconstruction done with cryo-EM electron density data, since images are selected
manually for mapping the electron densities. For biochemical assays, however, the
purity and concentration of GroEL samples are more critical. Crystallization of this
protein for nuclear-magnetic resonance (NMR) spectroscopy would require highly
pure samples of high concentration. From the findings of this research, it is not yet
possible to purify cyanobacterial GroEL to a level adequate for crystallographic
spectroscopy.
A number of measures were taken to improve clarity of the samples. With
the goal of cleaning crude lysate samples for better chromatography results, the
purpose of attempting an ammonium sulfate precipitation was to salt out competing
proteins before loading lysate samples onto the anion exchange column.
particular
interest was
the
removal
of a significant
percentage
Of
of the
phycobiliproteins that co-elute in the same fractions as GroEL in both anion
exchange and gel filtration chromatography.
In E. coli, over-expressed GroEL
remains soluble in solutions of up to 50% ammonium sulfate (Kamireddi, 1997).
Synechococcus WH8109 GroEL, which is not over-expressed in these experiments,
appeared to have variable solubility in ammonium sulfate. This is potentially due to
protein-protein
or
chaperone
binding
interactions
in
solution,
but
the
phycobiliprotein antennas also eluted in the same fractions as GroEL due to the size
of the phycobilisome complex and a similarity in charge. The comparison of these
characteristics for each of the pigment proteins has not been fully evaluated at this
time.
The incomplete precipitation of GroEL during each ammonium sulfate
precipitation step may also have been due, in part, to some dissociation of the
chaperonin complex during purification. The phycobiliproteins come out of solution
at around 40% ammonium sulfate.
The solubility of the phycobiliproteins is
affected by the presence of Triton-X100 in the lysis buffer, such that with TritonX100 in the lysis buffer the pigments pelleted firmly while samples without TritonX100 in the lysis buffer remained more soluble or float in solution, which made
separation of the chromophore-associated proteins from the supernatant very
difficult. The loss of GroEL at each ammonium sulfate step, however, prevented use
of this procedure before the anion exchange chromatography steps since too much
of the sample would be lost.
Instead of ammonium sulfate precipitation of competing proteins, AIEX
programs 2 and 3 were developed to clarify the lysate samples in a serial manner in
order to reduce excessive phycobiliprotein co-elution. While some of the pigmentassociated proteins were removed from the GroEL-containing fractions, the
complete removal of phycobiliproteins was unsuccessful. The second purification
trial also included a change in buffer composition. One possible explanation for the
late elution of GroEL from the size exclusion columns is that it had partially
dissociated. The fractions would indicate structures too large to be single subunits,
but potentially single barrel GroEL as opposed to the expected double-barrel
structure. To see if this was an avoidable situation, 10% (w/v) glycerol was added
to the buffers. Glycerol may protect the protein complex from breaking into smaller
pieces during lysis and chromatography steps. GroEL did not seem to elute from
size exclusion any earlier in purification trial #2, however. Electron micrographs of
the purified samples indicated 7subunit ring structures were present.
The
calibration of the column under different flow rates should be further explored to
53
determine whether these samples are indeed smaller than expected. An alternative
theory is that the complex elutes more slowly in the open state as opposed to the
closed state. 1mM ATP in the buffer should prevent stable adoption of the closed
state, which would necessitate association with the GroES complex. The closed state
complex should be much larger than the open state complex, and would presumably
have eluted in the earliest fractions.
SDS-PAGE and western blot analysis of samples purified by anion exchange
and size exclusion chromatography using anti-GroEL antibodies continued to
identify the doublet band for GroEL. This finding indicated both subunit species
remain in the soluble fraction of the lysate, but little more about the localization and
specificity of the two chaperonin complexes can be inferred. If one was localized
within the thylakoid and the other was cytoplasmic, French press disruption of
cellular membranes should release both into the soluble fraction, which makes the
hypothesis difficult to confirm by experimentation.
TEM imaging confirmed the presence of 7 subunit ring structures isolated
from the soluble fraction of the lysate, however, more efficient isolation of GroEL
should be possible. For future work on this purification, an alternative protocol
might include boiling the lysate samples for up to 20min. Protocols for purification
of GroEL from E. coli suggest this boiling step to dissociate GroEL from the insoluble
proteins and membranes, since GroEL is stable at high temperatures where other
proteins are not.
