and ,0.001 respectively). The difference between old cells in the wild type and L lineages was also significant (p = 0.04). Current concentration of medium did not show a significant difference although the difference was in the expected direction (all h pooled 0.033, all l pooled 0.019, p.0.05). Comparing the three strains, strain 2 (all selection-treatment pooled) had significantly greater proportion of old cells than the other strains (Monte Carlo method for comparison of more than 2 proportions, frequencies of old cells 0.010, 0.0455 and 0.023 respectively for strain 1, 2 and 3, p = 0.001). Effect of selection on cell morphology It can be argued that the cell division time asymmetry may be a result of stochastic morphological asymmetry. If the two daughter cells have a difference in cell length, the shorter of the two cells may take a longer time to grow and undergo next division. We therefore examined morphological asymmetry and its relation to time asymmetry. Morphological asymmetry index was defined as the difference in the lengths of the two sister cells divided by pre- division length of the mother cell. For all the three strains with and without selection, the division was morphologically more symmet- rical in low nutrient concentration than in high (Dataset S2). However there was no significant correlation between morpho- logical asymmetry and division time asymmetry indicating that morphological asymmetry was unlikely to be the cause of division time asymmetry. As a result of selection cell lengths significantly increased in H1 (p,0.0001), H2 (p = 0.002) and H3 (p,0.0001) whereas they remained indistinguishable from the wild types in L1, L2 and L3. Growth rates and growth yields As the Watve et al [10] Model predicted, asymmetric division is expected to increase growth rates and symmetric cell division may have an advantage of higher biomass conversion rates. Although the selection and monitoring of cell division symmetries was done on agar media, owing to the difficulty of calculating growth yields on agar media the growth rate and growth yield estimations were performed in shake flask cultures in liquid media. All the three wild type strains gave higher growth yields in l as compared to h but the growth rates were indistinguishable for two of the three strains. For strain 1 growth rate was higher in h as compared to l. In two of the three strains the growth rates increased as a result of selection both under low and high glucose concentration. In strain 1 the growth rate increased during selection in low substrate concentration but decreased in high substrate concentration. The correlation between index of asymmetry and growth yield for all the selected and wild type lines of all the three strains in both the nutrient conditions was found to be significantly negative in agreement with the model prediction (Figure 3) (t = 20.320, p = 0.031). However contrary to the model prediction no significant correlation was seen in the index of asymmetry and growth rates (Figure 4). Also no significant negative correlation between growth rates and growth yields was found (Figure 5) unlike what the growth rate growth yield trade-off [14,15] predicts. Cell polarity and division time asymmetry To check if morphological old pole cell is also functionally old in all the lineages, we compared the relative time taken by the old pole cell and the new pole cell for the subsequent division. In a pair wise comparison pooling over all conditions only in strain 2 there was significant difference in the division time of the old pole cell as compared to the corresponding new pole cell (paired t-test one tailed: n = 297, n1mean = 1.0147, n2mean = 0.9853, p = 0.032). Bacterial Division Symmetry Figure 3. Growth yield and index of division time asymmetry. Significant negative correlation was seen between growth yields and indices of asymmetry in the pooled data (t = 20.320, p = 0.031). Wild types are shown in red, H in blue and L in green. Crosses indicate low current concentration and solid circles high current concentration. doi:10.1371/journal.pone.0014516.g003 Segregating data according to treatments, in 12 out of 18 strain- treatment combinations, old poles cells divided slower than the new pole cells but only four of the differences were significant (W1l: n = 33, n1mean = 1.026,n2mean = 0.974, p = 0.04; W2h: n = 51, n1mean = 1.047,n2mean = 0.953, p = 0.036; W2l: n = 34, n1mean = 1.021, n2mean = 0.979, p = 0.045; L3h: n = 50, n1mean = 1.026, n2mean = 0.974, p = 0.032). In the remaining 6 a reversed pattern was seen and only one of the differences was significant (W3h: n = 42, n1 mean = 0.968, n2mean = 1.032, p = 0.03). These results indicate that the association of old pole with aging was only weakly and inconsistently significant in these lineages. Discussion Is cellular aging inevitable in E. coli? Our experiments demonstrate that asymmetry in cell division is not hard wired but is responsive to environmental conditions. The wild types showed phenotypic plasticity in cell division symmetry Figure 4. Cell division time asymmetry index and growth rate. No significant correlation was seen between cell division time asymmetry indices and growth rates. (t = 0.210, p = 0.112). Colour codes same as figure 3. doi:10.1371/journal.pone.0014516.g004 PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e14516