Table 1.
Strains and plasmids used in this study.
Table 2.
Primers used in this study.
Figure 1.
Biofilm formation of oxidative stress resistance mutants.
Biofilm formation was measured using crystal violet staining. The results show the means and standard deviations of a representative assay with triplicate samples. The experiment was repeated six times and all produced similar results. Statistical significance was analyzed using one-way analysis of variance (ANOVA). *P≤0.05, **P≤0.01, ****P≤0.0001.
Figure 2.
Effect of increased ahpC expression on biofilm formation.
Biofilm formation levels were determined with crystal violet staining after 24 h incubation of samples. The results show the means and standard deviations of a representative experiment with triplicate samples. The experiment was repeated six times and all produced similar results. The statistical differences between the wild type and each mutant were determined by t-test. NS: non-significant, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
Figure 3.
Determination of total ROS level (A) and lipid hydroperoxide (LPO; B) in the ahpC mutant, and reduced biofilm formation in the ahpC mutant by treatment with antioxidants (C).
The assays were carried out with 24 h old samples. Antioxidants were treated to a final concentration of 1nM. The results show the means and standard deviations of a representative experiment with triplicate samples. The assays were repeated at least three times and similar results were reproducible in all the experiments. Statistical significance was analyzed with t-test (Fig. 3A and 3B) and two-way ANOVA (Fig. 3C). *P≤0.05, ****P≤0.0001.
Figure 4.
Confocal microscopy analysis of biofilms of the wild type (A), the ahpC mutant (B), and the complementation strain (C).
Biofilms were grown 24 h and stained with the LIVE/DEAD Biofilm Viability kit (Life Technologies, US).