Psb31, a novel photosystem II associated protein in Synechocystis sp. PCC 6803 [Elektronische Ressource] / by Stefan Bennewitz
Description
Psb31, a novel photosystem II associated protein in Synechocystis sp. PCC 6803 Thesis in order to receive the academic degree doctor rerum naturalium (Dr.rer.nat.) submitted to the Rat der Biologisch-Pharmazeutischen Fakultät Friedrich Schiller Universität Jena by Diplom-Biochemiker Stefan Bennewitz rdborn on August 23 1979 in Altenburg, Germany Jena, January 2009 - 1 - Gutachter: 1. Prof. Dr. Ralf Oelmüller, FSU Jena 2. PD Dr. Thomas Pfannschmidt, FSU Jena 3. Dr. Terry Bricker, LSU Baton Rouge Tag der öffentlichen Verteidigung: 14.05.2009 - 2 - Tabble of Contents Table of Contents Abbreviations.................................................................................................................. 5 Measuring Units.............................................................................................................. 6 I Introduction................................................................................................................ 7 1.1 Cyanobacteria .................................................................................................... 7 1.2 Synechocystis sp. PCC 6803 .............................................................................. 8 1.3 Photosynthesis 9 1.4 Photosystem II ................................................................................................. 11 1.5 PSII reaction..
Sujets
Informations
| Publié par | friedrich-schiller-universitat_jena |
| Publié le | 01 janvier 2009 |
| Nombre de lectures | 17 |
| Langue | English |
| Poids de l'ouvrage | 3 Mo |
Extrait
Psb31, a novel photosystem II associated protein
inSynechocystissp. PCC 6803
Thesis in order to receive the academic degree doctor rerum naturalium (Dr.rer.nat.) submitted to the Rat der Biologisch-Pharmazeutischen Fakultät Friedrich Schiller Universität Jena
by
Diplom-Biochemiker Stefan Bennewitz
born on August 23rd1979 in Altenburg, Germany
Jena, January 2009
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Gutachter:
1.
2.
3.
Prof. Dr. Ralf Oelmüller, FSU Jena
PD Dr. Thomas Pfannschmidt, FSU Jena
Dr. Terry Bricker, LSU Baton Rouge
Tag der öffentlichen Verteidigung: 14.05.2009
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Tabble of Contents
Table of ContentsAbbreviations..................................................................................................................5Measuring Units.............................................................................................................. 6 I Introduction................................................................................................................ 7 1.1 Cyanobacteria .................................................................................................... 7 1.2Synechocystissp. PCC 6803 .............................................................................. 8 1.3 Photosynthesis.................................................................................................... 9 1.4 Photosystem II ................................................................................................. 11 1.5 PSII reaction..................................................................................................... 12 1.6 Accessory PSII proteins ................................................................................... 13 1.7 Sll1390 Psb31 ............................................................................................... 15 II Material and Methods .............................................................................................. 16 2.1 Materials .......................................................................................................... 16 2.1.1 Chemicals................................................................................................. 16 2.1.2 Enzymes and kits ..................................................................................... 16 2.1.3 Markers for electrophoresis ..................................................................... 17 2.1.4 General buffers and media ....................................................................... 17 2.1.5 Oligonucleotides ...................................................................................... 18 2.2 Methods............................................................................................................ 19 2.2.1 Bioinformatic analysis ................................................................................. 19 2.2.2 Strains ...................................................................................................... 19 2.2.3 Culture conditions.................................................................................... 21 2.2.4 DNA isolation .......................................................................................... 21 2.2.5 PCR .......................................................................................................... 22 2.2.6 Reverse transcriptase - PCR..................................................................... 22 2.2.7 Mixed culture experiments ...................................................................... 22 2.2.8 Gel electrophoresis................................................................................... 23 2.2.9 Antibody generation................................................................................. 23 2.2.10 Immunoprobing........................................................................................ 24 2.2.11 Purification of total membranes and PSII complexes.............................. 24 2.2.12 Two phase partitioning ............................................................................ 25 2.2.13 Separation of membrane complexes ........................................................ 25 2.2.14 Determination of protein concentration ................................................... 25 2.2.15 Determination of pigment concentrations................................................. 25 2.2.16 UV-visible absorption spectroscopy ........................................................ 26 2.2.17 77 K fluorescence spectroscopy............................................................... 26 2.2.18 Atomic absorption spectroscopy.............................................................. 26 2.2.19 Oxygen evolution and light saturation curves ......................................... 27 2.2.20 Fluorescence kinetics ............................................................................... 27 2.2.21 Photoinhibition and recovery ................................................................... 28 2.2.22 Blue light experiments ............................................................................. 28 2.2.23 Pull down experiments with truncated Psb31 expressed inE.coli.......... 29 2.2.24 Purification of the Psb31 HIS-tagged protein fromSynechocystis.......... 30
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III Results.................................................................................................................. 3.1. Bioinformatic analysis ..................................................................................... 3.2LocalizationofPsb31...................................................................................... 3.3 Lumenal Subunits ............................................................................................ 3.4 Construction of theΔpsb31deletion mutant.................................................... 3.5 Physiological analysis of theΔpsb31mutant .................................................. Growth assays ........................................................................................................... Oxygen evolution...................................................................................................... Fluorescence Kinetics ............................................................................................... 77 K Spectroscopy .................................................................................................... Pigment composition ................................................................................................ Protein composition .................................................................................................. 3.6 Mixed culture experiments ............................................................................. 3.7 Oxidative stress ............................................................................................... 3.8 Photoinhibition and recovery .......................................................................... 3.9Psb31expressionunderHL............................................................................ 3.10 Blue light treatment......................................................................................... 3.11 Pull down assays withE.colisynthesized truncated Psb31 ............................ 3.12 PsbV antibody ................................................................................................. 3.13 Purification of HIS-tagged Psb31 ................................................................... 3.14Psb31Doublemutants.................................................................................... IV Discussion ............................................................................................................ V-1 Summary .............................................................................................................. V-2 Zusammenfassung................................................................................................ VI Literature.............................................................................................................. VII Acknowledgements.............................................................................................. Curriculum vitae ........................................................................................................... Publications................................................................................................................... Ehrenwörtliche Erklärung .............................................................................................
