Changes in Cell Size and Shape during 50,000 Generations of Experimental Evolution with Escherichia coli

Abstract

Bacteria adopt a wide variety of sizes and shapes, with many species exhibiting stereotypical morphologies. How morphology changes, and over what timescales, is less clear. Previous work examining cell morphology in an experiment with Escherichia coli showed that populations evolved larger cells and, in some cases, cells that were less rod-like. That experiment has now run for over two more decades. Meanwhile, genome sequence data are available for these populations, and new computational methods enable high-throughput microscopic analyses. In this study, we measured stationary-phase cell volumes for the ancestor and 12 populations at 2,000, 10,000, and 50,000 generations, including measurements during exponential growth at the last time point. We measured the distribution of cell volumes for each sample using a Coulter counter and microscopy, the latter of which also provided data on cell shape. Our data confirm the trend toward larger cells while also revealing substantial variation in size and shape across replicate populations. Most populations first evolved wider cells but later reverted to the ancestral length-to-width ratio. All but one population evolved mutations in rod shape maintenance genes. We also observed many ghost-like cells in the only population that evolved the novel ability to grow on citrate, supporting the hypothesis that this lineage struggles with maintaining balanced growth. Lastly, we show that cell size and fitness remain correlated across 50,000 generations. Our results suggest that larger cells are beneficial in the experimental environment, while the reversion toward ancestral length-to-width ratios suggests partial compensation for the less favorable surface area-to-volume ratios of the evolved cells.IMPORTANCE Bacteria exhibit great morphological diversity, yet we have only a limited understanding of how their cell sizes and shapes evolve and of how these features affect organismal fitness. This knowledge gap reflects, in part, the paucity of the fossil record for bacteria. In this study, we revived and analyzed samples extending over 50,000 generations from 12 populations of experimentally evolving Escherichia coli to investigate the relation between cell size, shape, and fitness. Using this "frozen fossil record," we show that all 12 populations evolved larger cells concomitant with increased fitness, with substantial heterogeneity in cell size and shape across the replicate lines. Our work demonstrates that cell morphology can readily evolve and diversify, even among populations living in identical environments.

Keywords: Escherichia coli; cell death; cell size; experimental evolution; natural selection; organismal fitness.

FIG 1

Correlation between cell volume measurements obtained using microscopy and Coulter counter. Volumes obtained by microscopy are expressed in arbitrary units (a.u.) proportional to femtoliters (i.e., cubic micrometers); volumes obtained using the Coulter counter are expressed in femtoliters. Each point shows the grand median of three assays for clones sampled from the 12 evolving populations or of six assays for the two ancestral strains. Kendall’s coefficient τ = 0.5495, n = 38, and P < 0.0001.

FIG 2

Cell size trajectories for clones (A) and whole-population samples (B) obtained using a Coulter counter. Each quantile (5th, 25th, 50th, 75th, and 95th) represents the median of the corresponding quantile from six replicates of each ancestor (REL607 for Ara+ populations and REL606 for Ara− populations) and three replicates for cells sampled from each population. The asterisk indicates that the upper quantile for the 50,000-generation clone from population Ara−3 extends to ∼4.4 fl.

FIG 3

Tests of changes over time in average cell sizes of clones (A) and whole-population samples (B) from the 12 LTEE populations. Each point shows the grand mean of the grand median cell volumes calculated for each population. The 50,000-generation clone from population Ara−3 was an extreme outlier (Fig. 2A) and is excluded from panel A; however, the 50,000-generation whole-population sample from this population was not an outlier (Fig. 2B). Error bars are 95% confidence intervals, and brackets show the statistical significance (P value) based on one-tailed paired t tests. The last comparison in panel A remains significant even if one includes the outlier clone (P = 0.0090).

FIG 4

Average rate of cell volume increase. Slopes were calculated for each population over each of three intervals. Each point shows the grand mean for the 12 populations. Error bars are 95% confidence intervals, and brackets show the statistical significance (P value) based on one-tailed Wilcoxon tests, which account for the paired nature of the samples.

FIG 5

Cell sizes measured during exponential and stationary phases of ancestral strains and 50,000-generation clones from all 12 populations. Each point represents the median cell volume for one assay at either 2 h (exponential growth) or 24 h (stationary phase) in DM25. Horizontal bars are the means from the 3 replicate assays for each strain. The points for some individual replicates are not visible because some values were almost identical.

FIG 6

Correlation between cell sizes during exponential growth and in stationary phase. Each point represents the average over 3 replicates of the median cell volume in each growth phase using the data shown in Fig. 5. Kendall’s coefficient τ = 0.7582, n = 14, and P << 0.0001.

