max planck institute magdeburg cnv-meeting september 16 th 2011 redox sensors for photosystem...
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Max Planck Institute MagdeburgMax Planck Institute Magdeburg
CNV-Meeting September 16th 2011
Redox Sensors for Photosystem Regulation in Rhodospirillum rubrum
Anke Carius
MAX-PLANCK-INSTITUTDYNAMIK KOMPLEXER
TECHNISCHER SYSTEMEMAGDEBURG
Max Planck Institute for Dynamics of Complex Technical Systems Magdeburg/Germany
Max Planck Institute Magdeburg
Structure
• Introduction: Rhodospirillum rubrum
• Theory of known redox regulators
• Experiments & Results
• Outlook
Max Planck Institute Magdeburg
Introduction
Max Planck Institute Magdeburg
Rhodospirillum rubrum• Alpha-Proteobacterium• Versatile metabolism• Anoxygenic photosynthesis• Aerobic, semiaerobic and anaerobic
conditions
Max Planck Institute Magdeburg
The facultative phototrophic R. rubrum
anaerobic aerobicsemiaerobic
• Photosynthetic membrane (PM) expression is redox dependent
• Therewith it can be influenced by substrate and oxygen supply
• Reverse, PM reflect the culture redox potential
• PM can be easily detected at 880 nm in a photospectrometer
Fructose Succinate Fructose/SuccinateFructose Succinate Fructose/Succinate- O2
+ O2 PM repressed
PMexpressed
Max Planck Institute Magdeburg
Use of purple bacteria in science and industry
• Redox regulation and homeostasis science
• Microaerobic metabolism
• Precursor of mitochondria
• Biotechnological production of bacteriochlorophylls and carotenoids
• Various other potentially useful features:CO-utilization, CO2-fixation, PHB-production, H2
Max Planck Institute Magdeburg
Theory of known redox regulators
Max Planck Institute Magdeburg
• Activator Proteins: Enhance mRNA-Synthesis higher gene expression
• Repressor-Proteins: Inhibit mRNA-Synthesis reduced gene expression
• Antirepressor Proteins: Remove repressor allow gene expression
Genetic Regulators
RNA-Pol. DNA
DNA
RNA-Pol. DNA
Max Planck Institute Magdeburg
Thiolgroups as Redox Sensors:Sensor and Regulator in one Protein
SH
SH
Reducing conditions
SH
SH
S-S
S
S-…
S-…
S
SSS
S
- O2
+ O2
Oxidizing conditions
Max Planck Institute Magdeburg
CrtJ/PpsR: An Example
• Thiol-based redox sensor
• Repressor function
• Photosensitive antirepressor
R. rubrum posesses a PpsR-homologueMasuda et al. 2002,Repression of photosynthesis gene expression in Rhodobacter sphaeroides, Proc Natl Acad Sci USA, 10(99)
Max Planck Institute Magdeburg
Bauer et al. 2003. Signal Transduction By The Global Regulator RegB Is Mediated By A Redox Active Cysteine. EMBO J. 22, 4699-4780
The Two Component System: RegB/RegA
• RegB: Redoxsensorkinase• RegA: Response regulator, activator• Quinone pool based redox signal• Supposed to integrate cellular thiol redox potential
Control of gene expression
R. rubrum posesses no RegB/A-homologues… but a histidine kinase close to the photosynthetic gene cluster….
Max Planck Institute Magdeburg
Experiments & Results
Max Planck Institute Magdeburg
Chemical triggering of redoxsensors
Idea: Use of reductants to reduce thiol-groups Triggering of redox switch
Reducing Agents: Dithiothreitol (DTT), Glutathione (GSH) Oxidizing Agents: Diethylmaleate (DEM), ox. Glutathione (GSSG)
DTT: Reduces all thiol groups of proteinsGSH: Reduces some thiol group via specific interactionsDEM: Lowers the amount of reduced GSH in the cell
Max Planck Institute Magdeburg
Growth experiments
PM-Production in Rhodospirillum rubrum S1 on M2S-Medium with Glutathione
0
1
2
3
4
5
6
7
8
9
10
11
12
0 10 20 30 40 50 60 70
Time [h]O
D 6
60
0,40
0,45
0,50
0,55
0,60
0,65
0,70
0,75
0,80
0,85
0,90
0,95
1,00
Ratio
OD
880
/660
Control 660 1mM GSH 660
Control 880/660 1mM GSH 880/660
PM-Production in Rhodospirillum rubrum S1 on M2SF-Medium with
Glutathione
0
1
2
3
4
5
6
7
8
9
10
11
12
0 10 20 30 40 50 60 70 80
Time [h]
OD
660
0,40
0,45
0,50
0,55
0,60
0,65
0,70
0,75
0,80
0,85
0,90
0,95
1,00
Ratio
OD
880
/660
Control 660 1mM GSH 660 2mM GSH 660
Control 880/660 1mM GSH 880/660 2mM GSH 880/660
PM-Production in Rhodospirillum rubrum S1 on M2SF-Medium with oxidized Glutathione (GSSG)
0
1
2
3
4
5
6
7
8
9
10
11
12
0 10 20 30 40 50 60 70 80
Time [h]
OD
660
0,40
0,45
0,50
0,55
0,60
0,65
0,70
0,75
0,80
0,85
0,90
0,95
1,00
Ratio
OD
880
/660
Control 660 1mM GSSG 660 2mM GSSG 660
Control 880/660 1mM GSSG 880/660 2mM GSSG 880/660
PM-production in Rhodospirillum rubrum S1 on M2SF-Medium with Diethylmaleate (DEM)
0
1
2
3
4
5
6
7
8
9
10
11
12
0 10 20 30 40 50 60 70 80
Time [h]
OD
660
0,40
0,45
0,50
0,55
0,60
0,65
0,70
0,75
0,80
0,85
0,90
0,95
1,00
Rat
io O
D 8
80/6
60
Control 660 1mM DEM 660
Control 880/660 1mM DEM 880/660
PM-Production in Rhodospirillum rubrum S1 on M2S-Medium with Dithiothreitol (DTT)
0
1
2
3
4
5
6
7
8
9
10
11
12
0 20 40 60 80 100 120
Time [h]
OD
660
0,40
0,45
0,50
0,55
0,60
0,65
0,70
0,75
0,80
0,85
0,90
0,95
1,00
Ratio
OD
880
/660
Control 660 0,5mM DTT 6601mM DTT 660 2mM DTT 660Control 880/660 0,5mM DTT 880/6601mM DTT 880/660 2mM DTT 880/660
Max Planck Institute Magdeburg
PpsR in R. rubrum
• Results hint on a specific regulation via GSH
• Measurements of cytosolic GSH:
Active uptake of GSH !
• PpsR as a thiol redox sensor was deleted
Mutants were unable to produce PM!
PpsR in R. rubrum must be an activator protein
PpsR binds more DNA under reducing conditions
Max Planck Institute Magdeburg
Outlook
• Deletion mutant of histidine kinase close to photosynthetic genes
• Identification of PpsR binding motive
• Complementation of PpsR-deletion mutant
• Overexpression of PpsR in R. rubrum
Max Planck Institute Magdeburg
Thank you very much for your attention!