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.

Spatiotemporal Organization of the E. coli Transcriptome: Translation Independence and Engagement in Regulation.

RNA localization in eukaryotes is a mechanism to regulate transcripts fate. Conversely, bacterial transcripts were not assumed to be specifically localized. We previously demonstrated that E. coli mRNAs may localize to where their products localize in a translation-independent manner, thus challenging the transcription-translation coupling extent. However, the scope of RNA localization in bacteria remained unknown. Here, we report the distribution of the E. coli transcriptome between the membrane, cytoplasm, and poles by combining cell fractionation with deep-sequencing (Rloc-seq). Our results reveal asymmetric RNA distribution on a transcriptome-wide scale, significantly correlating with proteome localization and prevalence of translation-independent RNA localization. The poles are enriched with stress-related mRNAs and small RNAs, the latter becoming further enriched upon stress in an Hfq-dependent manner. Genome organization may play a role in localizing membrane protein-encoding transcripts. Our results show an unexpected level of intricacy in bacterial transcriptome organization and highlight the poles as hubs for regulation.

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.

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton.

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria.

Methods for studying RNA localization in bacteria.

The subcellular localization of RNA transcripts provides important insights into biological processes. Hence, understanding the mechanisms underlying RNA targeting is a high priority aim of modern cell biology. The advancements in imaging techniques, such as in situ hybridization and live-cell imaging, coupled with the evolution in optical microscopy led to the discovery that bacterial RNAs, despite the lack of nucleus, are specifically localized. Here we describe the methods used to study RNA localization in bacteria and their applications and discuss their advantages and limitations.

Protein targeting via mRNA in bacteria.

Proteins of all living organisms must reach their subcellular destination to sustain the cell structure and function. The proteins are transported to one of the cellular compartments, inserted into the membrane, or secreted across the membrane to the extracellular milieu. Cells have developed various mechanisms to transport proteins across membranes, among them localized translation. Evidence for targeting of Messenger RNA for the sake of translation of their respective protein products at specific subcellular sites in many eukaryotic model organisms have been accumulating in recent years. Cis-acting RNA localizing elements, termed RNA zip-codes, which are embedded within the mRNA sequence, are recognized by RNA-binding proteins, which in turn interact with motor proteins, thus coordinating the intracellular transport of the mRNA transcripts. Despite the rareness of conventional organelles, first and foremost a nucleus, pieces of evidence for mRNA localization to specific subcellular domains, where their protein products function, have also been obtained for prokaryotes. Although the underlying mechanisms for transcript localization in bacteria are yet to be unraveled, it is now obvious that intracellular localization of mRNA is a common mechanism to spatially localize proteins in both eukaryotes and prokaryotes. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Subcellular localization of RNA and proteins in prokaryotes.

The field of bacterial cell biology has been revolutionized in the last decade by improvements in imaging capabilities which have revealed that bacterial cells, previously thought to be non-compartmentalized, possess an intricate higher-order organization. Many bacterial proteins localize to specific subcellular domains and regulate the spatial deployment of other proteins, DNA and lipids. Recently, the surprising discovery was made that bacterial RNA molecules are also specifically localized. However, the mechanisms that underlie bacterial cell architecture are just starting to be unraveled. The limited number of distribution patterns observed thus far for bacterial proteins and RNAs, and the similarity between the patterns exhibited by these macromolecules, suggest that the processes that underlie their localization are inextricably linked. We discuss these spatial arrangements and the insights that they provide on processes, such as localized translation, protein complex formation, and crosstalk between bacterial machineries.

PafR, a novel transcription regulator, is important for pathogenesis in uropathogenic Escherichia coli.

The metV genomic island in the chromosome of uropathogenic Escherichia coli (UPEC) encodes a putative transcription factor and a sugar permease of the phosphotransferase system (PTS), which are predicted to compose a Bgl-like sensory system. The presence of these two genes, hereby termed pafR and pafP, respectively, has been previously shown to correlate with isolates causing clinical syndromes. We show here that deletion of both genes impairs the ability of the resulting mutant to infect the CBA/J mouse model of ascending urinary tract infection compared to that of the parent strain, CFT073. Expressing the two genes in trans in the two-gene knockout mutant complemented full virulence. Deletion of either gene individually generated the same phenotype as the double knockout, indicating that both pafR and pafP are important to pathogenesis. We screened numerous environmental conditions but failed to detect expression from the promoter that precedes the paf genes in vitro, suggesting that they are in vivo induced (ivi). Although PafR is shown here to be capable of functioning as a transcriptional antiterminator, its targets in the UPEC genome are not known. Using microarray analysis, we have shown that expression of PafR from a heterologous promoter in CFT073 affects expression of genes related to bacterial virulence, biofilm formation, and metabolism. Expression of PafR also inhibits biofilm formation and motility. Taken together, our results suggest that the paf genes are implicated in pathogenesis and that PafR controls virulence genes, in particular biofilm formation genes.

