In response to stress, some organisms effectively 'roll the dice,' selecting randomly from among various possible genetic programs. For example, in the bacteria Bacillus subtilis, individual cells respond to nutrient limitation differently: some become competent to take up DNA from the environment, others differentiate into a robust spore, while the rest continue to grow and divide. This ability to make random choices is crucial in microbial survival, and used throughout the development of multicellular organisms. The Elowitz lab will address the question of how genetic circuits allow otherwise identical cells to choose their fate randomly, using the soil bacteria Bacillus subtilis as a model system. The well-characterized genetic circuitry underlying the decision to sporulate will be analyzed using two complementary approaches. First, automated time-lapse fluorescence microscopy, together with quantitative image analysis, will be used to determine detailed gene expression dynamics in individual cells during the decision-making process. Second, synthetic biology techniques will be used to design, construct, and analyze simpler genetic circuits in Escherichia coli that generate similar responses. These synthetic circuits function as in vivo models of the more complex natural circuits they emulate. Mathematical modeling will inform all aspects of the research program, both in the analysis of data and formulation of hypotheses. The Broader Impact of the research will include the development of new curricula in systems biology and synthetic biology and the generation of tools for the research community, including software for single-cell analysis of time-lapse movies of bacterial growth.
Some cells respond to environmental conditions in a predictable way, following a well-defined stress response program. Others, however, choose to effectively ‘roll the dice.’ They select randomly from among various possible responses. For example, in the bacteria Bacillus subtilis, individual cells respond to nutrient limitation differently: some become competent to take up DNA from the environment, others differentiate into a robust spore, while the rest continue to grow and divide. In general, the ability to make random choices is crucial in microbial pathogenesis, and is used throughout the development of multicellular organisms. However, the mechanisms enabling such probabilistic responses generally remain unclear. In this project we combined time-lapse microscopy with computational image analysis to follow the dynamics of the genetic circuits that control probabilistic behaviors over time at the level of individual cells. Our results have provided a number of fundamental discoveries and insights: Key results: (1) We developed generally applicable methods for measuring single-cell gene expression dynamics in bacteria. These methods include the development of software for identifying cells in images, tracking cells over time, and quantifying changes in fluorescent protein expression. We used these methods to analyze several stress response systems. (2) We discovered that cells implement a cell autonomous "timer" circuit that allows them to defer a response to stress – turning into a dormant spore - for about 5 cell cycles. This type of deferred response is implemented with a circuit based on pulses of activation of a master regulator. We continue to work to understand the role that pulsing plays in enabling timer behavior. (3) We discovered that the general stress response system in Bacillus subtilis is activated in a sustained series of stochastic pulses, creating heterogeneity among genetically identical cells. We worked out the mechanism through which this system works, and we showed how it enables cells to effectively convert a constant input level into a dynamic frequency of intracellular pulses (number of pulses per unit time). This discovery reveals an unexpectedly dynamic and heterogeneous picture of cellular stress response. (4) We analyzed the dynamic mechanism through which cells integrate distinct types of stress. By carefully controlling the levels of environmental stresses over time, while analyzing cells using time-lapse microscopy, we discovered that the general stress response pathway in bacteria effectively responds not to the level of some environmental stresses but instead to the rate at which they are increasing, effectively allowing cells to anticipate future stresses. (5) We showed that stochastic fluctuations in cells can be analyzed dynamically to reveal regulatory interactions within a genetic circuit. In this sense, stochastic fluctuations can, counterintuitively, help enable researchers to better understand interactions between different types of cellular components. Together, these results are enabling us to understand how intrinsically random ‘noise’ in cells is integrated together with more deterministic responses to specify a whole range of cellular responses to environmental conditions. These results should provide a foundation for examining related phenomena in other cell types and conditions.