FIG 7

Representative micrographs of ancestors (REL606 and REL607) and evolved clones from each population at 50,000 generations. Phase-contrast images of cells from stationary-phase cultures were taken at ×100 magnification. Scale bars are 10 μm.

FIG 8

Average cell aspect ratios (length/width) of ancestral and evolved clones. Each point shows the mean ratio for the indicated sample. The lines show deviations in the aspect ratio from the ancestral state. The mean aspect ratios were calculated from three replicate assays in all but 4 cases (Ara−4 at 10,000 generations and Ara−2, Ara−4, and Ara−5 at 50,000 generations), which had two replicates each.

FIG 9

Tests of changes over time in cell aspect and surface-to-volume (SA/V) ratios. (A) Evolutionary reversal of cell aspect ratio. Each point is the grand mean of the cell aspect ratio (length/width) for the ancestors and evolved clones. n = 12, except at 50,000 generations, where n = 11 after excluding the outlier clone from the Ara−3 population. Errors bars are 95% confidence intervals, and brackets show the statistical significance (P value) based on two-tailed t tests. The tests were paired for clones sampled from the same population at the consecutive time points, and the Ara−3 population was excluded from the final test. (B) Monotonic decline in SA/V ratio over 50,000 generations. Each point shows the grand mean of the average ratio calculated for the ancestor and evolved clones. Error bars are 95% confidence intervals, and brackets show the statistical significance (P value) based on one-tailed paired t tests.

FIG 10

Average SA/V of ancestral and evolved clones. The surface area and volume of individual cells were calculated from microscopic images, as described in the text, and their ratio has arbitrary units proportional to micrometers. Each point shows the mean ratio for the indicated sample. The lines show deviations in the ratio from the ancestral state. The means were calculated from three replicate assays in all but 4 cases (Ara−4 at 10,000 generations and Ara−2, Ara−4, and Ara−5 at 50,000 generations), which had two replicates each.

FIG 11

Representative micrographs of cells from 2,000-generation (A) and 50,000-generation (B) clones of the Ara+5 population. Phase-contrast images were taken on an inverted microscope at a magnification of ×100. Scale bars are 10 μm. Arrows point to examples of nearly spherical cells in the earlier sample, which are not seen in the later one.

FIG 12

Parallel mutations in genes known to be involved in the maintenance of rod-shaped genes. Nonsynonymous mutations were found in all populations except Ara−5 by 50,000 generations. Populations Ara−2, Ara−4, Ara+3, and Ara+6 evolved hypermutable phenotypes between generations 2,000 and 10,000; populations Ara−1 and Ara−3 did so between generations 10,000 and 50,000. Hence, all synonymous mutations were found in lineages with a history of elevated point mutation rates.

FIG 13

Correlation between mean fitness relative to the LTEE ancestor and grand median cell volumes, both based on whole-population samples. Four points (Ara+6 at 10,000 generations and Ara−2, Ara−3, and Ara+6 at 50,000 generations) are absent due to missing fitness values reported by Wiser et al. (42). Kendall’s τ = 0.6066, n = 34, and P < 0.0001.

FIG 14

Representative micrograph of 50,000-generation Cit⁺ clone from population Ara−3 grown in DM0. As shown in Fig. 7, we observed translucent “ghost” cells in the only population that evolved the capacity to use citrate in the LTEE medium (DM25). This clone can also grow on citrate alone in the same medium except without glucose (DM0), which increased the proportion of presumably dead or dying ghost cells. Red arrows point to several ghost cells, some of which have darker punctate inclusions; white arrows point to several more typically opaque and presumably viable cells. Two insets show further magnified images of the highlighted regions. Scale bar is 10 μm.

FIG 15

Comparison of cell death in the ancestor and Cit⁺ clone. (A) Representative micrographs showing LIVE-DEAD staining of the LTEE ancestor (REL606) and the 50,000-generation Cit⁺ clone from population Ara−3 (REL11364), both grown in DM25. Scale bars are 10 μm. (B) Proportions of cells scored as alive (green) or dead (red), based on two-color stain assay. (C and D) Cell widths (C) and lengths (D) were calculated by multiplying the ShortAxis and LongAxis measurements (both in unit pixel) from the SuperSegger output by the conversion factor of 0.0664 μm/pixel. Colored boxplots show the distribution of cells scored as alive (green) or dead (red), based on a two-color stain assay. For each clone, we assayed cells from 5 biological replicates, which have been pooled for this figure.