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.

RNA localization in bacteria.

One of the most important discoveries in the field of microbiology in the last two decades is that bacterial cells have intricate subcellular organization. This understanding has emerged mainly from the depiction of spatial and temporal organization of proteins in specific domains within bacterial cells, e.g., midcell, cell poles, membrane and periplasm. Because translation of bacterial RNA molecules was considered to be strictly coupled to their synthesis, they were not thought to specifically localize to regions outside the nucleoid. However, the increasing interest in RNAs, including non-coding RNAs, encouraged researchers to explore the spatial and temporal localization of RNAs in bacteria. The recent technological improvements in the field of fluorescence microscopy allowed subcellular imaging of RNAs even in the tiny bacterial cells. It has been reported by several groups, including ours that transcripts may specifically localize in such cells. Here we review what is known about localization of RNA and of the pathways that determine RNA fate in bacteria, and discuss the possible cues and mechanisms underlying these distribution patterns.

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.

Roles for the Synechococcus elongatus RNA-Binding Protein Rbp2 in Regulating the Circadian Clock.

The cyanobacterial circadian oscillator, consisting of KaiA, KaiB, and KaiC proteins, drives global rhythms of gene expression and compaction of the chromosome and regulates the timing of cell division and natural transformation. While the KaiABC posttranslational oscillator can be reconstituted in vitro, the Kai-based oscillator is subject to several layers of regulation in vivo. Specifically, the oscillator proteins undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localized as a focus at or near the pole of the cell at night. Here, we report that the CI domain of KaiC, when in a hexameric state, is sufficient to target KaiC to the pole. Moreover, increased ATPase activity of KaiC correlates with enhanced polar localization. We identified proteins associated with KaiC in either a localized or diffuse state. We found that loss of Rbp2, found to be associated with localized KaiC, results in decreased incidence of KaiC localization and long-period circadian phenotypes. Rbp2 is an RNA-binding protein, and it appears that RNA-binding activity of Rbp2 is required to execute clock functions. These findings uncover previously unrecognized roles for Rbp2 in regulating the circadian clock and suggest that the proper localization of KaiC is required for a fully functional clock in vivo.

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.

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.

RNA localization in prokaryotes: Where, when, how, and why.

Only recently has it been recognized that the transcriptome of bacteria and archaea can be spatiotemporally regulated. All types of prokaryotic transcripts-rRNAs, tRNAs, mRNAs, and regulatory RNAs-may acquire specific localization and these patterns can be temporally regulated. In some cases bacterial RNAs reside in the vicinity of the transcription site, but in many others, transcripts show distinct localizations to the cytoplasm, the inner membrane, or the pole of rod-shaped species. This localization, which often overlaps with that of the encoded proteins, can be achieved either in a translation-dependent or translation-independent fashion. The latter implies that RNAs carry sequence-level features that determine their final localization with the aid of RNA-targeting factors. Localization of transcripts regulates their posttranscriptional fate by affecting their degradation and processing, translation efficiency, sRNA-mediated regulation, and/or propensity to undergo RNA modifications. By facilitating complex assembly and liquid-liquid phase separation, RNA localization is not only a consequence but also a driver of subcellular spatiotemporal complexity. We foresee that in the coming years the study of RNA localization in prokaryotes will produce important novel insights regarding the fundamental understanding of membrane-less subcellular organization and lead to practical outputs with biotechnological and therapeutic implications. This article is categorized under: RNA Export and Localization > RNA Localization Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Wisdom of the crowds: A suggested polygenic plan for small-RNA-mediated regulation in bacteria.

The omnigenic/polygenic theory, which states that complex traits are not shaped by single/few genes, but by situation-specific large networks, offers an explanation for a major enigma in microbiology: deletion of specific small RNAs (sRNAs) playing key roles in various aspects of bacterial physiology, including virulence and antibiotic resistance, results in surprisingly subtle phenotypes. A recent study uncovered polar accumulation of most sRNAs upon osmotic stress, the majority not known to be involved in the applied stress. Here we show that cells deleted for a handful of pole-enriched sRNAs exhibit fitness defect in several stress conditions, as opposed to single, double, or triple sRNA-knockouts, implying that regulation by sRNA relies on sets of genes. Moreover, analysis of RNA-seq data of Escherichia coli and Salmonella typhimurium exposed to antibiotics and/or infection-relevant conditions reveals the involvement of multiple sRNAs in all cases, in line with the existence of a polygenic plan for sRNA-mediated regulation.

