In contrast, the viable cell counts of the rpoN mutant continued to reduce during the whole period of culture (Figure 1B), suggesting that the rpoN mutation resulted in survival defects. The survival defect of the rpoN mutant in the static culture was observed
at both 37°C and 42°C (data not shown). These results show that RpoN affects the survival of C. jejuni under aeration-limited static culture conditions. Figure 1 Growth of the rpoN mutant under different aeration conditions. The C. jejuni strains were microaerobically cultured in MH broth at 42°C with shaking at 180 rpm (A) and without shaking (B). At the described time intervals, the optical density at 600 nm was measured, and viable cells were counted in static culture condition LCZ696 (without GDC941 shaking) by plating serially-diluted C. jejuni cultures on MH agar plates. The results are the mean ± standard deviation of three independent PI3K inhibitor experiments. ***: P < 0.001; the significance of results was statistically analyzed by two-way ANOVA analysis of variance with Bonferroni's post-tests at a 95% confidence interval using Prism software (version 5.01; GraphPad Software Inc., USA). Susceptibility of the rpoN mutant to osmotic stress Due to the hypersensitivity of Campylobacter to various osmolytes [34, 35], NaCl was used as an osmolyte to investigate the susceptibility of the
rpoN mutant to osmotic stress in this study. When grown on Mueller-Hinton (MH) agar plates containing a high concentration (0.8%) of NaCl, the rpoN mutant exhibited significant growth defects (Figure 2A). The colony size of the rpoN mutant on MH agar plates was extremely small even after incubation for two days compared to the wild type (data not shown), suggesting the rpoN mutant suffers
more osmotic stress than the wild type under the same stress condition. We used transmission electron microscopy (TEM) to investigate bacterial morphology under the osmotic stress. Interestingly, 79.3 ± 9.0% of rpoN mutant cells were abnormally elongated after exposure to osmotic stress. The rpoN mutant was approximately several times longer (approximately > 5 μm) than the wild type in the presence of 0.8% NaCl, MG-132 clinical trial and the morphological change in the rpoN mutant was restored by complementation (Figure 2B). Figure 2 Changes in viability and morphology under osmotic stress. (A) Viable cell counts of the rpoN mutant on MH agar pates containing 0.8% NaCl after incubation for 24 hr. Results are expressed as the mean ± standard deviation of three independent experiments. ***: P < 0.001; the significance of results was statistically analyzed by one-way ANOVA analysis of variance with Dunnett test at a 99.9% confidence intervals using Prism software (version 5.01; GraphPad Software Inc.).