Tyrosine phosphorylation-dependent localization of TmaR that controls activity of a major bacterial sugar regulator by polar sequestration

Abstract

The poles of Escherichia coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a previously unannotated protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization and activity of the general PTS protein Enzyme I (EI) by binding and polar sequestration of EI, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably, phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results suggest that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first, to our knowledge, E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release.

Regulation of major bacterial survival strategies by transcript sequestration in a membraneless organelle

“…2C). Notably, we have previously shown that the neighboring residue of E73, a tyrosine in position 72 (Y72), gets phosphorylated, a modification often associated with LLPS (19), and that this event is required for TmaR activity as EI regulator (13). Jointly, the current and previous results highlight the importance of the negative patch around positions 96-77 in TmaR, and suggest that both the phosphorylation and the charge it confers affect TmaR ability to undergo LLPS in vivo.…”

“…Of note, accumulating evidence suggest that many stages of the RNA life cycle in eukaryotes occur within biomolecular condensates (27). Further research is required for defining the protein and RNA content of TmaR condensates and the interactions among its constituents that enable the control of important pathways, sugar metabolism on one hand, as previously shown by us (13), and motility and biofilm formation on the other hand, as shown here.…”

“…We could also demonstrate that this ability is important for cell survival in mild acidic conditions, which E. coli often encounters in its natural habitats (13). We have further shown that TmaR controls the activity of the general protein of the phosphotransferase system (PTS), Enzyme I (EI), the main regulator of sugar utilization in most bacteria, by its polar sequestration and release (13). The dynamics of TmaR, mainly within the pole region, the lack of evidence for its membrane-anchoring, and the rapid change between clustered and diffused EI (14), intrigued us to posit that TmaR forms polar condensates, which compartmentalize EI, via LLPS.…”

“…We recently showed that TmaR (previously called YeeX) clusters to one pole in E. coli cells when phosphorylated on a tyrosine. We could also demonstrate that this ability is important for cell survival in mild acidic conditions, which E. coli often encounters in its natural habitats (13). We have further shown that TmaR controls the activity of the general protein of the phosphotransferase system (PTS), Enzyme I (EI), the main regulator of sugar utilization in most bacteria, by its polar sequestration and release (13).…”

“…Together, the timescale of TmaR cluster dynamics and their 1,6-hexanediol-induced dispersal suggest that they are formed by LLPS. EI, the major sugar utilization regulator, was shown to localize with the TmaR clusters and to be activated by its release from them, but not to cluster on its own or affect TmaR clustering (13). Hence, we investigated its dynamics in time-lapse microscopy and its response to hexanediol treatment.…”