4.0
Summary and Conclusion
Synechococcus WH8109 fills an ecological niche in the Sargasso Sea,
providing nutrients and playing a dominant role in the oceanic carbon cycle. This
thesis has preliminarily explored this organism for its merits as a model biological
system intended for laboratory research. The growth requirements, characteristics
of growth, and response to stress have been detailed. Furthermore, changes in
expression levels of key proteins during stress response motivate further
exploration of system. The challenges of using Synechococcus WH8109, however, lie
primarily in the fact that sequence data is current unavailable.
Sequencing has
concluded, but annotation is still underway (personal communication, Debbie
Lindell, May 2009).
Once this data becomes available, it will open the field of
research to many new options.
Because the sequence data for GroEL and GroES was not available at the time
of submission, genetic evaluation of this important stress response protein is
incomplete. The most closely related species of cyanobacteria with sequence data
available is Synechococcus CC9605. While overall these two genomes are most
closely related, it is hard to predict how homologous the groEL and groES genes will
be. GroEL-like chaperonins are highly conserved, showing nearly 80% identity
across all branches of bacteria.
Understanding the differences between the
cyanobacterial GroEL sequences and the well studied E. coli sequence, as well as
how these differences affect the protein structure may provide valuable insight into
the evolution of chaperonins and chaperonin specificity. For reference, a protein
alignment of E. coli K-12 GroEL (Accession number: YP_001732912.1) to the two
Synechococcus CC9605 GroEL protein sequences (Accession numbers: YP_380944.1,
YP_382463.1) showed 54% and 57% sequence identity, respectively.
Subtle
differences in GroEL morphology may influence substrate binding specificity and
cavity volume.
Not only would knowledge of the differences in GroEL structure be
interesting from an evolutionary point of view, but Synechococcus WH8109 is a
photosynthetic organism that could model cells in higher order plants.
The
commercial value of increasing plant tolerance to heat and oxidative stress would be
immense for agricultural applications.
Identifying how sequence and structure
differences allow plant GroEL to associate strongly with RuBisCO and thylakoid
membranes in photosynthetic organisms may be key to improving the efficiency of
photosynthesis and minimizing wasted energy due to excess repair of oxidative
damage. Understand how RuBisCO and the thylakoid membrane protein species
compete for folding assistance in the crowded cytoplasmic environment will help to
unlock some of the mysteries of the photosynthetic cell and its unique ability to
recycle damaged photosystem proteins.
This thesis takes steps to characterize the native GroEL protein from
Synechococcus WH8109, in order to elucidate the answers to these questions of
morphology and specificity. While progress is still underway on the clarification of
the purification process, many interesting observations were made.
GroEL
associates with soluble and insoluble proteins, and co-purifies with a significant
concentration of some phycobiliprotein species. Not enough experimentation has
been done to conclude that these proteins are substrates, and co-purify due to
binding. Similarity in the iso-electric point for both GroEL and the phycobilins as
well the formation of large complexes make chromatography a challenge.
The
correct procedure for separating these proteins warrants further exploration. Once
a pure enough sample has been achieved biochemical analysis and structure
determination should be pursued.
In conclusion, the steps taken to understand this biological system indicate it
would be adequate for use as a research strain. Synechococcus WH8109 could be
used to further explore the progression of phage infection and assembly of the
phage head within the host cells. WH8109's size allows it to be conveniently studied
by cryo-electron tomography. Visualizing the entire, undisrupted cell may lead to
answers about protein localization, and intracellular functions as of yet unknown.
The purification of native GroEL will make possible the study of what may be some
of the earliest molecular chaperones to evolve. And finally, this system can model
the photosynthetic cells of higher organisms, for applications in biofuel research,
and agriculture.
5.0
Future Work
There is still much to be done to fully understand the system at hand.
Synechococcus WH8109 has specific growth requirements, but further exploration
of its ability to tolerate changes in environment and to grow in different ecological
niches may facilitate its use as a laboratory strain. For example, in the absence of
sunlight, can WH8109 grow on an alternative carbon source?
How does the
presence of different or limited nutrients affect its growth and expression?
The ability to monitor transcriptional expression has not yet been perfected.
