A novel sugar-stimulated covalent switch in a sugar sensor.

The bgl sensory system is composed of a membrane-bound sugar sensor, BglF, and a transcriptional regulator, BglG. The sensor BglF has several enzymatic activities: in its nonstimulated state, it acts as BglG phosphorylase; in the presence of beta-glucoside in the growth medium, it acts as BglG dephosphorylase and as the beta-glucoside phosphotransferase. The same active site on BglF, Cys-24, is responsible for the phosphorylation of both the stimulating sugar and the BglG protein. BglF is composed of three domains, two hydrophilic and one hydrophobic. Our previous results suggested that catalysis of the sugar-stimulated functions depends on specific interactions between the B domain, which contains the active site cysteine, and the integral membrane C domain. We report here that the stimulating sugar triggers the formation of a disulfide bond between the active site cysteine and another cysteine in the membrane-embedded domain of BglF. Inability of a mutant BglF protein to form the disulfide bridge between the B and C domains correlates with its inability to catalyze the sugar-stimulated functions. The ability of the cysteine residue in BglF to bind covalently either to a phosphoryl group or to another cysteine residue, depending on the protein stimulation state, suggests a novel way to control signaling by alternative bond formation.

Dephosphorylation of the Escherichia coli transcriptional antiterminator BglG by the sugar sensor BglF is the reversal of its phosphorylation.

The Escherichia coli BglF protein catalyzes transport and phosphorylation of beta-glucosides. In addition, BglF is a membrane sensor which reversibly phosphorylates the transcriptional regulator BglG, depending on beta-glucoside availability. Therefore, BglF has three enzymatic activities: beta-glucoside phosphotransferase, BglG phosphorylase, and phospho-BglG (BglG-P) dephosphorylase. Cys-24 of BglF is the active site which delivers the phosphoryl group either to the sugar or to BglG. To characterize the dephosphorylase activity, we asked whether BglG-P can give the phosphoryl group back to Cys-24 of BglF. Here we provide evidence which is consistent with the interpretation that Cys-24-P is an intermediate in the BglG-P dephosphorylation reaction. Hence, the dephosphorylation reaction catalyzed by BglF proceeds via reversal of the phosphorylation reaction.

BglG, the transcriptional antiterminator of the bgl system, interacts with the beta' subunit of the Escherichia coli RNA polymerase.

The Escherichia coli BglG protein antiterminates transcription at two terminator sites within the bgl operon in response to the presence of beta-glucosides in the growth medium. BglG was previously shown to be an RNA-binding 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 beta' subunit of RNA polymerase as the target for BglG binding. The region that contains the binding site for BglG was mapped to the N-terminal region of beta'. The beta' subunit, produced in excess, prevented BglG activity as a transcriptional antiterminator. Possible roles of the interaction between BglG and the polymerase beta' subunit are discussed.

Characterization of the dimerization domain in BglG, an RNA-binding transcriptional antiterminator from Escherichia coli.

The Escherichia coli transcriptional antiterminator protein BglG inhibits transcription termination of the bgl operon in response to the presence of beta-glucosides in the growth medium. BglG is an RNA-binding protein that recognizes a specific sequence partially overlapping the two terminators within the bgl transcript. The activity of BglG is determined by its dimeric state which is modulated by reversible phosphorylation. Thus, only the nonphosphorylated dimer binds to the RNA target site and allows readthrough of transcription. Genetic systems which test dimerization and antitermination in vivo were used to map and delimit the region which mediates BglG dimerization. We show that the last 104 residues of BglG are required for dimerization. Any attempt to shorten this region from the ends or to introduce internal deletions abolished the dimerization capacity of this region. A putative leucine zipper motif is located at the N terminus of this region. The role of the canonical leucines in dimerization was demonstrated by their substitution. Our results also suggest that the carboxy-terminal 70 residues, which follow the leucine zipper, contain another dimerization domain which does not resemble any known dimerization motif. Each of these two regions is necessary but not sufficient for dimerization. The BglG phosphorylation site, His208, resides at the junction of the two putative dimerization domains. Possible mechanisms by which the phosphorylation of BglG controls its dimerization and thus its activity are discussed.

BglF, the Escherichia coli beta-glucoside permease and sensor of the bgl system: domain requirements of the different catalytic activities.

