Dephosphorylation of the
            <i>Escherichia coli</i>
            Transcriptional Antiterminator BglG by the Sugar Sensor BglF Is the Reversal of Its Phosphorylation

Chen, Qing; Postma, Pieter W.; Amster‐Choder, Orna

doi:10.1128/jb.182.7.2033-2036.2000

Cited by 17 publications

(15 citation statements)

References 28 publications

(16 reference statements)

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“…The A domain is phosphorylated by HPr; the phosphate is then transferred to the B domain and subsequently to ␤-glucosides; the membrane-spanning C domain presumably forms the sugar translocation channel and at least part of the sugar-binding site. Cys-24, residing in the B domain, was shown to be responsible for the phosphorylation of both the incoming sugar and the BglG protein and for the dephosphorylation of P-BglG (13,14). The mechanism that ensures correct delivery of the phosphoryl group to the right entity, sugar or protein, by the same active site is not known.…”

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Dynamic Membrane Topology of the Escherichia coli β-Glucoside Transporter BglF

Yagur‐Kroll

Amster‐Choder

2005

Journal of Biological Chemistry

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The Escherichia coli BglF protein, a permease of the phosphoenolpyruvate-dependent phosphotransferase system, catalyzes transport and phosphorylation of ␤-glucosides. In addition, BglF regulates bgl operon expression by controlling the activity of the transcriptional regulator BglG via reversible phosphorylation. BglF is composed of three domains; one is hydrophobic, which presumably forms the sugar translocation channel. We studied the topology of this domain by Cysreplacement mutagenesis and chemical modification by thiol reagents. Most Cys substitutions were well tolerated, as demonstrated by the ability of the mutant proteins to catalyze BglF activities. Our results suggest that the membrane domain contains eight transmembrane helices and an alleged cytoplasmic loop that contains two additional helices. The latter region forms a dynamic structure, as evidenced by the alternation of residues near its ends between faced-in and faced-out states. We suggest that this region, together with the two transmembrane helices encompassing it, forms the sugar translocation channel. BglF periplasmic loops are close to the membrane, the first being a reentrant loop. This is the first systematic topological study carried out with an intact phosphotransferase system permease and the first demonstration of a reentrant loop in this group of proteins.The Escherichia coli BglF protein (EII bgl ), an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS), 1 catalyzes concomitant transport and phosphorylation of ␤-glucosides across the cytoplasmic membrane (1). In addition to its ability to phosphorylate its sugar substrate, BglF phosphorylates the transcriptional regulator BglG in the absence of ␤-glucosides and dephosphorylates P-BglG upon addition of ␤-glucosides to the growth medium (2, 3). By controlling the phosphorylation state of BglG, BglF controls the dimeric state of BglG and, thus, its ability to bind RNA and antiterminate transcription of the bgl operon (4).BglF dimerizes spontaneously in the membrane, and its dimeric form can catalyze all the above mentioned activities (5). Several other enzymes II of PTS were shown to regulate activity of transcription factors. Two representative examples are SacX, a sucrose permease from Bacillus subtilis, which regulates the activity of the BglG homologue SacY by reversible phosphorylation (6, 7), and PstG, a glucose permease from E. coli, which regulates the activity of the global regulator Mlc by membrane sequestration (8, 9).The phosphate flux in PTS starts with a phosphoryl group, donated by phosphoenolpyruvate, which is passed through the general PTS proteins, enzyme I, and HPr, to the various sugar-specific permeases. Like many other PTS permeases, BglF is composed of three domains: the hydrophilic A and B domains (IIA bgl and IIB bgl ) and the hydrophobic C domain (IIC bgl ) (reviewed in Refs. 10 -12). The domains of BglF are covalently linked to one another in the order BCA. The A domain is phosphorylated by HPr; the phosphate is then t...

