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.

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 β-glucoside-specific permease, BglF, localizes to the cell membrane. EI, HPr and BglF control the β-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 β-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.

The Escherichia coli BglF protein is a sugar-sensor that controls the activity of the transcriptional antiterminator BglG by reversibly phosphorylating it, depending on ␤-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 ␤-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.

BglG, which regulates expression of the ␤-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 ␤-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 uptakeindependent 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.bgl system ͉ E. coli

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.

Expression of the bgl operon in Escherichia coli, induced by ␤-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 membranebound enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which reversibly phosphorylates BglG according to ␤-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.

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The Escherichia coli BglG protein antiterminates transcription at two terminator sites within the bgl operon in response to the presence of ␤-glucosides in the growth medium. BglG was previously shown to be an RNAbinding protein that recognizes a specific sequence located just upstream of each of the terminators and partially overlapping with them. We show here that BglG also binds to the E. coli RNA polymerase, both in vivo and in vitro. By using several techniques, we identified the ␤ subunit of RNA polymerase as the target for BglG binding. The region that contains the binding site for BglG was mapped to the Nterminal region of ␤. The ␤ subunit, produced in excess, prevented BglG activity as a transcriptional antiterminator. Possible roles of the interaction between BglG and the polymerase ␤ subunit are discussed.The bgl operon in Escherichia coli, induced by ␤-glucosides, is regulated by two of its gene products, BglG, a transcriptional regulator, and BglF, a membrane-bound sensor (1). Transcription from the bgl promoter initiates constitutively, but in the absence of inducer, most transcripts terminate prematurely at one of two -independent terminators within the operon; in the presence of inducer, BglG prevents termination at these sites (2, 3). The mechanism by which BglG antiterminates transcription differs from the antitermination mechanisms which operate in and the mechanisms of attenuation at amino acid biosynthetic operons, as well as at the pyrB1 and ampC operons (4). BglG is an RNA-binding protein that recognizes and binds to a specific sequence on the bgl transcript, which partially overlaps with each of the bgl terminators (5). BglF, the ␤-glucosides phosphotransferase, regulates the activity of BglG by reversible phosphorylation (6-9), which modulates BglG dimeric state (10). The phosphorylation and dimerization sites on BglG were recently mapped (11-13).Systems that resemble the bgl system were identified in different organisms (14). The most studied are the two sac systems in Bacillus subtilis, which regulate transcription of sacB and sacPA according to sucrose availability. Expression of these loci is regulated at the level of transcription antitermination by the two BglG homologues, SacY and SacT,. Similarly to BglG, SacY is reversibly phosphorylated in vivo (18). The RNA sequences recognized by SacY and SacT closely resemble the target site for BglG in the bgl transcript (19, 16) and also have the potential to fold into a stem-loop structure that partially overlaps with the respective terminators (5, 20). † It was suggested that the antiterminators of the BglG family block the formation of the terminator structures by stabilizing an alternative RNA conformation. The question then arises as to whether interaction with the nascent RNA chain is sufficient for implementing antitermination or additional interactions with the transcription machinery are required. In this paper we provide both in vivo and in vitro evidence for the interaction of BglG with the ␤Ј subunit of E. coli RNA poly...