The Escherichia coli BglF protein, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system, has several enzymatic activities. In the absence of beta-glucosides, it phosphorylates BglG, a positive regulator of bgl operon transcription, thus inactivating BglG. In the presence of beta-glucosides, it activates BglG by dephosphorylating it and, at the same time, transports beta-glucosides into the cell and phosphorylates them. BglF is composed of two hydrophilic domains, IIAbgl and IIBbgl, and a membrane-bound domain, IICbgl, which are covalently linked in the order IIBCAbgl. Cys-24 in the IIBbgl domain is essential for all the phosphorylation and dephosphorylation activities of BglF. We have investigated the domain requirement of the different functions carried out by BglF. To this end, we cloned the individual BglF domains, as well as the domain pairs IIBCbgl and IICAbgl, and tested which domains and which combinations are required for the catalysis of the different functions, both in vitro and in vivo. We show here that the IIB and IIC domains, linked to each other (IIBCbgl), are required for the sugar-driven reactions, i. e., sugar phosphotransfer and BglG activation by dephosphorylation. In contrast, phosphorylated IIBbgl alone can catalyze BglG inactivation by phosphorylation. Thus, the sugar-induced and noninduced functions have different structural requirements. Our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgl and IICbgl which occur upon the interaction of BglF with the sugar.

The different functions of BglF, the E. coli beta-glucoside permease and sensor of the bgl system, have different structural requirements.

The Escherichia coli BglF protein (EIIbgl) is an Enzyme II (EII) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) which catalyses transport and phosphorylation of beta-glucosides. In addition to its transport function, BglF serves as a beta-glucoside sensor which reversibly phosphorylates BglG, the transcription regulator of the bgl operon. Like many other PTS sugar permeases, the BglF protein is composed of three discrete functional and structural domains: IIAbgl and IIBbgl, which are hydrophilic, and IICbgl, which is hydrophobic. The domains of BglF are covalently linked to one another in the order BCA. The IIAbgl domain contains the first phosphorylation site, which accepts a phosphoryl group from the general PTS protein HPr and delivers it to the second phosphorylation site, located in the IIBbgl domain. This second site can deliver the phosphoryl group either to a beta-glucoside or to BglG. To elucidate the mechanism by which such different substrates can be phosphorylated by the same active site, we decided to try to separate the different phosphorylation activities catalyzed by BglF. To this end we rearranged the BglF domains and constructed IICBAbgl (scrambled-BglF). Scrambled-BglF behaved like wild-type BglF in its ability to be phosphorylated and to phosphorylate BglG in vitro and in vivo. However, it could not catalyze phosphorylation of beta-glucosides in vitro nor their phosphotransfer in vivo, and it could not catalyze BglG dephosphorylation in vitro or in vivo. Therefore, the two reactions induced by beta-glucosides, sugar phosphorylation and BglG dephosphorylation, seem to require a specific domain organization: IIBbgl should precede IICbgl. The order of the B and C domains is irrelevant for BglG phosphorylation, which occurs in the absence of beta-glucosides. Because the domain order affects the way that the domains are able to interact, our results suggest that catalysis of the sugar-induced functions depends on specific interactions between IIBbgl and IICbgl. In light of the previous assumption that domain order in EIIs is immaterial for their function, the finding that the order of the domains is important for the function of BglF as a sugar phosphotransferase raises two possibilities: (a) BglF differs from other EIIs in this regard; (b) BglF represents a subgroup of EIIs in which the requirement for a specific domain order correlates with the ability to transport a set of structurally related sugars.

BglF, the sensor of the bgl system and the beta-glucosides permease of Escherichia coli: evidence for dimerization and intersubunit phosphotransfer.

The Escherichia coli BglF protein, also designated EIIbgl, is an enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) that catalyzes transport and phosphorylation of beta-glucosides. In addition, BglF has the ability, unusual for an EII, to regulate the activity of a transcriptional regulator, BglG, by phosphorylating and dephosphorylating it according to beta-glucoside availability. Together, BglF and BglG constitute a novel sensory system. The membrane-bound sensor, BglF, has two phosphorylation sites: site 1 accepts a phosphoryl group from HPr and delivers it to site 2; site 2 delivers the phosphoryl group either to beta-glucosides or to BglG. Here, we provide several lines of evidence for the dimerization of BglF and for the occurrence of productive intersubunit phosphotransfer within the BglF dimers. (1) Two inactive BglF mutant proteins, one lacking phosphorylation site 1 and the other lacking site 2, complement one another to allow beta-glucoside utilization by bglF strains. (2) The pairs of mutant proteins complement one another in regulating BglG activity as a transcriptional antiterminator in vivo. (3) Only when they are present in the same membrane preparation do the mutant protein pairs efficiently transfer the phosphoryl group from HPr to beta-glucosides and to BglG in vitro. (4) Gentle extraction of cellular proteins followed by SDS-PAGE reveals the existence of BglF homodimers. A portion of the phosphorylated form of BglF can also be extracted from the membrane as a dimer. Dimerization is mediated by the membrane-bound IICbgl domain, as indicated by the dimerization of IICbgl by itself and of BglF derivatives that contain this domain. Since dimers persist in the presence of a reducing agent, they are apparently not held together by disulfide bonds. Rather, BglF dimerization might involve hydrophobic interactions between residues in the membrane-spanning domain. In addition, we show that BglF dimerization is not modulated by beta-glucosides and is therefore not part of the mechanism that diverts the phosphoryl group away from BglG to the transported sugar upon addition of beta-glucosides to the growth medium.

