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Functional analysis of paralogous thiol-disulfide oxidoreductases in Streptococcus gordonii.

ABSTRACT: Disulfide bonds are important for the stability of many extracellular proteins, including bacterial virulence factors. Formation of these bonds is catalyzed by thiol-disulfide oxidoreductases (TDORs). Little is known about their formation in Gram-positive bacteria, particularly among facultative anaerobic Firmicutes, such as streptococci. To investigate disulfide bond formation in Streptococcus gordonii, we identified five putative TDORs from the sequenced genome. Each of the putative TDOR genes was insertionally inactivated with an erythromycin resistance cassette, and the mutants were analyzed for autolysis, extracellular DNA release, biofilm formation, bacteriocin production, and genetic competence. This analysis revealed a single TDOR, SdbA, which exhibited a pleiotropic mutant phenotype. Using an in silico analysis approach, we identified the major autolysin AtlS as a natural substrate of SdbA and showed that SdbA is critical to the formation of a disulfide bond that is required for autolytic activity. Analysis by BLAST search revealed homologs to SdbA in other Gram-positive species. This study provides the first in vivo evidence of an oxidoreductase, SdbA, that affects multiple phenotypes in a Gram-positive bacterium. SdbA shows low sequence homology to previously identified oxidoreductases, suggesting that it may belong to a different class of enzymes. Our results demonstrate that SdbA is required for disulfide bond formation in S. gordonii and indicate that this enzyme may represent a novel type of oxidoreductase in Gram-positive bacteria.

SUBMITTER: Davey L

PROVIDER: S-EPMC3675578 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Functional analysis of paralogous thiol-disulfide oxidoreductases in Streptococcus gordonii.

Davey Lauren L Ng Crystal K W CKW Halperin Scott A SA Lee Song F SF

The Journal of biological chemistry 20130424 23

Disulfide bonds are important for the stability of many extracellular proteins, including bacterial virulence factors. Formation of these bonds is catalyzed by thiol-disulfide oxidoreductases (TDORs). Little is known about their formation in Gram-positive bacteria, particularly among facultative anaerobic Firmicutes, such as streptococci. To investigate disulfide bond formation in Streptococcus gordonii, we identified five putative TDORs from the sequenced genome. Each of the putative TDOR genes ...[more]

PMID: 23615907

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Project description:We previously identified a novel thiol-disulfide oxidoreductase, SdbA, in Streptococcus gordonii that formed disulfide bonds in substrate proteins and played a role in multiple phenotypes. In this study, we used mutational, phenotypic, and biochemical approaches to identify and characterize the redox partners of SdbA. Unexpectedly, the results showed that SdbA has multiple redox partners, forming a complex oxidative protein-folding pathway. The primary redox partners of SdbA that maintain its active site in an oxidized state are a surface-exposed thioredoxin family lipoprotein called SdbB (Sgo_1171) and an integral membrane protein annotated as CcdA2. Inactivation of sdbB and ccdA2 simultaneously, but not individually, recapitulated the sdbA mutant phenotype. The sdbB-ccdA2 mutant had defects in a range of cellular processes, including autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release. AtlS, the natural substrate of SdbA produced by the sdbB-ccdA2 mutant lacked activity and an intramolecular disulfide bond. The redox state of SdbA in the sdbB-ccdA2 mutant was found to be in a reduced form and was restored when sdbB and ccdA2 were knocked back into the mutant. In addition, we showed that SdbB formed a disulfide-linked complex with SdbA in the cell. Recombinant SdbB and CcdA2 exhibited oxidase activity and reoxidized reduced SdbA in vitro Collectively, our results demonstrate that S. gordonii uses multiple redox partners for oxidative protein folding.IMPORTANCEStreptococcus gordonii is a commensal bacterium of the human dental plaque. Previously, we identified an enzyme, SdbA, that forms disulfide bonds in substrate proteins and plays a role in a number of cellular processes in S. gordonii Here, we identified the redox partners of SdbA. We showed that SdbA has multiple redox partners, SdbB and CcdA2, forming a complex oxidative protein-folding pathway. This pathway is essential for autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release in S. gordonii These cellular processes are considered to be important for the success of S. gordonii as a dental plaque organism. This is the first example of an oxidative protein-folding pathway in Gram-positive bacteria that consists of an enzyme that uses multiple redox partners to function.

Project description:UnlabelledStreptococcus gordonii is a commensal inhabitant of the human oral cavity. To maintain its presence as a major component of oral biofilms, S. gordonii secretes inhibitory molecules such as hydrogen peroxide and bacteriocins to inhibit competitors. S. gordonii produces two nonmodified bacteriocins (i.e., Sth1 and Sth2) that are regulated by the Com two-component regulatory system, which also regulates genetic competence. Previously we found that the thiol-disulfide oxidoreductase SdbA was required for bacteriocin activity; however, the role of SdbA in Com signaling was not clear. Here we demonstrate that ΔsdbA mutants lacked bacteriocin activity because the bacteriocin gene sthA was strongly repressed and the peptides were not secreted. Addition of synthetic competence-stimulating peptide to the medium reversed the phenotype, indicating that the Com pathway was functional but was not activated in the ΔsdbA mutant. Repression of bacteriocin production was mediated by the CiaRH two-component system, which was strongly upregulated in the ΔsdbA mutant, and inactivation of CiaRH restored bacteriocin production. The CiaRH-induced protease DegP was also upregulated in the ΔsdbA mutant, although it was not required for inhibition of bacteriocin production. This establishes CiaRH as a regulator of Sth bacteriocin activity and links the CiaRH and Com systems in S. gordonii. It also suggests that either SdbA or one of its substrates is an important factor in regulating activation of the CiaRH system.ImportanceStreptococcus gordonii is a noncariogenic colonizer of the human oral cavity. To be competitive in the oral biofilm, S. gordonii secretes antimicrobial peptides called bacteriocins, which inhibit closely related species. Our previous data showed that mutation of the disulfide oxidoreductase SdbA abolished bacteriocin production. In this study, we show that mutation of SdbA generates a signal that upregulates the CiaRH two-component system, which in turn downregulates a second two-component system, Com, which regulates bacteriocin expression. Our data show that these systems are also linked in S. gordonii, and the data reveal that the cell's ability to form disulfide bonds is sensed by the CiaRH system.

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.

Dataset Information

Functional analysis of paralogous thiol-disulfide oxidoreductases in Streptococcus gordonii.

Publications

Functional analysis of paralogous thiol-disulfide oxidoreductases in Streptococcus gordonii.

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