Bacterial viability and oxidative stress
Plating conditions generate an oxidative stress
We have investigated the first events that occur when exponentially grown cells are transferred from a liquid medium (Luria-Bertani [LB]) to a solid medium (LB agar [LBA]). We observed an initial lag phase of 180 min for the wild type MG1655 without any apparent growth. This lack of growth was independent of the bacterial physiological state (either the stationary or the exponential phase), the solid medium composition, or the number of cells on the plate, but it was dependent on the bacterial genotype. Using lacZ-reporter fusions and two-dimensional electrophoresis analysis, we observed that when cells from exponential-phase cultures were plated on LBA, several global regulons, like heat shock regulons (RpoH, RpoE, CpxAR) and oxidative-stress regulons (SoxRS, OxyR, Fur), were immediately induced. Our results indicate that in order to grow on plates, bacteria must not only adapt to new conditions but also perceive a real stress (Cuny et al., 2007). This stress might have deleterious effects on cells and could explain at least in part the phenomenon of VBNC cells. Results support the idea that TiO2 photocatalysis generates damage which later becomes deleterious during recovery on plates. We found this to be partly due to DNA attack via hydroxyl radicals generated by the Fenton reaction during recovery (Gogniat and Dukan, 2007). In summary, oxidative stress performed by cells during the recovery period may explain partially the presence of VBNC cells after hypochlorous acid exposure (Dukan et al., 1997 ; 1999), TiO2 photocatalysis exposure (Gogniat et al., 2006) or the unability to form colonies on plate of the oxyR mutant during stasis (Dukan and Nystrom, 1999).
Global expression monitoring by 2D gel analysis. MC4100 cells were grown in M9 + 2% glucose and sampled in exponential phase (OD600=0.5) and plate on M9A 2 % glucose containing methionine 35S on the surface. After 10 minutes of incorporation in liquid (A) or on plate (B) cells were collected and 2D analysis performed. (A) repressed proteins in liquid compared to solid “circle”, (B) induced proteins in solid compare to liquid “circle”.