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Dynamic Membrane Topology of the Escherichia coli β-Glucoside Transporter BglF

Yagur‐Kroll

Amster‐Choder

2005

Journal of Biological Chemistry

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“…Cys-24, located in the B domain of BglF, is involved in the catalysis of both activities (10,13). To address the question of how the opposing functions of BglF are orchestrated, we attempted to isolate BglF mutants that exhibit normal ability to phosphorylate BglG but no ability or a strongly reduced ability to dephosphorylate it, i.e., mutants that inactivate BglG normally but are defective in BglG activation in the presence of the stimulating sugar.…”

Section: Isolation Of Bglf Mutants Impaired In Bglg Activation But Nomentioning

confidence: 99%

“…In any case, in the absence of ␤-glucosides, BglF directly phosphorylates BglG; upon ␤-glucoside addition, BglF dephosphorylates BglG in a manner that does not depend on the general PTS proteins (2, 10) (see Discussion). Notably, the same active site in BglF, Cys24, phosphorylates both the sugar and the BglG protein and also accepts the phosphate from phospho-BglG (P-BglG) (10,13). The mechanism by which the sugar shifts the balance and triggers BglF to switch from the nonstimulated mode to the stimulated mode is still poorly understood.…”

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Genetic Dissection of the Divergent Activities of the Multifunctional Membrane Sensor BglF

Monderer-Rothkoff

Amster‐Choder

2007

J Bacteriol

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BglF catalyzes ␤-glucoside phosphotransfer across the cytoplasmic membrane in Escherichia coli. In addition, BglF acts as a sugar sensor that controls expression of ␤-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 ␤-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.Proteins that execute different and/or opposing reactions depending on environmental conditions provide an opportunity to explore molecular switches. By and large, phosphorylation and dephosphorylation of proteins are carried out by separate factors, although there are proteins that catalyze both activities. One example is the group of kinases of the bacterial two-component systems that often act also as phosphatases of their cognate response regulators, e.g., EnvZ and NRII, but apparently they do so by stimulating autodephosphorylation by the regulators (35, 55). Another example is provided by proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Most phosphorylation reactions carried out by PTS proteins are reversible, and the direction of the reaction being catalyzed depends on carbohydrate availability (5, 56). This report focuses on BglF, which serves as a paradigm for a group of PTS sugar permeases that control expression of sugar utilization genes by reversibly phosphorylating transcriptional antiterminators depending on the availability of their cognate sugars (reviewed in references 1 and 67). BglF, the ␤-glucoside phosphotransferase from Escherichia coli, negatively regulates expression of the bgl (␤-glucosides utilization) operon in the ab...

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“…The A domain is phosphorylated at H547 by HPr; the phosphate is then transferred to C24 in the B domain and subsequently to the incoming ␤-glucosides (8, 28). In addition, BglF regulates the transcriptional antiterminator BglG, depending on ␤-glucoside availability, by reversibly phosphorylating it via the same residue that phosphorylates the incoming sugar, C24 (2,3,4,8,9).The membrane domain of PTS permeases is assumed to form the sugar translocation channel. The nature of the channel and the mechanism that guarantees coupling between translocation of PTS sugars and phosphorylation are poorly understood (10,20).…”

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Spatial Arrangement of the β-Glucoside Transporter fromEscherichia coli

2009

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The Escherichia coli BglF protein, a sugar permease of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), catalyzes concomitant transport and phosphorylation of ␤-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.The phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) is a central system in bacteria that controls preferential use of carbon sources. In addition to catabolite repression and inducer exclusion, the PTS is responsible for the translocation of a variety of energetically favorable carbohydrates across the cytoplasmic membrane and for their concomitant phosphorylation (10). It consists of two general PTS proteins, enzyme I and HPr, and a number of sugar-specific permeases. The phosphorylation cascade starts with enzyme I, which accepts a phosphoryl group from phosphoenolpyruvate and passes it on to HPr; the latter molecule phosphorylates the sugar-specific permeases. BglF (also designated EII bgl ) is a PTS permease that catalyzes phosphotransfer of ␤-glucosides into Escherichia coli cells (13). Like many other PTS permeases, BglF is composed of three conserved domains (for reviews, see references 27 and 22): the hydrophilic A and B domains and the membrane C domain (corresponding to the IIA bgl , IIB bgl , and IIC bgl domains, respectively). The A domain is phosphorylated at H547 by HPr; the phosphate is then transferred to C24 in the B domain and subsequently to the incoming ␤-glucosides (8, 28). In addition, BglF ...

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Dephosphorylation of the Escherichia coli Transcriptional Antiterminator BglG by the Sugar Sensor BglF Is the Reversal of Its Phosphorylation

Cited by 17 publications

References 28 publications

Dynamic Membrane Topology of the Escherichia coli β-Glucoside Transporter BglF

Dynamic Membrane Topology of the Escherichia coli β-Glucoside Transporter BglF

Genetic Dissection of the Divergent Activities of the Multifunctional Membrane Sensor BglF

Spatial Arrangement of the β-Glucoside Transporter fromEscherichia coli

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