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!