Cesar, S. | Search bioRxiv

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↗

bioRxiv2020

Bacterial evolution in high osmolarity environments

Bacteria must maintain a cytosolic osmolarity higher than that of their environment in order to take up water. High osmolarity environments therefore present a formidable stress to bacteria. To explore the evolutionary mechanisms by which bacteria adapt to high osmolarity environments, we selected Escherichia coli in media with a variety of osmolytes and concentrations for 250 generations. Adaptation was osmolyte-dependent, with sorbitol stress generally resulting in increased fitness in conditions with higher osmolarity, while selection in high concentrations of proline resulted in increased fitness specifically on proline. Consistent with these phenotypes, sequencing of the evolved populations showed that passaging in proline resulted in specific mutations in an associated metabolic pathway that increases the ability to utilize proline for growth, while evolution in sorbitol resulted in mutations in many different genes that generally improve growth in high osmolarity conditions at the expense of growth at low osmolarity. High osmolarity decreased growth rate but increased mean cell volume compared with growth on proline as the sole carbon source, demonstrating that osmolarity-induced changes in growth rate and cell size follow an orthogonal relationship from the classical Growth Law relating cell size and nutrient quality. Isolates from a sorbitol-evolved population that capture the likely temporal sequence of mutations revealed by metagenomic sequencing demonstrate a tradeoff between growth at high and low osmolarity. Our study highlights the utility of experimental evolution for dissecting complex cellular networks and environmental interactions, particularly in the case of behaviors that can involve both specific and general metabolic stressors. ImportanceFor bacteria, maintaining higher internal solute concentrations than the environment allows cells to take up water. As a result, survival is challenging in high osmolarity environments. To investigate how bacteria adapt to high osmolarity environments, we evolved Escherichia coli in a variety of high osmolarity solutions for hundreds of generations. We found that evolved populations adopted different strategies to improve their growth depending on the osmotic passaging condition, either generally adapting to high osmolarity conditions or better metabolizing the osmolyte as carbon source. Single-cell imaging demonstrated that enhanced fitness was coupled to faster growth, and metagenomic sequencing revealed mutations that reflect growth tradeoffs across osmolarities. Our study demonstrates the utility of long-term evolution experiments for probing adaptation during environmental stress.

microbiology↗