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Max Planck Institute Magdeburg

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

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Max Planck Institute Magdeburg

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  1. CNV-Meeting September 16th2011 MAX-PLANCK-INSTITUT DYNAMIK KOMPLEXER TECHNISCHER SYSTEME MAGDEBURG Redox Sensors forPhotosystem Regulation in Rhodospirillumrubrum Anke Carius Max Planck Institute for Dynamics of Complex Technical Systems Magdeburg/Germany Max Planck Institute Magdeburg

  2. Structure Introduction: Rhodospirillumrubrum Theoryofknownredoxregulators Experiments & Results Outlook

  3. Introduction

  4. Rhodospirillumrubrum Alpha-Proteobacterium Versatile metabolism Anoxygenicphotosynthesis Aerobic, semiaerobicandanaerobicconditions

  5. The facultativephototrophicR. rubrum + O2 PM expressed PM repressed - O2 anaerobic semiaerobic aerobic • Photosyntheticmembrane (PM) expressionisredoxdependent • Therewithitcanbeinfluencedbysubstrateandoxygensupply • Reverse, PM reflectthecultureredox potential • PM canbeeasilydetectedat 880 nm in a photospectrometer

  6. Useofpurplebacteria in scienceandindustry Redoxregulationandhomeostasisscience Microaerobicmetabolism Precursorofmitochondria Biotechnological productionofbacteriochlorophyllsandcarotenoids Variousotherpotentiallyusefulfeatures: CO-utilization, CO2-fixation, PHB-production, H2

  7. Theoryofknownredoxregulators

  8. Genetic Regulators RNA-Pol. RNA-Pol. DNA DNA DNA • Activator Proteins: EnhancemRNA-Synthesis highergeneexpression • Repressor-Proteins: InhibitmRNA-Synthesis  reducedgeneexpression • Antirepressor Proteins: Remove repressor  allowgeneexpression

  9. ThiolgroupsasRedox Sensors:Sensor and Regulator in one Protein Oxidizingconditions - O2 SH S-S S-… SH SH S S SH + O2 S-… S S Reducingconditions S S

  10. CrtJ/PpsR: An Example • Thiol-basedredoxsensor • Repressorfunction • Photosensitiveantirepressor R. rubrumposesses a PpsR-homologue Masudaet al. 2002, Repression ofphotosynthesisgeneexpression in Rhodobactersphaeroides, ProcNatlAcadSci USA, 10(99)

  11. The TwoComponent System: RegB/RegA R. rubrumposessesnoRegB/A-homologues … but a histidinekinaseclosetothephotosyntheticgenecluster…. Bauer et al. 2003. Signal TransductionBy The Global Regulator RegBIsMediatedBy A RedoxActiveCysteine. EMBO J. 22, 4699-4780 Controlofgeneexpression RegB: Redoxsensorkinase RegA: Response regulator, activator Quinonepoolbasedredoxsignal Supposedtointegratecellularthiolredox potential

  12. Experiments & Results

  13. Chemical triggeringofredoxsensors Idea: Useofreductantstoreducethiol-groups Triggeringofredoxswitch ReducingAgents: Dithiothreitol (DTT), Glutathione (GSH) OxidizingAgents: Diethylmaleate (DEM), ox. Glutathione (GSSG) DTT: Reduces all thiolgroupsofproteins GSH: Reducessomethiolgroup via specificinteractions DEM: Lowerstheamountofreduced GSH in thecell

  14. Growth experiments

  15. PpsR in R. rubrum Resultshint on a specificregulation via GSH Measurementsofcytosolic GSH: Activeuptakeof GSH ! PpsRas a thiolredoxsensor was deleted Mutantswereunabletoproduce PM! PpsR in R. rubrummust be an activatorprotein PpsRbindsmore DNA underreducingconditions

  16. Outlook Deletionmutantofhistidinekinaseclosetophotosynthetic genes IdentificationofPpsRbindingmotive ComplementationofPpsR-deletionmutant OverexpressionofPpsR in R. rubrum

  17. Thankyouverymuchforyourattention!

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