MinD-RNase E interplay controls localization of polar mRNAs in E. coli.

The E. coli transcriptome at the cell's poles (polar transcriptome) is unique compared to the membrane and cytosol. Several factors have been suggested to mediate mRNA localization to the membrane, but the mechanism underlying polar localization of mRNAs remains unknown. Here, we combined a candidate system approach with proteomics to identify factors that mediate mRNAs localization to the cell poles. We identified the pole-to-pole oscillating protein MinD as an essential factor regulating polar mRNA localization, although it is not able to bind RNA directly. We demonstrate that RNase E, previously shown to interact with MinD, is required for proper localization of polar mRNAs. Using in silico modeling followed by experimental validation, the membrane-binding site in RNase E was found to mediate binding to MinD. Intriguingly, not only does MinD affect RNase E interaction with the membrane, but it also affects its mode of action and dynamics. Polar accumulation of RNase E in ΔminCDE cells resulted in destabilization and depletion of mRNAs from poles. Finally, we show that mislocalization of polar mRNAs may prevent polar localization of their protein products. Taken together, our findings show that the interplay between MinD and RNase E determines the composition of the polar transcriptome, thus assigning previously unknown roles for both proteins.

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

TmaR, the only known pole-localizer protein in Escherichia coli, was shown to cluster at the cell poles and control localization and activity of the major sugar regulator in a tyrosine phosphorylation-dependent manner. Here, we show that TmaR assembles by phase separation (PS) via heterotypic interactions with RNA in vivo and in vitro. An unbiased automated mutant screen combined with directed mutagenesis and genetic manipulations uncovered the importance of a predicted nucleic-acid-binding domain, a disordered region, and charged patches, one containing the phosphorylated tyrosine, for TmaR condensation. We demonstrate that, by protecting flagella-related transcripts, TmaR controls flagella production and, thus, cell motility and biofilm formation. These results connect PS in bacteria to survival and provide an explanation for the linkage between metabolism and motility. Intriguingly, a point mutation or increase in its cellular concentration induces irreversible liquid-to-solid transition of TmaR, similar to human disease-causing proteins, which affects cell morphology and division.

Heterotypic phase separation of Hfq is linked to its roles as an RNA chaperone.

Hfq, an Sm-like protein and the major RNA chaperone in E. coli, has been shown to distribute non-uniformly along a helical path under normal growth conditions and to relocate to the cell poles under certain stress conditions. We have previously shown that Hfq relocation to the poles is accompanied by polar accumulation of most small RNAs (sRNAs). Here, we show that Hfq undergoes RNA-dependent phase separation to form cytoplasmic or polar condensates of different density under normal and stress conditions, respectively. Purified Hfq forms droplets in the presence of crowding agents or RNA, indicating that its condensation is via heterotypic interactions. Stress-induced relocation of Hfq condensates and sRNAs to the poles depends on the pole-localizer TmaR. Phase separation of Hfq correlates with its ability to perform its posttranscriptional roles as sRNA-stabilizer and sRNA-mRNA matchmaker. Our study offers a spatiotemporal mechanism for sRNA-mediated regulation in response to environmental changes.

Evolutionarily conserved mechanism for membrane recognition from bacteria to mitochondria.

The mechanisms controlling membrane recognition by proteins with one hydrophobic stretch at their carboxyl terminus (tail anchor, TA) are poorly defined. The Escherichia coli TAs of ElaB and YqjD, which share sequential and structural similarity with the Saccharomyces cerevisiae TA of Fis1, were shown to localize to mitochondria. We show that YqjD and ElaB are directed by their TAs to bacterial cell poles. Fis1(TA) expressed in E. coli localizes like the endogenous TAs. The yeast and bacterial TAs are inserted in the E. coli inner membrane, and they all show affiliation to phosphatidic acid (PA), found in the membrane of the bacterial cell poles and of the yeast mitochondria. Our results suggest a mechanism for TA membrane recognition conserved from bacteria to mitochondria and raise the possibility that through their interaction with PA, and TAs play a role across prokaryotes and eukaryotes in controlling cell/organelle fate.

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

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.

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

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.

A Search for Ribonucleic Antiterminator Sites in Bacterial Genomes: Not Only Antitermination?

BglG/LicT-like proteins are transcriptional antiterminators that prevent termination of transcription at intrinsic terminators by binding to ribonucleic antiterminator (RAT) sites and stabilizing an RNA conformation which is mutually exclusive with the terminator structure. The known RAT sites, which are located in intergenic regions of sugar utilization operons, show low sequence conservation but significant structural analogy. To assess the prevalence of RATs in bacterial genomes, we employed bioinformatic tools that describe RNA motifs based on both sequence and structural constraints. Using descriptors with different stringency, we searched the genomes of Escherichiacoli K12, uropathogenic E. coli and Bacillus subtilis for putative RATs. Our search identified all known RATs and additional putative RAT elements. Surprisingly, most putative RATs do not overlap an intrinsic terminator and many reside within open reading frames (ORFs). The ability of one of the putative RATs, which is located within an antiterminator-encoding ORF and does not overlap a terminator, to bind to its cognate antiterminator protein in vitro and in vivo was confirmed experimentally. Our results suggest that the capacity of RAT elements has been exploited during evolution to mediate activities other than antitermination, for example control of transcription elongation or of RNA stability.

