1. Max Planck Institute for Dynamics of Complex Technical Systems
CNV-Meeting September 16th 2011
Redox Sensors for Photosystem Regulation in Rhodospirillum rubrum
Anke Carius
MAX-PLANCK-INSTITUT
DYNAMIK KOMPLEXER
TECHNISCHER SYSTEME
MAGDEBURG
Max Planck Institute for Dynamics of Complex Technical Systems
Magdeburg/Germany
Max Planck Institute Magdeburg

2. Structure Introduction: Rhodospirillum rubrum
Theory of known redox regulators
Experiments & Results
Outlook

3. Introduction

4. Rhodospirillum rubrum
Alpha-Proteobacterium
Versatile metabolism
Anoxygenic photosynthesis
Aerobic, semiaerobic and anaerobic conditions

5. The facultative phototrophic R. rubrumPM
expressed PM
repressed
- O2
anaerobic
semiaerobic
aerobic
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

6. 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

7. Theory of known redox regulators

8. Genetic Regulators Activator Proteins: Enhance mRNA-Synthesis
 higher gene expression
Repressor-Proteins: Inhibit mRNA-Synthesis
 reduced gene expression
Antirepressor Proteins: Remove repressor
 allow gene expression
RNA-Pol.
DNA
DNA
RNA-Pol.
DNA

9. Thiolgroups as Redox Sensors: Sensor and Regulator in one Protein
Oxidizing conditions
- O2 S SH S
S-… SH
+ O2
S-S
Reducing conditions

10. R. rubrum posesses a PpsR-homologue
CrtJ/PpsR: An Example
Thiol-based redox sensor
Repressor function
Photosensitive antirepressor
R. rubrum posesses a PpsR-homologue
Masuda et al. 2002,
Repression of photosynthesis gene expression in Rhodobacter sphaeroides, Proc Natl Acad Sci USA, 10(99)

11. The Two Component System: RegB/RegA
RegB: Redoxsensorkinase
RegA: Response regulator, activator
Quinone pool based redox signal
Supposed to integrate cellular thiol redox potential
R. rubrum posesses no RegB/A-homologues
… but a histidine kinase close to the photosynthetic gene cluster….
Bauer et al Signal Transduction By The Global Regulator RegB Is Mediated By A Redox Active Cysteine. EMBO J. 22,
Control of gene expression

12. Experiments & Results

13. 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 proteins
GSH: Reduces some thiol group via specific interactions
DEM: Lowers the amount of reduced GSH in the cell

14. Growth experiments

15. Mutants were unable to produce PM!
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

16. 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

17. Thank you very much for your attention!