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32 32 38 42 44 45 45 47 49 51 51 53 53 55 57 58 58 59 62 63 67 70 80 82 84 90 91 92 93
Abbreviations
BSA cDNA Chl DCBQ -DCMU DUF477 DM DMSODNA dNTP EDTA HCl HL HEPES IPTG LED
mRNAOD PAGE PCR PSI
PSII RNA RC SD SDS-PAG Tris WT
E
Albumine bovine serum Complementary deoxyribose nucleic acid Chlorophyll 2,6 dichloro-p-benzoquinone 3-(3,4-dichlorophenyl)-1,1-dimethylureaDomain of unknown function number 477 Dodecyl maltoside Dimethyl sulfoxide Deoxyribose nucleic acid Desoxynucleoside triphosphate Ethylenediaminetetraacetate Hydrochloric acid High light N-2-Hydroxyethylpiperazin-N`-2-ethanolsulfonic acid Isopropylthio-β-D-galactoside Light-emitting diode
Messenger ribonucleic acid Optical density Polyacrylamide gel electrophoresis Polymerase Chain Reaction Photosystem I Photosystem II Ribonucleic acid Reaction center Standard derivation Sodium dodecyl sulfate polyacrylamide gel electrophoresis Tris-(hydroxymethyl)-aminomethaneWild type
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kb
kDa
h
K
Minute
Nanometer
l
M
Base pairs
Degree celsius
Kilo base pairs
Kilodalton
Hour
Degree kelvin
bp
°C
Angström
Å
6 --
Weight per volume
Measuring Units
Second
Micromole
Lightintensity in microeinstein per square meter per second
Rotations per minute
sec
rpm
nm μE/m2 s
min
w/v
μmol
Measuring Units
Litre
Molar concentration
I
1.1
Introduction
Cyanobacteria
Introduction
Cyanobacteria are a large and diverse group of photosynthetic prokaryotes also re
ferred to as blue-green algae. Cyanobacteria are resilient and tough and can be found in a
wide variety of environments including fresh water, marine, terrestrial environments and
extremes like hot springs and rock surfaces in Antarctica and deserts. As
photoautotrophic organisms, cyanobacteria utilize light as energy to reduce inorganic
CO2energy rich carbohydrates. Fossilized cyanobacteria have been dated as far backinto
as 2.8 billion years and they are thought to be responsible for the transformation of the
earths atmosphere from reducing to oxidizing by producing molecular oxygen. Most
cyanobacteria contain thylakoids, an extensive internal membrane system containing the
components of photosynthesis and respiration. The mechanism of cyanobacterial
photosynthesis is remarkably similar to the one in higher plants. In fact, the
semiautonomous eukaryotic chloroplast is likely the result of an endosymbiosis of a
cyanobacterium by a non-photosynthetic protoeukaryotic cell. Higher plants chloroplasts
still contain coding DNA that is organized and regulated in a bacterial manner. Over time
a significant number of chloroplast genes were transferred to the host nucleus so the
chloroplast no longer contains enough information to be completely free of the nucleus.
Many chloroplast proteins are therefore synthesized in the plant cell cytoplasm and
transported into the plastid. This requires a highly sophisticated interaction between the
plant cell organelles. In contrast, cyanobacteria have a relative simple genetic structure
compared to higher plants and are therefore ideal to study oxygenic photosynthesis.
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Introduction
Cyanobacteria have attracted attention these days with their potential to serve as
food stock or alternative food fuel sources. The best known example of commercially
used cyanobacteria isArthrospira platensis, widely known as Spirulina. Its potential as
food stock was described as early as 1983 by Orio Ciferri and it is widely available as
food supplement in health stores. Some species of cyanobacteria have the potential to
produce hydrogen and others have high lipid content. It has been estimated that
cyanobacteria could generate 100 times more biodiesel per biomass compared to any
plant system (Rittmann, 2008). There is clearly a need for more detailed studies of
cyanobacterial physiology, energy transduction
demonstrate their utility for bioenergy production.