SacY, a transcriptional antiterminator from Bacillus subtilis, is regulated by phosphorylation in vivo.

SacY antiterminates transcription of the sacB gene in Bacillus subtilis in response to the presence of sucrose in the growth medium. We have found that it can substitute for BglG, a homologous protein, in antiterminating transcription of the bgl operon in Escherichia coli. We therefore sought to determine whether, similarly to BglG, SacY is regulated by reversible phosphorylation in response to the availability of the inducing sugar. We show here that two forms of SacY, phosphorylated and nonphosphorylated, exist in B. subtilis cells and that the ratio between them depends on the external level of sucrose. Addition of sucrose to the growth medium after SacY phosphorylation in the cell resulted in its rapid dephosphorylation. The extent of SacY phosphorylation was found to be proportional to the cellular levels of SacX, a putative sucrose permease which was previously shown to have a negative effect on SacY activity. Thus, the mechanism by which the sac sensory system modulates sacB expression in response to sucrose involves reversible phosphorylation of the regulator SacY, and this process appears to depend on the SacX sucrose sensor. The sac system is therefore a member of the novel family of sensory systems represented by bgl.

BglG, the response regulator of the Escherichia coli bgl operon, is phosphorylated on a histidine residue.

We have shown previously that the activity of BglG, the response regulator of the bgl system, as a transcriptional antiterminator is modulated by the sensor BglF, which reversibly phosphorylates BglG. We show here that the phosphoryl group on BglG is present as a phosphoramidate, based on the sensitivity of phosphorylated BglG to heat, hydroxylamine, and acidic but not basic conditions. By analyzing the products of base-hydrolyzed phosphorylated BglG by thin-layer chromatography, we show that the phosphorylation occurs on a histidine residue. This result supports the notion that the bgl system is a member of a new family of bacterial sensory systems.

BglF, the sensor of the E. coli bgl system, uses the same site to phosphorylate both a sugar and a regulatory protein.

The Escherichia coli BglF protein is a sugar permease that is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). It catalyses transport and phosphorylation of beta-glucosides. In addition to its ability to phosphorylate its sugar substrate, BglF has the unusual ability to phosphorylate and dephosphorylate the transcriptional regulator BglG according to beta-glucoside availability. 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. BglF has two phosphorylation sites. The first site accepts a phosphoryl group from the PTS protein HPr; the phosphoryl group is then transferred to the second phosphorylation site, which can deliver it to the sugar. We provide both in vitro and in vivo evidence that the same phosphorylation site on BglF, the second one, is in charge not only of sugar phosphorylation but also of BglG phosphorylation. Possible mechanisms that ensure correct phosphoryl delivery to the right entity, sugar or protein, depending on environmental conditions, are discussed.

The localization of the phosphorylation site of BglG, the response regulator of the Escherichia coli bgl sensory system.

BglG, the response regulator of the bgl sensory system, was recently shown to be phosphorylated on a histidine residue. We report here the localization of the phosphorylation site to histidine 208. Localization of the phosphorylated histidine was carried out in two steps. We first engineered BglG derivatives with a specific protease (factor Xa) cleavage site that allowed asymmetric splitting of each prephosphorylated protein to well defined peptides, of which only one was labeled by radioactive phosphate. This allowed the localization of the phosphorylation site to the last 111 residues. Subsequently, we identified the phosphorylated histidine by mutating each of the three histidines located in this region to an arginine and following the ability of the resulting mutants to be in vivo regulated and in vitro phosphorylated by BglF, the bgl system sensor. Histidine 208 was the only histidine which failed both tests. The use of simple techniques to map the phosphorylation site should make this protocol applicable for the localization of phosphorylation sites in other proteins. The bgl system represents a new family of sensory systems. Thus, the mapping reported here is an important step toward the definition of the functional domains involved in the transduction of a signal by the components that constitute systems of this novel family.

