Inducible switching between phenotypes is a common strategy of bacteria to adapt to fluctuating environments. Here, we analyze the switching kinetics of a paradigmatic inducible system, the arabinose utilization system in E. coli. Using time-lapse fluorescence microscopy of microcolonies in a microfluidic chamber, which permits sudden up- and down-shifts in the inducer arabinose, we characterize the single-cell gene expression dynamics of the araBAD operon responsible for arabinose degradation. While there is significant, inducer-dependent cell-to-cell variation in the timing of the on-switching, the off-switching triggered by sudden removal of arabinose is homogeneous and rapid. We find that rapid off-switching does not depend on internal arabinose degradation. Because the system is regulated via the internal arabinose level sensed by AraC, internal arabinose must be rapidly depleted by leakage or export from the cell, or by degradation via a non-canonical pathway. We explored whether the poorly characterized membrane protein AraJ, which is part of the arabinose regulon and has been annotated as a possible arabinose efflux protein, is responsible for rapid depletion. However, we find that AraJ is not essential for rapid switching to the off-state. We develop a mathematical model for the arabinose system, which quantitatively describes both the heterogeneous on-switching and the homogeneous off-switching. The model also predicts that mutations which disrupt the positive feedback of internal arabinose on the production of arabinose uptake proteins change the heterogeneous on-switching behavior into a homogeneous, graded response. We construct such a mutant and confirm the graded response experimentally. Taken together, our results indicate that the physiological switching behavior of this sugar utilization system is asymmetric, such that off-switching is always rapid and homogeneous, while on-switching is slow and heterogeneously timed at sub-saturating inducer levels.
The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner-take-all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single-cell resolution. Using time-lapse microscopy in perfused microchannels, we analyzed the competition between N-acetyl-glucosamine and sorbitol, as representative PTS sugars, by measuring both the expression of their utilization systems and the concomitant impact of sugar utilization on growth rates. We find two distinct regimes: hierarchical usage of the carbohydrates, and co-expression of the genes for both systems. Simulations of a mathematical model incorporating asymmetric sugar quality reproduce our metabolic phase diagram, indicating that under conditions of nonlimiting phosphate flux, co-expression is due to uncoupling of both sugar utilization systems. Our model reproduces hierarchical winner-take-all behaviour and stochastic co-expression, and predicts the switching between both strategies as a function of available phosphate flux. Hence, experiments and theory both suggest that PTS sugar utilization involves not only switching between the sugars utilized but also switching of utilization strategies to accommodate prevailing environmental conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.