Summary auto-generated
This study investigates the role of disulfide bond formation in Erwinia carotovora by isolating and characterizing dsbA and dsbC genes from two subspecies. The researchers created mutants deficient in these genes and analyzed their effects on exoenzyme secretion, activity, and virulence. The dsbA mutants showed reduced activity and secretion of pectate lyase (PelC) and endopolygalacturonase (Peh), both of which require disulfide bonds for proper folding. However, cellulase (CelV), which lacks cysteine residues, was still secreted normally in dsbA mutants and showed unexpected two- to threefold increased activity through transcriptional upregulation. Protease secretion remained unaffected in the dsbA mutant. The Eca dsbA mutant was non-motile, suggesting disulfide formation is essential for motility in this strain. All dsb mutants exhibited reduced virulence in potato tuber assays. These results suggest a periplasmic stress signal transduction system exists that responds to defects in disulfide bond formation and regulates transcription of genes encoding secreted proteins through a feedback mechanism.
Key findings
- DsbA and DsbC proteins are required for proper folding and secretion of disulfide-bond-dependent exoenzymes like pectate lyase and endopolygalacturonase in E. carotovora
- Cellulase (CelV), which lacks cysteine residues, is secreted normally in dsbA mutants but shows increased transcriptional expression, indicating the secretion apparatus functions independently of disulfide bond formation
- DsbA is essential for motility in E. carotovora subsp. atroseptica but not in E. carotovora subsp. carotovora
- Disulfide bond defects trigger a feedback regulation system that downregulates transcription of pectinase genes while upregulating cellulase genes
- All dsb mutants display significantly reduced virulence in potato tuber infections
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Abstract
The dsbA genes, which encode major periplasmic disulfide-bond-forming proteins, were isolated from Erwinia carotovora subsp. carotovora (Ecc) and Erwinia carotovora subsp. atroseptica (Eca), and the dsbC gene, encoding another periplasmic disulfide oxidoreductase was isolated from Ecc. All three genes were sequenced and mutants deficient in these genes were created by marker exchange mutagenesis. The Ecc mutants were severely affected in activity and secretion of pectate lyase, probably due to the absence of functional PelC, which is predicted to require disulfide bond formation to achieve its correct conformation prior to secretion across the outer membrane. Similarly, endopolygalacturonase, also predicted to possess disulfide bonds, displayed reduced activity. The major Ecc cellulase (CelV) does not contain cysteine residues and was still secreted in dsbA-deficient strains. This observation demonstrated unequivocally that the localization and activity of the individual components of the Out apparatus are independent of disulfide bond formation. Surprisingly, cellulase activity was shown to be increased approx. two- to threefold in the DsbA mutant. This phenomenon resulted from transcriptional up-regulation of celV gene expression. In contrast, transcription of both pelC and peh were down-regulated in dsbA-deficient strains when compared to the wild-type. Protease (Prt) activity and secretion were unaffected in the Ecc dsbA mutant. Prt activity was considerably reduced in the double dsbA dsbC mutant. However Prt was secreted normally in this strain. The Eca dsbA mutant was found to be non-motile, suggesting that disulfide bond formation is essential for motility in this strain. All of the dsb mutants showed reduced tissue maceration in planta. These results suggest that a feedback regulation system operates in Ecc. In this system, defects in periplasmic disulfide bond formation act as a signal which is relayed to the transcription machinery regulating gene expression in diverse ways.