1.2
Synechocystissp. PCC 6803
pathways
and
metabolisms
to
Synechocystis PCC 6803 is an important model organism for photosynthesis sp.
research (figure 1and reactions are similar to the processes). Its photosynthetic structures
in higher plants. There are glucose tolerant strains allowing heterotrophic growth and
thus the study of photosynthetic knock-out mutants. Genetic manipulation of
Synechocystissp. PCC 6803 is possible due to its ability to uptake and incorporate DNA
via highly efficient double homologous recombination (Williams, 1988). Coupled with its
doubling time of 6-10 hours under optimal photoautothrophic conditions, homozygous
mutants can be generated very fast inSynechocystis6803.
The unicellular cyanobacteriumSynechocystissp. PCC 6803 was the third
prokaryote and first photosynthetic organism whose genome was completely sequenced
(Kaneko et al., 1996). Today there have been more than 40 cyanobacterial genomes
sequenced. Their size ranges from the most primitiveProchlorococcus with less strains
than 2 Mb to the nitrogen fixingNostoc punctiformePCC 73102 with a genome of 9 Mb.
Synechocystis6803 has a genome of 3.9 Mb and approximately 3600 genes.
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Introduction
Figure 1 -ocystischneSy PCC 6803: sp.stiySenhccosy be grown in liquid BG11 media or on Agar can containing plates.
1.3
Photosynthesis
Photosynthesis is a series of enzyme catalyzed reactions in which light energy is
converted into chemical energy by living organisms. It is performed by many different
organisms, namely plants, algae and certain bacteria. The best known form of
photosynthesis is oxygenic photosynthesis where water is oxidized to molecular oxygen.
It is considered the most important biochemical pathway as most organisms depend on
the oxygen and reduced carbon produced. Photosynthesis occurs in two phases, the light
dependent and the light independent dark reactions or Calvin-Benson Cycle. The first
phase of photosynthesis utilizes light energy to oxidize water and frees molecular
oxygen, protons and electrons.
The electrons are transferred through the photosynthetic electron transfer chain to
donate a reducing equivalent to NADP and form the cellular reductant NADPH.
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phototrophs are represented in four bacterial phyla - purple bacteria, green sulfur bacteria,
green gliding bacteria, and gram positive bacteria. Contrary to cyanobacteria these
anoxygenic photosynthesis and use light energy to extract electrons from molecules other
than water such as H2S and therefore do not generate molecular oxygen. Anoxygenic
While cyanobacteria perform oxygenic photosynthesis, some bacteria perform
carbon dioxide + water + light energy→ waterglucose + oxygen +
6 CO2 H 122O + photons→C6H12O6+ 6 O2+ 6 H2O +
reduction and to make precursors of carbohydrates.
A simplified equation of photosynthesis:
Introduction
ATP and NADPH are used in the Calvin-Benson-Cycle to fix CO2 chemical by
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Protons liberated into the thylakoid lumen during water oxidation form an
generating electrons used to reduce the mobile electron carrier, plastoquinone.
synthase. The cytochromeb6f complex transfers electrons from the membrane soluble
electrochemical gradient over the thylakoid membrane which utilized by the ATP
reduced plastoquinone to the soluble copper protein plastocyanin. Additional protons are
translocated into the lumen during this process.
to have evolved before oxygenic photosynthetic organisms (Blankenship, 2002). For the
organisms have only one reaction center, either a type I or a type II and they are thought
cyanobacteria.
purpose of this thesis, I will only refer to oxygenic photosynthesis performed by
The light reactions require a series of specialized multi-subunit pigment-protein
(PSII) complex is responsible for water oxidation during oxygenic photosynthesis
complexes which form the photosynthetic electron transport chain. The photosystem II
Introduction
The photosystem I (PSI) uses light energy to transfer the electrons from thylakoid
lumen located plastocyanin to the stromal (cytoplasmic in cyanobacteria) ferredoxin.
With the help of ferredoxin NADP+ the electro reductase
generate the reductant NADPH.
1.4
Photosystem II
n is transferred to NADP+ to
Photosystem II is a large multisubunit structure in thylakoid membranes that
catalyzes light-driven charge separation accompanied by water splitting during oxidative
photosynthesis (figure 2). The cyanobacterial PSII complex acts as a dimer where each
monomer contains at least 20 protein subunits and 77 cofactors including chlorophylls,
pheophytins, carotenoids, lipids, plastquinones, manganese, calcium, chloride and both
non-heme and heme iron (Loll et al., 2005).
Figure 2 Architecture of the PSII dimer:Isolated fromThermosynechococcus elongatesat a resolution of 3.5 Å adapted from (Ferreira et al., 2004).
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