This is discussed in further detail in Appendix C, however the ability to manipulate
the genetic system of WH8109 will be critical for its success as a model organism.
The biological functions and systems may be better understood via recombinant
manipulation. The ability to introduce exogenous genes into this system as well as
the ability to express genes from WH8109 in a different system will make a
significant difference in our ability to understand how these organisms work.
The photosynthetic capacity of the Synechococcus WH8109 is key to its
survival. Under stress conditions, evaluation of photosynthetic output may help
understand the energetic of the system. Alternative energy research would benefit
from the detailed understanding of how these abundant species are able to cope
with damage and stress and how the photosystem is modified in times of extra
energetic requirement.
Perfection
of the purification process is paramount for the further
characterization of the Synechococcus WH8109 native proteins.
Highly pure
samples are required for biochemical analysis and this is not yet possible. Once it is
possible, however, sensitive measurement of the kinetics and specificity of
important proteins may be done.
Finally, the ability to image proteins within cells will be a major advance in
the current technology. To see localization or trafficking of proteins in situ without
genetic manipulation would be applicable to not only basic scientific research, but
also the imaging of proteins associated with disease. The applications of this type of
technology would be far reaching. Getting a better grasp on the system described in
the thesis may be a first step to allowing for the visualization of proteins and
reconstruction of the protein complexes within a single cell.
6.0
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7.0
Appendices
Appendix A: Sea Water Media for Laboratory Growth of Synechococcus
Artificial Sea Water Medium (ASW):
Recipe was slightly modified from recipe in Wyman et al. (1985) and Lindell et al.
(1998).
In 0.22 m filtered MilliQ (Millipore) water, make pH 8.0 media with:
428mM NaCl
9.8mM MgCl2-6H20
6.7mM KC1
8.8mM NaNO3
14.2mM MgSO4 (Anhydrous)
3.4mM CaCl2-2H20
0.13mM K2 HPO 4 (Anhydrous)
5.9mM NaHCO3
9.1mM Tris Base
SN Media:
In 3L of 0.2gm filtered sea water from Nahant, which was stored in 20L
polycarbonate carboys (Nalgene), 1L of 0.22ptm filtered MilliQ water was added.
Both ASW and SN medium were autoclave in acid washed glass Erlenmeyer flasks
on Autoclave's Fluid setting for 20min/L of media, or 20min total for volumes less
than 1L. Before inoculating media with growing culture or frozen stock, the
following salt and trace metal solutions were added to the media.
Salts:
Each salt solution of less than 0.5L was autoclaved for 15min on Fluid setting.
2.5mL/L media of 300g/L NaNO3
2.6mL/L media of 6.1g/L K2 HPO4 (Anhydrous)
2.8mL/L media of 2.0g/L Na2 EDTA
2.6mL/L media of 4.0g/L Na2CO3
1mL/L media 1000x Trace Solution (as described below)
1000x Cyano Trace Metal Stock Solution:
Stock solution was autoclaved for 20min on Fluid setting.
0.77mM ZnSO 4
7mM MnCl2
0.14mM CoCl2
1.6mM Na2MoO4
30mM Citric Acid
5mM Iron Citrate
Appendix B: "Weigele Bubblers"
"Weigele Bubblers" were designed initially by Peter Weigele and modified over
time to fit the needs of the cultures and experiments. The bubbler apparatus
consists of a polycarbonate bottle with polypropylene cap (Nalgene), a gas
dispersion tube (ChemGlass), silicone tubing (Cole-Parmer), fittings (Cole-Parmer),
and 0.2pm filters (Pall). The cap has 2-3 holes: one for the aeration equipment, one
for making a Pasteur loop, and an additional optional hole that may be used for
sample collection.
