Systems Biology at Washington University

FIBR


Formation of Disulfide Bonds in Synechocystis 6803:

Proteomic studies have shown that extra-cytoplasmic spaces (lumen and periplasm) in cyanobacteria have numerous cysteine containing proteins. Function of these proteins depends on the redox state of cysteines. Therefore, proteins involved in dithiol-disulfied transitions would be expected to play an important role in the maintenance of redox poise. However, presence of a system for disulfide bond formation in cyanobacteria is unknown. Well-defined pathways are present for the formation of disulfide bonds in non-photosynthetic organisms.

Key Findings: Disulfide bond formation is a catalyzed process in cyanobacteria A novel protein unique to the oxygenic photosynthetic organisms is involved in disulfide formation This novel Cyanobacterial protein can functionally complement the mutants deficient in disulfide formation in E. coli. Optimal growth of Synechocystis 6803 is dependent on the function of this protein and it is essential under reducing condition Future Goals: Identify proteins whose function modulates with the redox state of cysteines and understand their overall impact on the redox poise under changing environmental conditions







by Abhay Singh

A system for disulfide bond formation in E. coli The thiols present in newly secreted proteins are oxidized by the DsbA, a 21 kDa periplasmic protein with a canonical CXXC motif in its active site. DsbA is reoxidized by a membrane localized protein thiol-oxidoreductase, DsbB. DsbB donates electron to molecular oxygen via ubiquinone using the components of electron transport chain. Together, the proteins couple intracellular biochemical reactions to the formation of disulfide bonds. Two additional proteins, a soluble thioredoxin like protein DsbC and a cytoplasmic membrane protein DsbD, are involved in the isomerization of incorrectly paired cysteines. The active site cysteines of DsbC are maintained in a functional reduced state by DsbD which shuttles the reducing equivalents from NADPH and thioredoxin reductase
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Disulfide Bond Formation is a Catalyzed Process in Cyanobacteria

by Abhay Singh

Formation of disulfide bond in the E. coli alkaline phosphatase (AP) exported to the periplasmic space of Synechocystis 6803. To express E. coli AP in Synechocystis 6803, phoA gene was fused to different regions of mntB gene that allowed its localization either in the cytoplasm or in the membrane facing cytoplasm or periplasm. MntB is membrane protein present in the plasma membrane. In addition, expression of mntB gene can be controlled since it is expressed only under the manganese limiting conditions along with mntA and mntC. The AP activity of wild type and three strains expressing E. coli AP of Synechocystis 6803 was measured after 15 h growth in manganese limited BG11. As can be seen, E. coli AP is activity only when present in the periplasmic space.
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A Novel Protein Unique to the Oxygenic Photosynthetic Organisms

by Abhay Singh

A schematic diagram showing two domains of the SynDsbAB proteins in Synechocystis 6803 similar to E. coli DsbA and DsbB proteins. The filled boxes represent the trans-membrane helices of SynDsbAB. The canonical CXXC present in both domains are indicated.
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Functionally Complement the Mutants Deficient in Disulfide Formation

E. coli ΔdsbA and ΔdsbB strains exhibit a number of phenotypes caused by a generalized inability to form disulfide bonds. On minimal media, both strains are non-motile due to their inability to introduce a single disulfide required for proper folding of the flagellar motor protein FlgI. Therefore, we used E. coli mutants to check whether SynDsbAB can restored the defect in disulfide formation. 1-2 µl of cells containing SyndsbAB were spread onto M63 containing 0.3% agar and 0.4% glucose as carbon source. After 24 h incubation on minimal plates containing 0.3% agar, both ΔdsbA and ΔdsbB were non-motile. Transformation of ΔdsbA and ΔdsbB strains with SynDsbAB resulted in the restoration of motility in these strains.
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A Protein Essential under Reducing Condition

by Abhay Singh

Synechocystis 6803 lacking SynDsbAB showed significant growth impairment when compared to wild type under photoautotrophic condition under normal light intensities (30 µmol photons m^-2s^-1). Glucose can often supplement the growth defects of cyanobacterial mutant strains deficient in photosynthesis. When glucose (5 mM) was included in the growth medium, all mutant strains died rapidly. In addition, when a membrane-permeable reductant DTT (0.2 mM) was added, mutant strains were hyper-sensitive to DTT and after 24 h, all cells died. In contrast, addition of DTT had no effect on the growth of wild type Synechocystis 6803. Addition of the membrane-permeable diazine compound diamide (10 µM) restored the growth of mutant strains marginally. This is in marked contrast to mutant strains of E. coli and yeast, where addition of oxidants had appreciable improvement in the growth patterns of mutant strains defective in disulfide bond formation (A) Wild-type (square), ΔSyndsbAB (triangle), ΔSyndsbA (circle), and ΔSyndsbB (cross) under photoautotrophic conditions. (B) Growth of wild-type (square), ΔSyndsbAB (triangle), ΔSyndsbA (circle), and ΔSyndsbB (cross) under mixotrophic conditions (5 mM glucose). (C) Effect of 0.2 mM DTT and (D) 10 µM diamide on the growth of wild-type (square), ΔSyndsbAB (triangle), ΔSyndsbA (circle), and ΔSyndsbB (cross).
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