Phenotypic Heterogeneity in Sugar Utilization by E. coli Is Generated by Stochastic Dispersal of the General PTS Protein EI from Polar Clusters

Abstract

Although the list of proteins that localize to the bacterial cell poles is constantly growing, little is known about their temporal behavior. EI, a major protein of the phosphotransferase system (PTS) that regulates sugar uptake and metabolism in bacteria, was shown to form clusters at the Escherichia coli cell poles. We monitored the localization of EI clusters, as well as diffuse molecules, in space and time during the lifetime of E. coli cells. We show that EI distribution and cluster dynamics varies among cells in a population, and that the cluster speed inversely correlates with cluster size. In growing cells, EI is not assembled into clusters in almost 40% of the cells, and the clusters in most remaining cells dynamically relocate within the pole region or between the poles. In non-growing cells, the fraction of cells that contain EI clusters is significantly higher, and dispersal of these clusters is often observed shortly after exiting quiescence. Later, during growth, EI clusters stochastically re-form by assembly of pre-existing dispersed molecules at random time points. Using a fluorescent glucose analog, we found that EI function inversely correlates with clustering and with cluster size. Thus, activity is exerted by dispersed EI molecules, whereas the polar clusters serve as a reservoir of molecules ready to act when needed. Taken together our findings highlight the spatiotemporal distribution of EI as a novel layer of regulation that contributes to the population phenotypic heterogeneity with regard to sugar metabolism, seemingly conferring a survival benefit.

Tyrosine phosphorylation-dependent localization of TmaR, a novelE. colipolar protein that controls activity of the major sugar regulator by polar sequestration

“…Hence, the activity of EI inversely correlates with the level of TmaR in the cell. These results strengthen our previous results, which suggested that clustering of EI negatively correlates with EI activity (15).…”

“…Although EI-mCherry activity has been demonstrated previously (13) and shown to be comparable to that of wild-type EI (15), not only did we verify this again (see SI Appendix, Fig. S9), but we also asked if EI-mCherry fluorescent signal correlates with EI-mCherry level.…”

“…An additional assay that we used to compare the effect of TmaR subcellular level on PTS sugars uptake relied on the use of a strain expressing mCerulean, a cyan fluorescent protein (CFP), from the N-acetyl glucosamine (NAG) promoter (P NAG ) and EI-mCherry (31). NAG has been used in the past as a representative of PTS sugars, whose utilization is positively regulated by EI, since induction of P NAG by NAG is consistent with the positive feedback mechanism suggested for promoters of PTS sugar utilization genes (31) (15). Hence, production of mCerulean from P NAG implies that EI is active and enables estimation of EI activity by fluorescence microscopy.…”

“…The general PTS protein EI initiates the phosphorylation cascade that controls hierarchical uptake and utilization of preferred carbohydrates from complex environments (28). The soluble EI protein has previously been shown in our lab to shift between assembling into polar clusters and diffusing in the cytoplasm (13)(14)(15), but the factor responsible for its polar localization remained unknown (13,14). We, therefore, asked whether TmaR is involved in determining the subcellular localization of EI.…”

“…The PTS-imported sugars enter glycolysis, whose product, phosphoenolpyruvate (PEP), phosphorylates EI, making EI an important link between glycolysis and sugar uptake (12). We have previously shown that the general PTS proteins localize to the E. coli cell poles (13), although their localization depends on yet unknown factors (14), that during growth, EI polar clusters form stochastically from pre-existing dispersed molecules, and that EI clustering negatively correlates with EI function (15). Still, conclusive proof for polar localization as an inhibitory mechanism of EI function is lacking and the identity of the factor that captures EI at the pole remained unknown.…”