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

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

Introduction

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

The facultative phototrophic R. rubrum PM 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

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

Theory of known redox regulators

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

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

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)

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. 2003. Signal Transduction By The Global Regulator RegB Is Mediated By A Redox Active Cysteine. EMBO J. 22, 4699-4780 Control of gene expression

Experiments & Results

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

Growth experiments

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

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

Thank you very much for your attention!