Making Bubbler Lids:
For the 1L and 500mL bottles, the caps are the same size and require a size
%-28 UNF tap, tap wrench, and 3/16"drill bit. Begin by pre-drilling 2 holes (and a
3rd if desired) with the 3/16" bit. Insert straight into the cap by turning the tap and
wrench through the pre-drilled hole. Turn the tap wrench at least 5 full turns into
the cap and then twist straight out following the same thread path. This will give
the plastic cap a threaded hole for the connectors for sealed connections. In one
hole, insert the female luer bulkhead with %-28 UNF thread into the cap with hose
attachment end facing into the lid cavity. On the bulkhead attach the second hose
attachment. This will attach to the aerator and pump connection. The aerators are
made by cutting the dispersion tube and fitting the sharp end with 'A" inner
diameter tubing and a barbed fitting. This will connect to the hose connection
pointing into the cap of the lid via 1/8" tubing. On the outside hose connector,
attach 1/8" inner diameter tubing with 0.2 m filter (Pall). In the second hole, insert
a barbed fitting hose adaptor. To this, you will attach a 1/8" inner diameter tube
that will serve as the Pasteur loop. See Figure 29 below for the format of a fully
constructed 500mL bottle and components.
The lids for 2L bottles can be ordered with 3 holes and connectors already in
place. One of the holes must be sealed with a screw cap. The other two holes must
be threaded with a tap wrench and will have similar connectors to those described
above, but of the larger size. The dispersion tube should be cut long enough to reach
to within a quarter of an inch of the bottom of the bottle. This can be extended with
tubing if necessary.
Cleaning the Bottles:
The bubblers require acid washing to remove metals and potential organic
contaminants.
Synechococcus WH8109 is particularly susceptible to metal
....
..
. ...............
........
...........
poisoning from iron. It is almost impossible to prevent iron from the air from
entering the sea water media, but excess contaminant will prevent growth. MilliQ
water is the only water used to clean the bottles and make media to prevent metal
leached from water pipes from contaminating the media and bottles. Acid washing
is performed filling the bottles with 10% HCl (v/v) and letting them soak for at least
2 hours, but preferentially overnight. We use a stock of 10% HCl specifically for acid
washing, and reuse the acid after rinsing the bottles. The HCI is changed
approximately every 9-12 months. After acid washing, the bottles are rinsed
thoroughly with MilliQ water 6 times. After rinsing, the bottles are filled with MilliQ
water and sealed. The water rinse should last at least 24 hours, but can be longer.
This dilutes and dissolves any excess acid left in the bottle after rinsing. When a
bottle is needed for a new culture, it is emptied of the water, the filter is attached,
and the whole system is autoclaved for 20min on the Fast setting of the autoclave.
After autoclaving, the bottle can be left on the shelf until needed. Filters should only
be autoclaved twice before disposal. After use, the bottle is cleaned with MilliQ
water and residual cell detritus is scrubbed from the aerator and insides of the
bottle using a cyano-dedicated brush. The bottles are left to soak in Micro detergent
for at least 24 hours. The micro is rinsed 6 times from the bottles and then the acid
wash process begins again.
Air Pumps:
An air supply column has been constructed for the incubator. This is an air
pump with valves that can be opened when connected to a bottle. Cultures are
aerated to the point that bubbles break the surface of the media, but not too
vigorously. In our set-up, this is not well regulated for uniformity. However, too
much agitation by bubbling will shear the cells and result in lysis.
Figure 28: Synechococcus WH8109 Cultures
Figure 29: "Weigele Bubbler" Blueprint
A - Aerator Connection: Dispersion Tube, %" tubing, bulkhead connector.
B - Filter Connection: 2 Bulkhead connectors, %" tubing, PALL 0.22pm filter.
C - Pasteur Loop.
D - Air supply.
E - Luer and connector for lid connection to aerator and filter. Inside and outside of lid indicated.
F - 2L Bottle lid with holes.
G - SOOmL bottle assembled.
68
Appendix C: Evaluation of Transcriptional Expression
Initially, the goal of this research included characterization of both the
transcriptional and translation response of Synechococcus WH8109 to stress
conditions. Here, I have detailed the experimental design and primers designed for
sequencing the genes of interest from Synechococcus WH8109.
To characterize the transcriptional response to heat and infection stress, I
proposed to compare the expression levels of wild-type cells to the levels measured
in cells subjected to multiple heat shock conditions and to cells through a time
course of infection using RT-PCR.
To accomplish this, I selected genes whose
products are known to participate in photosynthesis, stress response, and carbon
fixation, in order to compare them to several endogenous control genes. Because
WH8109's sequence has not been released yet, I used an alignment of homologous
genes
from
Synechococcus species:
WH8102,
CC9605,
WH5701,
RCC307,
PCC7002,and WH7803, and Synechococcus elongatus species PCC 7942 and PCC
6301, to make sequencing primers based on the most homologous regions of each
gene.
