Figure 4
Survival and P rpoS activity in P . putida KT2440 and its Δ ppk and Δ ppx mutant derivatives. (A) Propidium iodide (PI) test to estimate cell viability in P. putida KT2440 (wt) and the Δppk and Δppx mutants. Stationary-phase cell suspensions grown on M9 minimal medium with either 0.2% (w/v) glucose or succinate were stained with PI and analysed by flow cytometry as detailed in the Methods section. Box plots represent the median value and the 1st and 3rd quartiles of the geometric mean values of quadruplicate determinations from three independent cultures, and asterisks identify significant differences at the P < 0.05 (*) or P < 0.01 (**) levels as assessed with the Mann–Whitney U test. (B) The ability of P. putida KT2440 (wt) and the Δppk and Δppx mutants to form colonies when transferred into fresh medium was evaluated by plating appropriate dilutions of the stationary-phase cell suspensions onto LB plates. Bars represent the mean value ± SD of three measurements from at least three independent experiments, and the asterisk identifies a significant difference at the P < 0.05 level (ANOVA). (C) Scheme of the relevant elements of the P rpoS translational fusion borne by plasmid pMCH4. The T0 transcriptional terminator from phage λ is denoted as T. Elements in this outline are not drawn to scale. (D) Expression of the P rpoS → rpoS‘-’lacZ translational fusion (β-galactosidase activity) monitored in P. putida KT2440 (wt) and the Δppk and Δppx mutants. Cells were grown on M9 minimal medium with 0.2% (w/v) glucose and 150 μg/ml kanamycin, and harvested during exponential growth (E) or in the stationary phase (S). Bars represent the mean value of the reporter activity ± SD of duplicate measurements from at least three independent experiments. Asterisks identify significant differences at the P < 0.05 level (ANOVA).