Lenski, R. | bioRxiv Science

bioRxiv2020

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

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. Here, 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 timepoint. 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 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. ImportanceBacteria 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. Here, we revive and analyze 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.

evolutionary biology↗

bioRxiv2020

Environmental and physiological factors affecting high-throughput measurements of bacterial growth

Bacterial growth in nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multi-well plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth. Abstract ImportanceHow starved bacteria adapt to and multiply in replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allows for accurate growth-rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli. Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic-acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.

microbiology↗