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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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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


Structure

Introduction: Rhodospirillumrubrum

Theoryofknownredoxregulators

Experiments & Results

Outlook


Introduction


Rhodospirillumrubrum

Alpha-Proteobacterium

Versatile metabolism

Anoxygenicphotosynthesis

Aerobic, semiaerobicandanaerobicconditions


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


Useofpurplebacteria in scienceandindustry

Redoxregulationandhomeostasisscience

Microaerobicmetabolism

Precursorofmitochondria

Biotechnological productionofbacteriochlorophyllsandcarotenoids

Variousotherpotentiallyusefulfeatures:

CO-utilization, CO2-fixation, PHB-production, H2


Theoryofknownredoxregulators


Genetic Regulators

RNA-Pol.

RNA-Pol.

DNA

DNA

DNA

  • Activator Proteins: EnhancemRNA-Synthesis

    highergeneexpression

  • Repressor-Proteins: InhibitmRNA-Synthesis

     reducedgeneexpression

  • Antirepressor Proteins: Remove repressor

     allowgeneexpression


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


CrtJ/PpsR: An Example

  • Thiol-basedredoxsensor

  • Repressorfunction

  • Photosensitiveantirepressor

R. rubrumposesses a PpsR-homologue

Masudaet al. 2002,

Repression ofphotosynthesisgeneexpression in Rhodobactersphaeroides, ProcNatlAcadSci USA, 10(99)


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


Experiments & Results


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


Growth experiments


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


Outlook

Deletionmutantofhistidinekinaseclosetophotosynthetic genes

IdentificationofPpsRbindingmotive

ComplementationofPpsR-deletionmutant

OverexpressionofPpsR in R. rubrum


Thankyouverymuchforyourattention!


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