Mechanisms of asymmetric segregation Our studies do not address the question of how asymmetric segregation is achieved. However, considerable progress has been made in understanding how damaged proteins are asymmetrically segregated in daughter cells and protein aggregation appears to be one of the mechanisms of cell division asymmetry [5–7]. If protein aggregation is central to bacterial aging, the process of aggregation should also be flexible and responsive to environmental stimuli. Although protein aggregation has not been studied under variable nutritional environments, flexibility of the process in terms of reversibility and disaggregation has been demonstrated [26]. The Heat shock proteins play a role in protein aggregation and the expression of HSPs is known to be different in the stationary phase [27] as compared to the growth phase raising the possibility that protein aggregation may be responsive to the nutritive environ- ment. There are other possible mechanisms too. Polyphosphate accumulation in response to nutrient stress has been shown to affect polar localization of proteins and this relocation response may bring greater symmetry in component distributions on starvation [28]. On the other hand it is questionable whether polar localization is central to aging in bacteria. In rod shaped bacteria including E. coli the poles and polarisation of protein aggregates may play an important role. But spherical bacteria that divide in more than one plane do not have distinct poles. If poles are indeed central to cellular aging, cocci should be immune to aging [13]. Currently there is no published data on cell division symmetry or aging in spherical bacteria. Our preliminary observations show cell division asymmetry in staphylococci (data not shown). Even in E. coli protein aggregates are not always associated with the old pole [5,7]. Unlike Stewart et al [4] and Lindner et al [5], we find only marginal association of poles with cell division times. It is possible that the extent of aggregation rather than polarity plays a major role in asymmetric segregation and the extent of association of aggregates with old poles may be different in different strains. An important issue in the context of selection is whether there is a cost involved in mechanisms of protein aggregation or aggregation is an entropy-driven process and its prevention or disaggregation needs extra cost. Contradictory claims have been made regarding the costs of aggregation and disaggregation [7,26]. The cost will certainly influence the direction and strength of selective forces. Our finding that commitment to asymmetric division appears faster and more consistent suggests that there could be greater cost in symmetric division so that mutants that lack this costly facility rapidly get selected when it is not needed. It is necessary at this stage that research on the proximate causes (mechanisms) of asymmetric division and ultimate causes (differential selective advantages in different conditions) follow a coherent path. Materials and Methods Media compositions Composition of Glucose mineral medium: Na2HPO4 0.1 gm%, KNO3 0.3 gm%, (NH4)2SO4 0.05 gm%, NaCl 0.05 gm%. Glucose: For high concentration medium: 10 mg/ml; for dilute medium: 0.1 mg/ml. All media were prepared in distilled water and pH was adjusted to 7 and sterilized by autoclaving at 115uC for 20 minutes. For the observations of cell division slides of the media mentioned above were used with 2% agar. All incubations as well as microscopy was done at 28uC. Determination of growth rates and growth yields Growth rates and growth yields were determined in liquid culture in triplicates as follows. Growth rates were calculated in Bacterial Division Symmetry shake flask cultures based on the difference in the log absorbance per unit time at mid-exponential growth phase when the slope of the growth curve was maximum. Growth yields were calculated by harvesting cells by centrifugation from 40 ml broth at the beginning of stationary phase. Total cell protein was estimated by Folin-Lowry method and initial and residual glucose was estimated by using Di-nitro Salicylic acid method. Growth yield was expressed as total cell protein produced per unit of glucose consumed. Selection under high and low nutrient concentrations Continuous subculturing of all the cell lineages was done on agar plates with media composition mentioned above. An advantage of using solid medium was that any contaminant could be easily detected and avoided. The subculturing was done from 10–15 colonies. The number of generations per subculturing was estimated from the estimated mean number of cells per colony. On high nutrient concentration the estimated number of generations was 22 and on low nutrient concentration 19 to 20. For convenience of inoculation, one subculture prior to microscopy was done in liquid medium of the same composition. After 2000 generations of subculturing the 16S rDNA sequences of all lineages were matched with the wild types to confirm the identity. Microscopy and cell division observations Photomicrography was used to observe cell division and developing microcolonies. A broth culture in its mid-exponential phase was poured on a sterile slide coated with agar medium with the same composition as the broth. Although the nutrient composition in broth and on the slide was the same and the cells were in mid exponential phase, a short lag after transferring from broth to slide was observed presumably owing to transfer from a liquid to a solid surface. After the lag subsequent divisions and microcolony development could be monitored by time lapse photomicrography. Time-lapse microscopy and imaging was performed to observe cell divisions in developing micro-colonies on Zeiss Imager (Axio Imager Z1) microscope with the magnification of 636 OEL objective and Differential Interference Contrast (DIC) technique in transmitted light till the microcolonies reached 16 to 32 cells stage, i e. 4 to 5 generations (Figure S1). For all the samples cell division time asymmetry indices on a minimum sample size of 45 pairs of sister cells with a maximum of 8 from a single microcolony were recorded. Cell lengths were measured using ImageJ software calibrated for Axio-Vision microscopy software. Experimental design Three strains of E. coli (E. coli ATCC 113-3D, E. coli KL16 and E. coli 2563 obtained from National Collection of Industrial Microorganisms, NCL Pune, referred as strains 1, 2 and 3 respectively) were grown in liquid culture at high (10 mg/ml) and low (0.1 mg/ml) glucose concentrations and at mid-exponential growth phase used to inoculate slides for time-lapse microscopy as described above. The experiment was performed in four replicates and the index of division time asymmetry under high and low glucose concentrations was calculated. Each strain was subjected to the selection procedure as mentioned above on high and low nutrient concentrations simultaneously. At estimated 1000 and 2000 generations of selection, the selected strains were again tested for index of division time asymmetry on both the nutrient concentrations. At the end of 2000 generations growth rate and growth yield were determined again on both the nutrient concentrations. PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e14516