PCR was successful for every gene, but the primers were not specific enough
to identify the correct sequence of the gene of interest. After repeated attempts to
extract the appropriate gel bands according to the expected size, sequencing results
were fragmented at best. Included in this appendix are the genes of interest and the
sequencing primers I used for PCR. The strategy for this experiment was:
1. Extract gDNA
2. PCR genes of interest using homology-based primers
3. Sequence genes of interest using sequencing primers
4. Design RT-PCR probes
5. Test system
6. Measure transcript levels of genes of interest under different conditions
Table 2: Genes of Interest
Cellular Role or Function
Table 3: PCR Primers
Gene
Primer (5'4
->3)
Length
(bp)
G/C Content
ctgtttctctcagcgtctcc
Caac ccaa at cc
20
17
0.55
0.588
Tm
('C)
Expected
Product
Len h b
GroEL
Forward
Reverse
GroES
Forward
Reverse
297
55.1
53.8
clpB
Forward
Reverse
rbcL
Forward
Reverse
545
catgttgctgcacatccacc
c aactc aactt atctccttcc
20
24
0.55
0.5
57.2
57.2
rbcS
Forward
Reverse
cpeA
Forward
Reverse
cpeB
Forward
Reverse
482
ccgtcgtcaccactgttgt
ca c
at c atc
18
21
0.58
0.57
57.7
58.7
psbJ
Forward
Reverse
195
cggcaaaaaatccccgtatccc
ca c aacc acgc
22
16
0.545
0.75
58.8
60
gspE
Forward
Reverse
rnpA
Forward
Reverse
rnpB
Forward
Reverse
367
gcactccccgcctcc
ca aact a aa act c c
15
21
0.8
0.523
59.4
55.3
Table 4: Sequencing Primers
Gene
Primer (5' -> 3')
Length
(bp)
G/C Content Tm
(OC)
Expected
Product
I.Pnuth (hnI
GroEL
Forward
Reverse
130
psbJ
Forward
Reverse
cttcctgatggccgtcctgc
aggagccatagaagaacaatcccacaac
20
28
0.65
0.536
60.8
63.2
rnpA
Forward
Reverse
261
rnpB
Forward
Reverse
catctatctgggatcgccgttac
caggcttgctgggtaacg
23
18
0.522
0.611
57
56.4
Appendix D: Mass Spectrometry Preliminary Results
MS Analysis Gel (from Figure 27) Results provided by the Koch Institute for
Integrative Cancer Research at MIT, Proteomics Core Facility:
The results presented herein were generated from five samples, each containing a
gel piece that was excised from a Coomassie-stained gel ("Gel 1"). The proteins in
each sample were subjected to in-gel reduction with dithiothreitol and alkylation
with iodoacetamide, followed by in-gel digestion with trypsin, as per standard
protocols. LC-MS analyses were performed with the QSTAR mass spectrometer
installed at the KI Proteomics Core facility. Database searches, using the Mascot
database search software, were carried out against all bacterial protein sequences in
the UnireflOO protein database.
The proteins identified in each sample are shown in the table on page 2 of this
report. For each protein, the number of peptides that were uniquely matched to the
particular protein sequence with scores greater or equal to the Mascot peptide
identity threshold is listed, along with the calculated molecular weight for the
protein. Although reliable protein identifications can be made from single peptides,
in general the more peptides that are matched to a protein, the more confident one
is that the identification is not random but actually correct. In addition to the
proteins shown in the table, keratin, a common contaminant and trypsin, used in
protein digestion, during which it partially autolyzes, were also present in some of
the samples.
