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Bacterial viability and oxidative stress
Protein aggregation and recovery on plate
Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. We demonstrated the presence of protein aggregates in an exponentially grown E. coli culture. Our results led us to speculate that protein aggregates may function as a temporary “trash organelle” for cellular detoxification (Maisonneuve et al., 2008a).
Silver-stained 2D gel analysis of proteins in pellets and supernatant. Panels show protein patterns in LP, SP, ILP, ISP, and supernatant obtained after 2D gel electrophoresis and visualized by silver staining.
In light of these observations, protein aggregates could be considered damage to cells that is able to pass from one generation to the next. Based on the assumption that the amount of aggregate protein could represent an aging factor, we monitored this amount in a bacterial culture during senescence. In doing so, we observed (i) a significant increase in the amount of aggregate protein over time,
Aggregate protein accumulates over time. (A) Coomassie-stained SDS/PAGE gel showing the relative amount of aggregate protein (per mg of soluble protein) at three time points during exponential (2, 4 and 6 hours) or stationary phase (10, 24 and 48 hours). At each point, samples were prepared from equal OD amounts of cells. Panel (B) shows representative results from relative aggregate content for each time during exponential (2, 4 and 6 hours) or stationary phase (10, 24, 30, 36 and 48 hours) quantified using Quantity One software (BioRad).
(ii) a proportional relationship between the amount of aggregate protein and the level of VBNC cells, (iii) a larger amount in VBNC cells than in culturable cells, (iv) a heterogeneous distribution of different amounts within a homogenous population of culturable cells entering stasis, and (v) that the initial amount of aggregate protein within a culturable population conditioned the VBNC cell rate of the culture. Together, the results presented in this study suggest that protein aggregates represent one aging factor leading to VBNC cell formation (Maisonneuve et al., 2008b). For this study, we got a highlight in Microbes (nov. 2008).
(A) Determination of reproductive ability (CFU) and cell integrity in the Low Density (LD, shaded bar) and High Density (HD, unshaded bar) cell populations from a 48 hours stationary-phase culture. (B) Coomassie-stained SDS/PAGE gel showing the relative amount of aggregate protein (per mg of soluble protein) from LD and HD cells prepared from an equal OD amount of cells. (C) Relative aggregate amount from LD an HD cells quantified using Quantity One software (BioRad). (D) Determination of reproductive ability (CFU) and cell integrity in the Low Density (LD, shaded bar) and High Density (HD, unshaded bar) cell populations from a 10 hours culture. (E) Coomassie-stained SDS/PAGE gel showing the relative amount of aggregate protein (per mg of soluble protein) from LD and HD cells prepared from an equal OD amount of cells. (F) Relative aggregate amount from LD an HD cells quantified using Quantity One software (BioRad).