Translation-independent localization of mRNA in E. coli.

Understanding the organization of a bacterial cell requires the elucidation of the mechanisms by which proteins localize to particular subcellular sites. Thus far, such mechanisms have been suggested to rely on embedded features of the localized proteins. Here, we report that certain messenger RNAs (mRNAs) in Escherichia coli are targeted to the future destination of their encoded proteins, cytoplasm, poles, or inner membrane in a translation-independent manner. Cis-acting sequences within the transmembrane-coding sequence of the membrane proteins are necessary and sufficient for mRNA targeting to the membrane. In contrast to the view that transcription and translation are coupled in bacteria, our results show that, subsequent to their synthesis, certain mRNAs are capable of migrating to particular domains in the cell where their future protein products are required.

Spatial and temporal organization of the E. coli PTS components.

The phosphotransferase system (PTS) controls preferential use of sugars in bacteria. It comprises of two general proteins, enzyme I (EI) and HPr, and various sugar-specific permeases. Using fluorescence microscopy, we show here that EI and HPr localize near the Escherichia coli cell poles. Polar localization of each protein occurs independently, but HPr is released from the poles in an EI- and sugar-dependent manner. Conversely, the ß-glucoside-specific permease, BglF, localizes to the cell membrane. EI, HPr and BglF control the ß-glucoside utilization (bgl) operon by modulating the activity of the BglG transcription factor; BglF inactivates BglG by membrane sequestration and phosphorylation, whereas EI and HPr activate it by an unknown mechanism in response to ß-glucosides availability. Using biochemical, genetic and imaging methodologies, we show that EI and HPr interact with BglG and affect its subcellular localization in a phosphorylation-independent manner. Upon sugar stimulation, BglG migrates from the cell periphery to the cytoplasm through the poles. Hence, the PTS components appear to control bgl operon expression by ushering BglG between the cellular compartments. Our results reinforce the notion that signal transduction in bacteria involves dynamic localization of proteins.

Modulation of transcription antitermination in the bgl operon of Escherichia coli by the PTS.

BglG, which regulates expression of the beta-glucoside utilization (bgl) operon in Escherichia coli, represents a family of RNA-binding transcriptional antiterminators that positively regulate transcription of sugar utilization genes in Gram-negative and Gram-positive organisms. BglG is negatively regulated by the beta-glucoside phosphotransferase, BglF, by means of phosphorylation and physical association, and it is positively regulated by the general phosphoenolpyruvate phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr. We studied the positive regulation of BglG both in vitro and in vivo. Here, we show that although EI and HPr are essential for BglG activity, this mode of activation does not require phosphorylation of BglG by HPr, as opposed to the phosphorylation-mediated activation of many BglG-like antiterminators in Gram-positive organisms. The effect of EI and HPr on BglG is not mediated by BglF. Nevertheless, the release of BglG from BglF, which is stimulated by the extracellular sugar in a sugar uptake-independent manner, is a prerequisite for BglG activation. Taken together, the results indicate that activation of BglG is a 2-stage process: a sugar-stimulated release from the membrane-bound sugar sensor followed by a phosphorylation-independent stimulatory effect exerted by the general PTS proteins.

Modulation of monomer conformation of the BglG transcriptional antiterminator from Escherichia coli.

The BglG protein positively regulates expression of the bgl operon in Escherichia coli by binding as a dimer to the bgl transcript and preventing premature termination of transcription in the presence of beta-glucosides. BglG activity is negatively controlled by BglF, the beta-glucoside phosphotransferase, which reversibly phosphorylates BglG according to beta-glucoside availability, thus modulating its dimeric state. BglG consists of an RNA-binding domain and two homologous domains, PRD1 and PRD2. Based on structural studies of a BglG homologue, the two PRDs fold similarly, and the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have recently shown that the affinity between PRD1 and PRD2 of BglG is high, and a fraction of the BglG monomers folds in the cell into a compact conformation, in which PRD1 and PRD2 are in close proximity. We show here that both BglG forms, the compact and noncompact, bind to the active site-containing domain of BglF, IIB(bgl), in vitro. The interaction of BglG with IIB(bgl) or BglF is mediated by PRD2. Both BglG forms are detected as phosphorylated proteins after in vitro phosphorylation with IIB(bgl) and are dephosphorylated by BglF in vitro in the presence of beta-glucosides. Nevertheless, genetic evidence indicates that the interaction of IIB(bgl) and BglF with the compact form is seemingly less favorable. Using in vivo cross-linking, we show that BglF enhances folding of BglG into a compact conformation, whereas the addition of beta-glucosides reduces the amount of this form. Based on these results we suggest a model for the modulation of BglG conformation and activity by BglF.

