Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used in this study.
Figure 1.
Redox state of HP0231 in WT, mutants: dsbI::aph, hp0231::cat and complemented strains.
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) controls were separated by 18% SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against HP0231 was performed. Each lane contains proteins isolated from the same amount of bacteria.
Figure 2.
The H. pylori N6 hp0231::cat mutant is DTT sensitive.
Exponentially growing H. pylori wt and hp0231::cat cultures were ten-fold serial diluted and spotted on BA plates without (panel A) or with (panel B) 8 mM DTT, and incubated at 37°C. The mutant shows reduced growth after 3 days of incubation on plates containing DTT.
Figure 3.
Motility of H. pylori N6 strains: wt, hp0231::cat and hp0231::cat complemented in trans by puWM397 (hp0231+).
Bacterial motility was monitored after 4 days of incubation on 0.3% MH-agar plates containing 10% FCS. The hp0231 mutant strain is non-motile.
Figure 4.
Comparison of H. pylori N6 cells (wt, hp0231::cat and hp0231::cat complemented in trans by puWM397 (pHel2/hp0231) morphology by transmission electron microscopy (TEM).
A) WT, B) hp0231::cat and C) hp0231::cat/hp0231+ (hp0231::cat complemented in trans by wild type copy of hp0231). D) The diagram illustrates mean lengths and standard deviations of 100 bacterial cells in nm. Error bars with the different letters indicate a significant difference (p<0.01) in length between the H. pylori N6 wt cells and the mutant strain, and the mutant and the complementant strain (Welch ANOVA followed by post hoc Tukey's test).
Figure 5.
HP0231 restores the E. coli dsbA− wild type phenotype in three independent functional assays.
As a negative control E. coli dsbA::aph was transformed with an empty pHEL2 vector. Panel A DTT sensitivity assay; panel B alkaline phosphatase assay. The bars represent average activity of three independent experiments with wild type set to 100% activity; error bars with the different letters indicate a significant difference (p<0.01) in relative alkaline phosphatase activity between the H. pylori N6 wt cells and the mutant strains, and the complementant strains (in ANOVA followed by post hoc Tukey's test). Alkaline phosphatase activity of wild type and dsb mutants and complementants strains was performed in M63 minimal media; Panel C motility assay.
Figure 6.
The reaction mixture contained 150 µM insulin in potassium phosphate buffer, pH 7.0 and 2 mM EDTA. The reaction was performed in the absence () or presence of 10 µM EcDsbA (
), 10 µM HP0231 (
). Reactions started by adding DTT to the final concentration of 1 mM. The changes in the absorbance at 650 nm as a function of time were measured. Three independent experiments were performed.
Figure 7.
Redox equilibrium of H. pylori HP0231 with glutathione.
The fraction of reduced (R) HP0231 was determined using the specific HP0231 fluorescence at 324 nm.