Samples correspond to circled gel bands in Figure 27 as follows:
Left top: la
Center top: lb
Center bottom: 1c
Right middle: id
Right top: le
Sample I a
Q3AMG3
Cal.
escr1Icn
Aceso
Phyc~titlsonle linter pot~eptkie r%- Tax-Synect'acoccus sp. CC96D5
RevID-O23AVMG3
SYN.SC
iv M"lq
595-21
2
Calc
it Lique
Samplelb
nesc11p'k~n
Acession
Q3AIC4
Ferredoxln--NADP reductase n-1 Tax-Synechacocci-i sp. Cc9Eo15
RePID-03AIC4 SYNSC
410
________Saawleic
c-preyerthrrl cfass
I alpha Ctain r- 1 Tax-STieclwococcus SF. CC9&3I5
SYN-SC
03AMIH9
18074
10
'C-phy)cerythrin class 11 aip~'a chain n-I Tax-Syrechcccus sp.C9F
SYNSC
17%-3
7
Phycocyln, bella subm,
It n-i I ax-Synec1Iommucs sp. CC9EGS5
1813-
7
O~M1 C-ph"crythrin SYNSC
I1 beta MIain r-1 Tax-Synecf ,ccoaccura Sp.
CC9&05
(3 M1Re ,pID-23AIHI1 Class
20_43
D3
5
cAM6 Phycocyin, alpha subunit n-1 Tax-S IectIocooc-iI sp.
CC9605
17319
6
______RevID-Q3MH-9
023AMH2
______ReplD-Q3AMH~2
QZAM15
SYNSC
______RetDID-03M15
SYNSC
_______ReIDID-Q3AM1E
~3~O Re
C-phrycerythri
class
I Deta chain n-1 7ax-Synefcoc=L6 sp. CC9E405
PID-QMM41
SYNSO
143
Q3H6
Min~ n-i1 Tax-Syrechcoccwis sp. CC9-#EDE
QAJ6 Alloplycocyainapha
ReplD-03AMH6 SYNSC
17515
______
03AI-J50 Aloprfyew~yaulin beta subLntt n-1 7ax-Synectiococcus sp. CC9EC15
SYNSC
______
_______
~Sample
0Q3AJE9
Id___
lA
______
Possible pihvobilsanle lInker ,xitypeptide n- 1 T ax-Srleefioemcu
ReD>ID-Q~3)UE9 SYNSC
_______CC96D5
sp
PhycoIIlscrne rod-core
polypepile
cpcG I...-RC
2E15.
n-1
sp.[Inter
CC9GD5
RepID-Q3AMB~1
SVNSC
~3AH1 -ptccerythri cdaSS 11 beta cIhaln n-1 Tax-Synedmoccws sp. CC9&15
03AHB1
_______Tax-Syvnechooocus
SYNSC
______RepID-03MMI1
FflA3 1
C-p--ycoerythlt class 2 subunit alpha r -2 Tax-Synechicmecs
SYNPX
_______RepID-PdiEA2
~Sample
_______
le
29672
1
#9Lnique
peptIdes
9
___
3
2DC43
2
17946
1
______
#ec~tonCl-4Lflque
Dectides,
__MW_
TranSketase n- I Tax-S~~iec Mcoccus sp. CC9605
02 SYNSC
72597
1iD
Acetate-CoA ligase n-1 77ax-Syneehococcus sp. CC9Ear,
72190
4~
_______RepIO-Q3AN
03AKIS
__
.3
31734
Accession
Q3,AND2
_5
___
O-sipwCaic.
Accession
19433
17496
_______ReplD-Q3MArir-
_______
iu
aer:
Mwescpan
Acoession
_____ReDID-Q3AX18
SYNSCI
SEC 16a Gel (from Figure 27) Results provided by the Koch Institute for Integrative
Cancer Research at MIT, Proteomics Core Facility:
The results presented herein were generated from six samples, each containing a gel
piece that was excised from a Coomassie-stained gel ("Gel 2"). The proteins in each
sample were subjected to in-gel reduction with dithiothreitol and alkylation with
iodoacetamide, followed by in-gel digestion with trypsin, as per standard protocols.
LC-MS analyses were performed with the QSTAR mass spectrometer installed at the
KI Proteomics Core facility. Database searches, using the Mascot database search
software, were carried out against all bacterial protein sequences in the UnireflOO
or Swiss-Prot protein database.
The proteins identified in each sample are shown in the table on page 2 of this
report. For each protein, the number of peptides that were uniquely matched to the
particular protein sequence with scores greater or equal to the Mascot peptide
identity threshold is listed, along with the calculated molecular weight for the
protein. Although reliable protein identifications can be made from single peptides,
in general the more peptides that are matched to a protein, the more confident one
is that the identification is not random but actually correct. In addition to the
proteins shown in the table, keratin, a common contaminant and trypsin, used in
protein digestion, during which it partially autolyzes, were also present in some of
the samples.
