Single Cell Kinetics of Phenotypic Switching in the Arabinose Utilization System of E. coli

Abstract

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.

Reciprocal Regulation ofl-Arabinose andd-Xylose Metabolism in Escherichia coli

“…Both sugar utilization systems exhibit bimodal responses (11)(12)(13)(14)(15)(16), and our initial goal was to understand how catabolite repression affected the response of individual cells. Our data show that the response to the two sugars is multimodal and that the shape of this distribution is determined by the reciprocal regulation of these two sugar utilization systems.…”

“…Both the arabinose and xylose genes are known to exhibit a bimodal/heterogeneous induction response to their cognate sugar (11)(12)(13)(14)(15)(16). Of particular note, a previous study by Afroz and coworkers investigated the expression of the arabinose and xylose metabolic genes in E. coli in response to their cognate sugar (11).…”

Phenotypic Heterogeneity, a Phenomenon That May Explain Why Quorum Sensing Does Not Always Result in Truly Homogenous Cell Behavior

“…Yet another recent example for heterogeneous expression of genes linked to metabolic traits concerns the arabinose utilization system in E. coli (63,64). With quantitative time-lapse microscopy and by challenging E. coli cells with very low concentrations of arabinose, a time delay in the response of single cells to arabinose was discovered.…”

Screening of an Escherichia coli promoter library for a phenylalanine biosensor

“…Thus, the design of suitable biosensors would provide a convenient tool for strain analysis and screening, and might be especially useful for the identification of non-intuitive targets for strain improvement (Binder et al 2012;Mahr et al 2015;Mustafi et al 2012). However, our study revealed that several parameters require careful consideration to set up a screening procedure for effector-responsive promoters, including the adaption to growth medium, the uptake and catabolism of effector molecules (Luo et al 2014;Payne 1977), the activation of the gene expression machinery (Bintu et al 2005b;Carey et al 2013;Fritz et al 2014;Mäkelä et al 2013), the maturation of fluorescent proteins (Craggs 2009;Iizuka et al 2011), and last but not least, the composition of the particular growth medium. Here, we used M9 minimal medium due to its lack of other amino acids and carbon source and its very low auto-fluorescence (Miller 1972).…”