Fig 1.
Phylogenetic tree of Dsb catalytic domains.
Background colors indicate families: HP0231-like and C8J_1298-like (violet and cyan, respectively), DsbG (green), and DsbC (wheat). Each family is accompanied by a sequence logo representing typical residues in CXXC and XcP motifs (please note that the logos were calculated based on larger data sets). Numbers near branches denote support values. Representative proteins mentioned in the text are indicated with red dots.
Fig 2.
Biochemical features of C8J_1298 resemble those of EcDsbC.
Purified EcDsbA and/or EcDsbC were used as controls. (A) Insulin reduction assay. The reaction was performed in the absence (black) or presence of 10 μM of each protein: EcDsbA (blue), EcDsbC (green), C8J_1298 (red). The data presented is the average of three independent experiments, with two technical repetitions (n = 3). (B) In vitro C8J_1298 oxidative activity assay using reduced unfolded RNase A (ruRnase A). (C) In vitro C8J_1298 isomerase activity assay using scrambled RNase A (scRnase A). (D) Determination of the oligomeric state of C8J_1298 versions using glutaraldehyde. Purified C8J_1298 was cross-linked in the presence of different concentration of glutaraldehyde. Figs B, C, D present representative result based on three independent experiments (n = 3).
Fig 3.
Redox state of C8J_1298 in various strains of C. jejuni 81116.
(A) Strains lacking membrane Dsb proteins: ΔdsbD (AB2), ΔdsbI (AG4) and ΔdsbB (AG3). (B) Strain lacking CjDsbA1 (KBO1). Bacterial cultures were treated with 10% TCA, followed by alkylation with AMS. Cellular proteins including the reduced (red; DTT treated, modified with AMS) and the oxidized (ox; non-modified with AMS) wild type controls were separated by 14% SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against C8J_1298 was performed. Each lane contains proteins isolated from the same amount of bacteria. (C) Table and (D) plot presents equlibrium beetwen reduced and oxidized forms of C8J_1298 in various C. jejuni 81116 strains. Proportion of reduced and oxidized forms of protein was estimated using ImageLab™ (Bio-Rad). Result is the average of three independent experiments, with two technical repetitions (n = 3). The figure presents a representative result.
Fig 4.
Construction of ΔdsbA1, ΔdsbA1Δc8j_1298 deficient strains and double mutant complemented strains (ΔdsbA1Δc8j_1298/chrA1, ΔdsbA1Δc8j_1298/chr1298).
C. jejuni proteins (whole cell lysates) were separated by 12% SDS-PAGE and electrotransferred onto nitrocellulose membrane. Specific rabbit sera with antibodies against (A) CjDsbA1 or (B) C8J_1298 were used to verify the absence or presence CjDsbA1 or C8J_1298 in C. jejuni 81116 cells, respectively.
Fig 5.
Impact of the lack of C8J_1298 on various cell physiological properties measured by spot-titer method.
C. jejuni 81116 strains in exponential growth phase were 10-fold diluted and spotted on BA plates with DTT (A), CdCl2 (B) or CuCl2 (C). (D) Viability assay after exposure on hydrogen peroxide was performed with C. jejuni 81116 strains. Bacterial cultures in exponential growth phase were incubated with hydrogen peroxide one hour, then 10-fold diluted and spotted on BA plates. Three independent experiments with two technical repetitions were performed (n = 3). The figures present a representative result.
Fig 6.
C8J_1298 activity in E. coli dsb deficient strains.
(A) Motility assay: Exponentially growing E. coli strains were spotted on soft agar plates and incubated 24h at 37°C. C8J_1298 restored motility in both dsbA and dsbAdsbB deficient strains Table and plot present diameter of growth on soft agar plates of tested strains. Presented average is the result of three independent experiments (n = 3). (B) Copper sensitivity assay: Exponentially growing E. coli strains were 10-fold diluted and spotted on BHI-agar plates supplemented with arabinose and CuCl2. Presence of C8J_1298 only slightly increased resistance to copper toxicity in E. coli dsbC lacking strain. (C) Mucoid phenotype assay: E. coli strains were cultivated on M63 minimal medium. C8J_1298 restored mucoid phenotype in mdoG deficient E. coli strain. In all assays three independent experiments were performed (n = 3) and bacterial cultures growing without arabinose supplementation were used as controls. The figures present a representative result.
Fig 7.
Redox state of C8J_1298 in (A) E. coli strains deficient in proteins of oxidative Dsb pathway. (B) E. coli strain deficient in EcDsbC protein Bacterial cultures were treated with 10% TCA, followed by alkylation with AMS. Cellular proteins including the reduced (red; DTT treated, modified with AMS) and the oxidized (ox; non-modified with AMS) wild type controls were separated by 14% SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against C8J_1298 was performed. Each lane contains proteins isolated from the same amount of bacteria. (C) Table and (D) plot presents equlibrium beetwen reduced and oxidized forms of C8J_1298 in various E. coli strains. Proportion of reduced and oxidized forms of protein was estimated using ImageLab™ (Bio-Rad). Result is the average of three independent experiments (n = 3). (*) unspecific serum reaction. The figure presents a representative result.
Table 1.
Strains used in this study.
Table 2.
Plasmids used in this study.