A fraction of the BglG transcriptional antiterminator from Escherichia coli exists as a compact monomer.

Expression of the bgl operon in Escherichia coli, induced by beta-glucosides, is positively regulated by BglG, a transcriptional antiterminator. In the presence of inducer, BglG dimerizes and binds to the bgl transcript to prevent premature termination of transcription. The dimeric state of BglG is determined by BglF, a membrane-bound enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which reversibly phosphorylates BglG according to beta-glucoside availability. BglG is composed of an RNA-binding domain followed by two homologous PTS regulation domains (PRD1 and PRD2). The predicted structure of dimeric LicT, a BglG homologue from Bacillus subtilis, suggests that the two PRDs adopt a similar structure and that the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have shown recently that the PRD1 and PRD2 domains of BglG can form a stable heterodimer. We report here, based on in vitro and in vivo cross-linking experiments, that a fraction of BglG is present in the cell in a compact form in which PRD1 and PRD2 are in close proximity. The compact form is present mainly in the BglG monomers. Our results imply that the monomer-dimer transition involves a conformational change. The possible role of the compact form in preventing untimely induction of the bgl operon is discussed.

Interactions between the PTS regulation domains of the BglG transcriptional antiterminator from Escherichia coli.

The E. coli BglG protein inhibits transcription termination within the bgl operon in the presence of beta-glucosides. BglG represents a family of transcriptional antiterminators that bind to RNA sequences, which partially overlap rho-independent terminators, and prevent termination by stabilizing an alternative structure of the transcript. The activity of BglG is determined by its dimeric state, which is modulated by reversible phosphorylation catalyzed by BglF, a PTS permease. Only the non-phosphorylated BglG dimer binds to RNA and allows read-through of transcription. BglG is composed of three domains: an RNA-binding domain followed by two domains, PRD1 and PRD2 (PTS regulation domains), which are similar in their sequence and folding. Based on the three-dimensional structure of dimeric LicT, a BglG homologue from Bacillus subtilis, the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have shown before that PRD2 mediates homodimerization very efficiently. Using genetic systems and in vitro techniques that assay and characterize protein-protein interactions, we show here that the PRD1 dimerizes very slowly, but once it does, the homodimers are stable. These results support our model that formation of BglG dimers initiates with PRD2 dimerization followed by zipping up of two BglG monomers to create the active RNA-binding domain. Moreover, our results demonstrate that PRD1 and PRD2 heterodimerize efficiently in vitro and in vivo. The affinity among the PRDs is in the following order: PRD2-PRD2 > PRD1-PRD2 > PRD1-PRD1. The interaction between PRD1 and PRD2 offers an explanation for the requirement of conserved residues in PRD1 for the phosphorylation of PRD2 by BglF.

The BglF sensor recruits the BglG transcription regulator to the membrane and releases it on stimulation.

The Escherichia coli BglF protein is a sugar-sensor that controls the activity of the transcriptional antiterminator BglG by reversibly phosphorylating it, depending on beta-glucoside availability. BglF is a membrane-bound protein, whereas BglG is a soluble protein, and they are both present in the cell in minute amounts. How do BglF and BglG find each other to initiate signal transduction efficiently? Using bacterial two-hybrid systems and the Far-Western technique, we demonstrated unequivocally that BglG binds to BglF and to its active site-containing domain in vivo and in vitro. Measurements by surface plasmon resonance corroborated that the affinity between these proteins is high enough to enable their stable binding. To visualize the subcellular localization of BglG, we used fluorescence microscopy. In cells lacking BglF, the BglG-GFP fusion protein was evenly distributed throughout the cytoplasm. In contrast, in cells producing BglF, BglG-GFP was localized to the membrane. On addition of beta-glucoside, BglG-GFP was released from the membrane, becoming evenly distributed throughout the cell. Using mutant proteins and genetic backgrounds that impede phosphorylation of the Bgl proteins, we demonstrated that BglG-BglF binding and recruitment of BglG to the membrane sensor requires phosphorylation but does not depend on the individual phosphorylation sites of the Bgl proteins. We suggest a mechanism for rapid response to environmental changes by preassembly of signaling complexes, which contain transcription regulators recruited by their cognate sensors-kinases, under nonstimulating conditions, and release of the regulators to the cytoplasm on stimulation. This mechanism might be applicable to signaling cascades in prokaryotes and eukaryotes.