Direct Correlation between Motile Behavior and Protein Abundance in Single Cells

Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.Author Summary: Cell-to-cell variations in protein numbers due to random fluctuations at the molecular level lead to cell-to-cell variations in behavior. To maintain predictable responses, signaling networks have evolved robustness against noise, but in some situations phenotypic diversity in a clonal population can be beneficial as a bet hedging or division of labor strategy. Investigating of how random molecular fluctuations affect cell behavior requires to measure biological parameters at different scales. Here, we report a new experiment that allows the measure of both protein numbers and behavior in cells that are free to move in their environment. Using Escherichia coli, a model system for the study of cellular behavior, we investigated the effects variations in the numbers of the chemo-receptor modification enzymes on single-cell swimming behavior. We found that the mean and variance of the behavior can be adjusted independently in the population by adjusting protein expression. This mechanism allows for the genetic selection of phenotypic diversity without disrupting correlations in protein expression that are important for the overall robustness of the chemotaxis system.