Coupled Transcription-Translation in Prokaryotes: An Old Couple With New Surprises.

Coupled transcription-translation (CTT) is a hallmark of prokaryotic gene expression. CTT occurs when ribosomes associate with and initiate translation of mRNAs whose transcription has not yet concluded, therefore forming "RNAP.mRNA.ribosome" complexes. CTT is a well-documented phenomenon that is involved in important gene regulation processes, such as attenuation and operon polarity. Despite the progress in our understanding of the cellular signals that coordinate CTT, certain aspects of its molecular architecture remain controversial. Additionally, new information on the spatial segregation between the transcriptional and the translational machineries in certain species, and on the capability of certain mRNAs to localize translation-independently, questions the unanimous occurrence of CTT. Furthermore, studies where transcription and translation were artificially uncoupled showed that transcription elongation can proceed in a translation-independent manner. Here, we review studies supporting the occurrence of CTT and findings questioning its extent, as well as discuss mechanisms that may explain both coupling and uncoupling, e.g., chromosome relocation and the involvement of cis- or trans-acting elements, such as small RNAs and RNA-binding proteins. These mechanisms impact RNA localization, stability, and translation. Understanding the two options by which genes can be expressed and their consequences should shed light on a new layer of control of bacterial transcripts fate.

Spatial arrangement of the beta-glucoside transporter from Escherichia coli.

The Escherichia coli BglF protein, a sugar permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), catalyzes concomitant transport and phosphorylation of beta-glucosides across the cytoplasmic membrane. Despite intensive studies of PTS permeases, the mechanism that couples sugar translocation to phosphorylation and the nature of the translocation apparatus are poorly understood. Like many PTS permeases, BglF consists of a transmembrane domain, which in addition to transmembrane helices (TMs) contains a big cytoplasmic loop and two hydrophilic domains, one containing a conserved cysteine that phosphorylates the incoming sugar. We previously reported that the big hydrophilic loop, which connects TM VI to TM VII, contains regions that alternate between facing-in and facing-out states and speculated that it is involved in creating the sugar translocation channel. In the current study we used [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET), a membrane-impermeative thiol-specific reagent, to identify sites that are involved in sugar transport. These sites map to the regions that border the big loop. Using cross-linking reagents that penetrate the cell, we could demonstrate spatial proximity between positions at the center of the big loop and the phosphorylation site, suggesting that the two regions come together to execute sugar phosphotransfer. Additionally, positions on opposite ends of the big loop were found to be spatially close. Cys accessibility analyses suggested that the sugar induces a change in this region. Taken together, our results demonstrate that the big loop participates in creating the sugar pathway and explain the observed coupling between translocation of PTS sugars from the periplasm to the cytoplasm and their phosphorylation.

Genetic dissection of the divergent activities of the multifunctional membrane sensor BglF.

BglF catalyzes beta-glucoside phosphotransfer across the cytoplasmic membrane in Escherichia coli. In addition, BglF acts as a sugar sensor that controls expression of beta-glucoside utilization genes by reversibly phosphorylating the transcriptional antiterminator BglG. Thus, BglF can exist in two opposed states: a nonstimulated state that inactivates BglG by phosphorylation and a sugar-stimulated state that activates BglG by dephosphorylation and phosphorylates the incoming sugar. Sugar phosphorylation and BglG (de)phosphorylation are both catalyzed by the same residue, Cys24. To investigate the coordination and the structural requirements of the opposing activities of BglF, we conducted a genetic screen that led to the isolation of mutations that shift the balance toward BglG phosphorylation. We show that some of the mutants that are impaired in dephosphorylation of BglG retained the ability to catalyze the concurrent activity of sugar phosphotransfer. These mutations map to two regions in the BglF membrane domain that, based on their predicted topology, were suggested to be implicated in activity. Using in vivo cross-linking, we show that a glycine in the membrane domain, whose substitution impaired the ability of BglF to dephosphorylate BglG, is spatially close to the active-site cysteine located in a hydrophilic domain. This residue is part of a newly identified motif conserved among beta-glucoside permeases associated with RNA-binding transcriptional antiterminators. The phenotype of the BglF mutants could be suppressed by BglG mutants that were isolated by a second genetic screen. In summary, we identified distinct sites in BglF that are involved in regulating phosphate flow via the common active-site residue in response to environmental cues.