Modulation of the dimerization of a transcriptional antiterminator protein by phosphorylation.

The transcriptional antiterminator protein BglG inhibits transcription termination of the bgl operon in Escherichia coli when it is in the nonphosphorylated state. The BglG protein is now shown to exist in two configurations, an active, dimeric nonphosphorylated form and an inactive, monomeric phosphorylated form. The migration of BglG on native polyacrylamide gels was consistent with it existing as a dimer when nonphosphorylated and as a monomer when phosphorylated. Only the nonphosphorylated dimer was found to bind to the target RNA. When the dimerization domain of the lambda repressor was replaced with BglG, the resulting chimera behaved like an intact lambda repressor in its ability to repress lambda gene expression, which suggests that BglG dimerizes in vivo. Repression by the lambda-BglG hybrid was significantly reduced by BglF, the BglG kinase, an effect that was relieved by conditions that stimulate dephosphorylation of BglG by BglF. These results suggest that the phosphorylation and the dephosphorylation of BglG regulate its activity by controlling its dimeric state.

Regulation of activity of a transcriptional anti-terminator in E. coli by phosphorylation in vivo.

Expression of the bgl operon of Escherichia coli is regulated in vitro by phosphorylation and dephosphorylation of a positive regulatory protein, BglG, which functions in its nonphosphorylated state as a transcriptional antiterminator. The degree of phosphorylation of BglG in vivo was shown to be dependent on the cellular levels of BglF protein, which is both the BglG kinase and phosphatase. The degree of phosphorylation of BglG also depended on the presence or absence of a beta-glucoside, the inducer of operon expression. Addition of inducer to cells in growth medium resulted in rapid dephosphorylation of phosphorylated BglG. The bgl operon is thus regulated by a sensory system that modulates gene expression by protein phosphorylation and dephosphorylation in response to the external levels of inducer.

Protein phosphorylation regulates transcription of the beta-glucoside utilization operon in E. coli.

We have investigated the interaction between BglF and BglG, two proteins that regulate expression of the E. coli bgl operon. BglF is both a negative regulator of operon expression and a phosphotransferase involved in uptake of beta-glucosides. BglG is a positive regulator that functions as a transcriptional antiterminator. We show here that BglF is phosphorylated by the soluble components of the phosphotransferase system: Enzyme I, HPr, and the phosphate donor phosphoenolpyruvate. Phosphorylated BglF can then transfer phosphate either to beta-glucosides or to wild-type BglG. Mutant BglG derivatives, which give constitutive expression of the bgl operon, show little or no phosphorylation by BglF. Hence BglF exerts its negative effect on operon expression by phosphorylating BglG, blocking its action as an antiterminator. BglG is dephosphorylated only in the presence of both BglF and beta-glucosides. Based on these results, we propose the following mechanism: In the absence of beta-glucosides, BglG is phosphorylated by BglF and is inactive in antitermination. Addition of inducer stimulates BglF to dephosphorylate BglG, allowing BglG to function as a positive regulator of operon expression. Beta-Glucosides are then phosphorylated and transported into the cell by BglF.

Efficient and accurate in vitro processing of simian virus 40-associated small RNA.

Nuclei were isolated from simian virus 40 (SV40)-infected cells with a hypotonic, detergent-free buffer and incubated in vitro in a high-ionic-strength buffer containing [alpha-32P]UTP. The labeled viral RNAs produced were analyzed by gel electrophoresis together with 3-h-labeled viral RNAs extracted from SV40-infected cells. The in vitro-synthesized RNA contained a major RNA species of 62 to 64 nucleotides that appeared on the gel at the same position as in vivo-synthesized SV40-associated small RNA (SAS-RNA). Analyses of the in vitro-synthesized 62- to 64-nucleotide RNA by hybridization to restriction fragments and by the use of an SAS-RNA deletion mutant clearly identified it as SAS-RNA. The intensity of the band of the in vitro-synthesized SAS-RNA increased with an increase in the labeling time or when a short pulse was followed by a chase. Moreover, the SAS-RNA band disappeared when ITP replaced GTP in the transcription reaction mixture. These results indicate that SAS-RNA is processed from a precursor molecule and that an RNA secondary structure could be an element recognized by the processing enzyme.