Samples correspond to circled gel bands in Figure 27 as follows:
Leftmost: 2a
Right top: 2b
Right 2nd from top: 2c
Right middle: 2d
Right 2nd from bottom: 2e
Right bottom: 2f
Results indicate GroES is present, which, until now, was unknown. Anti-E. coli
GroES antibodies did not recognize Synechococcus WH8109 GroES.
Sample 2a
Accession
Description
aw
pnide
Q3ALA9
Putative
CC9605 uncharac:erized
ReplD=Q3ALA9 protein
SYNSCn=1 Tex=Synechococcus sp.
Ferredcxin-NADP
reductase n=1 Tax=Synechococcus sD CC9305
ReplD=Q3AIC4 SY\ISC
43903
7
425, 7
2
Q3AIC4
Oie peptide ir rommon between the ahove two prnreins
Sample 2b
Accession
Description
c.
pniqe
Q3AGA1
Ferredcxin--nitrite
reductase n= I Tax=SVnechococcus sp. CC9605
ReplD-Q3AGAI SvNSC
56909
5
A4CWY5
Ferredcxin-nitrite
reductase n= I Tax-SVnechococcu3 sp. VH 7805
ReplD=A4CWY5 SYNPV
56561
2
34380
2
Sample 2c
Accession
HEM3 SYNS9
Description
PorpI'ohilinogern
OS=Synechncoccuij
GN=hemC PE= dRaminase
SV=1
p (strain CC9902)
There were ro sigificant prctein hits ident ficd from the scarch of alI bacterial pro-c ns (357949S protein
seqiences i-i rhe unireflo clItaare, whilt from the search of the murh smil er Swiss-Prnt dataahe
(232399 bacterial protein sequences) the abcve prtein was iden"Atied with qcod contidence. Alth:ugh tte
prUbability tlId a y peptide hit ib d IdIIUII eveit is irIvereIy propoUtioaIdi LUitit Size o the pULeinI Uda.JdSe
sea-cled, nevertheless the above result otained from searching Swiss-Prot aopears to b-e significant.
Sample 2d
Accession
Q3AICl-,4
Description
Ferredcxin-NADP reductase n=1 Tax=Synechococcus sp. CC9305
RepID-Q3AIC4
SYNJSC
Caic.
IVI
apeptides
unique
43903
7
Calc.
# unique
S1733
3
wvc
e ide
10768
3
Sample 2e
Accession
Description
Q3AKA4
Elongation factor Ts n=I Tax=Synochococcus sp. CC0605
ReplD=EFTS SYNSC
03AR5
Geranygeranyl pyrophosphate synthase n-I Tax-Synechococcus
sp. CC9605
Accession
A5GiBO
ReplD=Q3AIB5 SYNSC
Sample 2f
Description
IC kDa chaperonin
n-I Tax-Synechococczus s3. WH 7e03
RepiD=CH10
SYNPW
IVW
24139
peptides
4
8.0
Acknowledgments
There are so many people who have made this project possible that I barely know
where to begin in thanking them. First and foremost, I would like to thank Jonathan King
for his guidance and support. I would also like to thank John Essigmann for his advice and
help throughout my years at MIT. Finally, I want to thank Eric Alm for agreeing to mentor
the project as my faculty supervisor.
I am also incredibly grateful for the daily help of my colleagues. I would like to
thank Desislava Raytcheva for her patience and constant willingness to help. I would
especially like to thank Ligia Acosta-Sampson for her valiant effort to help me through the
purification of GroEL and teach me the fine art of FPLC. I would like to thank Jason Holder
for his valuable advice on graduate education, expert Adobe Photoshop skill, and letting me
know it is okay if I cannot quite "wrestle the beast to the ground alone." I would like to
thank Kelly Knee and Dan Goulet for technical advice and assistance. I would like to thank
Jeannie Chew, Althea Hill, and Cameron Haase-Pettingell for all of their help with keeping
the lab stocked with supplies and plenty of Syn 5, and helping keep the growth of WH8109
running smoothly. And finally, I'd like to thank everyone else in the King Lab for their
support and friendly faces every day.