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Overproduction or absence of the periplasmic protease DegP severely compromises bacterial growth in the absence of the dithiol: disulfide oxidoreductase DsbA.

ABSTRACT: Facultative phototrophic bacterium Rhodobacter capsulatus DsbA-null mutants are proficient in photosynthesis but are defective in respiration especially in enriched growth medium at 35 degrees C. They also exhibit severe pleiotropic phenotypes extending from motility defects to osmofragility and oxidative stresses. In this work, using a combined proteomics and molecular genetics approach, we demonstrated that the respiratory defect of R. capsulatus DsbA-null mutants originates from the overproduction of the periplasmic protease DegP, which renders them temperature-sensitive for growth. The DsbA-null mutants reverted frequently to overcome this growth defect by decreasing, but not completely eliminating, their DegP activity. In agreement with these findings, we showed that overproduction of DegP abolishes the newly restored respiratory growth ability of the revertants in all growth media. Structural localizations of the reversion mutations in DegP revealed the regions and amino acids that are important for its protease-chaperone activity. Remarkably although R. capsulatus DsbA-null or DegP-null mutants were viable, DegP-null DsbA-null double mutants were lethal at all growth temperatures. This is unlike Escherichia coli, and it indicates that in the absence of DsbA some DegP activity is required for survival of R. capsulatus. Absence of a DegQ protease homologue in some bacteria together with major structural variations among the DegP homologues, including a critical disulfide bond-bearing region, correlates well with the differences seen between various species like R. capsulatus and E. coli. Our findings illustrate the occurrence of two related but distinct periplasmic protease families in bacterial species.

SUBMITTER: Onder O

PROVIDER: S-EPMC2401338 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Overproduction or absence of the periplasmic protease DegP severely compromises bacterial growth in the absence of the dithiol: disulfide oxidoreductase DsbA.

Onder Ozlem O Turkarslan Serdar S Sun David D Daldal Fevzi F

Molecular & cellular proteomics : MCP 20080102 5

Facultative phototrophic bacterium Rhodobacter capsulatus DsbA-null mutants are proficient in photosynthesis but are defective in respiration especially in enriched growth medium at 35 degrees C. They also exhibit severe pleiotropic phenotypes extending from motility defects to osmofragility and oxidative stresses. In this work, using a combined proteomics and molecular genetics approach, we demonstrated that the respiratory defect of R. capsulatus DsbA-null mutants originates from the overprodu ...[more]

PMID: 18174153

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Project description:BackgroundBacterial Dsb enzymes are involved in the oxidative folding of many proteins, through the formation of disulfide bonds between their cysteine residues. The Dsb protein network has been well characterized in cells of the model microorganism Escherichia coli. To gain insight into the functioning of the Dsb system in epsilon-Proteobacteria, where it plays an important role in the colonization process, we studied two homologs of the main Escherichia coli Dsb oxidase (EcDsbA) that are present in the cells of the enteric pathogen Campylobacter jejuni, the most frequently reported bacterial cause of human enteritis in the world.Methods and resultsPhylogenetic analysis suggests the horizontal transfer of the epsilon-Proteobacterial DsbAs from a common ancestor to gamma-Proteobacteria, which then gave rise to the DsbL lineage. Phenotype and enzymatic assays suggest that the two C. jejuni DsbAs play different roles in bacterial cells and have divergent substrate spectra. CjDsbA1 is essential for the motility and autoagglutination phenotypes, while CjDsbA2 has no impact on those processes. CjDsbA1 plays a critical role in the oxidative folding that ensures the activity of alkaline phosphatase CjPhoX, whereas CjDsbA2 is crucial for the activity of arylsulfotransferase CjAstA, encoded within the dsbA2-dsbB-astA operon.ConclusionsOur results show that CjDsbA1 is the primary thiol-oxidoreductase affecting life processes associated with bacterial spread and host colonization, as well as ensuring the oxidative folding of particular protein substrates. In contrast, CjDsbA2 activity does not affect the same processes and so far its oxidative folding activity has been demonstrated for one substrate, arylsulfotransferase CjAstA. The results suggest the cooperation between CjDsbA2 and CjDsbB. In the case of the CjDsbA1, this cooperation is not exclusive and there is probably another protein to be identified in C. jejuni cells that acts to re-oxidize CjDsbA1. Altogether the data presented here constitute the considerable insight to the Epsilonproteobacterial Dsb systems, which have been poorly understood so far.

Project description:Chlamydia trachomatis is an obligate intracellular bacterium with a distinctive biphasic developmental cycle that alternates between two distinct cell types; the extracellular infectious elementary body (EB) and the intracellular replicating reticulate body (RB). Members of the genus Chlamydia are dependent on the formation and degradation of protein disulfide bonds. Moreover, disulfide cross-linking of EB envelope proteins is critical for the infection phase of the developmental cycle. We have identified in C. trachomatis a homologue of the Disulfide Bond forming membrane protein Escherichia coli (E. coli) DsbB (hereafter named CtDsbB) and-using recombinant purified proteins-demonstrated that it is the redox partner of the previously characterised periplasmic oxidase C. trachomatis Disulfide Bond protein A (CtDsbA). CtDsbA protein was detected in C. trachomatis inclusion vacuoles at 20 h post infection, with more detected at 32 and similar levels at 44 h post infection as the developmental cycle proceeds. As a redox pair, CtDsbA and CtDsbB largely resemble their homologous counterparts in E. coli; CtDsbA is directly oxidised by CtDsbB, in a reaction in which both periplasmic cysteine pairs of CtDsbB are required for complete activity. In our hands, this reaction is slow relative to that observed for E. coli equivalents, although this may reflect a non-native expression system and use of a surrogate quinone cofactor. CtDsbA has a second non-catalytic disulfide bond, which has a small stabilising effect on the protein's thermal stability, but which does not appear to influence the interaction of CtDsbA with its partner protein CtDsbB. Expression of CtDsbA during the RB replicative phase and during RB to EB differentiation coincided with the oxidation of the chlamydial outer membrane complex (COMC). Together with our demonstration of an active redox pairing, our findings suggest a potential role for CtDsbA and CtDsbB in the critical disulfide bond formation step in the highly regulated development cycle.

Dataset Information

Overproduction or absence of the periplasmic protease DegP severely compromises bacterial growth in the absence of the dithiol: disulfide oxidoreductase DsbA.

Publications

Overproduction or absence of the periplasmic protease DegP severely compromises bacterial growth in the absence of the dithiol: disulfide oxidoreductase DsbA.

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