Catabolite control of Escherichia coli regulatory protein BglG activity by antagonistically acting phosphorylations.

In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.     

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

SUMMARY

The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components.     

How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria.

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.     

How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria

The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens.     

Structure and conformational stability of the enzyme I of Streptomyces coelicolor explored by FTIR and circular dichroism

The bacterial phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS), formed by a cascade of several proteins, couples the translocation and phosphorylation of specific sugars across cell membranes. The structure and thermal stability of the first protein (enzyme I, EI) of the PTS in Streptomyces coelicolor is studied by using far-UV circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) at pH 7.0. The deconvolution of FTIR spectra indicates that the protein is mainly composed by a 35% of alpha-helical structure and 30% of beta-sheet. The thermal denaturation curves, as followed by both techniques, show only a midpoint at 330 K. This thermal denaturation behaviour is different to that observed in other members of the EI family.     

Glucose transport by a mutant of Streptococcus mutans unable to accumulate sugars via the phosphoenolpyruvate phosphotransferase system.

Streptococcus mutans transports glucose via the phosphoenolpyruvate (PEP)-dependent sugar phosphotransferase system (PTS). Earlier studies indicated that an alternate glucose transport system functions in this organism under conditions of high growth rates, low pH, or excess glucose. To identify this system, S. mutans BM71 was transformed with integration vector pDC-5 to generate a mutant, DC10, defective in the general PTS protein enzyme I (EI). This mutant expressed a defective EI that had been truncated by approximately 150 amino acids at the carboxyl terminus as revealed by Western blot (immunoblot) analysis with anti-EI antibody and Southern hybridizations with a fragment of the wild-type EI gene as a probe. Phosphotransfer assays utilizing 32P-PEP indicated that DC10 was incapable of phosphorylating HPr and EIIAMan, indicating a nonfunctional PTS. This was confirmed by the fact that DC10 was able to ferment glucose but not a variety of other PTS substrates and phosphorylated glucose with ATP and not PEP. Kinetic assays indicated that the non-PTS system exhibited an apparent Ks of 125 microM for glucose and a Vmax of 0.87 nmol mg (dry weight) of cells-1 min-1. Sugar competition experiments with DC10 indicated that the non-PTS transport system had high specificity for glucose since glucose transport was not significantly by a 100-fold molar excess of several competing sugar substrates, including 2-deoxyglucose and alpha-methylglucoside. These results demonstrate that S. mutans possesses a glucose transport system that can function independently of the PEP PTS.     

The helical structure propensity in the first helix of the histidine phosphocarrier protein of Streptomyces coelicolor

The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), formed by a cascade of several proteins, mediates the uptake and phosphorylation of carbohydrates, and it is also involved in signal transduction. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or proteins fragments able to interfere the first step of the protein cascade: the phosphorylation of the HPr protein by the enzyme EI. We designed a peptide comprising the active site and the first alpha-helix of HPr of S. coelicolor; we also obtained a fragment of HPr by protein engineering methods, comprising the first forty-eight residues and thus, containing the amino acids of the shorter peptide. Both fragments were disordered in aqueous solution, with a similar percentage of helical structure ( approximately 7 %), and an identical free energy of helix formation. In 40 % TFE, both fragments acquired native-like helical structure, stabilized by non-native hydrophobic interactions, as shown by the 2D-NMR assignments of the shorter peptide, and the presence of similar NOE contacts in both fragments. These findings, with the kinetic results in other members of the HPr family, highlight the importance of short- and long-range interactions during the folding reaction of HPr proteins. Based on the residual helical population, hypothesis about the inhibition capacity of the PTS by both fragments are discussed.     

Replacing the general energy-coupling proteins of the phospho-enol-pyruvate: sugar phosphotransferase system of Salmonella typhimurium with fructose-inducible counterparts results in the inability to utilize nonphosphotransferase system sugars

A Salmonella typhimurium mutant lacking Enzyme I and HPr, general proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but producing homologues EI(Fructose) and FPr constitutively, did not grow in minimal medium supplemented with non-PTS sugars (melibiose, glycerol, and maltose) in the absence of any trace of Luria-Bertani broth; adding cyclic AMP allowed growth. On melibiose, rapid growth began only when melibiose permease activity had reached a threshold level. Wild-type cultures reached this level within about 2 h, but the mutant only after a 12-14 h lag period, and then only when cyclic AMP had been added to the medium. On a mixture of melibiose and a PTS sugar, permease was undetectable in either the wild type or mutant until the PTS sugar had been exhausted. Permease then appeared, increasing with time, but in the mutant it never reached the threshold allowing rapid growth on melibiose unless cyclic AMP had been added. On rich medium supplemented with melibiose or glycerol, the mutant produced lower (30%) levels of melibiose permease or glycerol kinase compared with the wild type. We propose that poor phosphorylation of the regulatory protein Enzyme IIA(Glucose), leading to constitutive inducer exclusion and catabolite repression in this strain, accounts for these results.     

Regulation of sugar uptake via the phosphoenolpyruvate-dependent phosphotransferase systems in Bacillus subtilis and Lactococcus lactis is mediated by ATP-dependent phosphorylation of seryl residue 46 in HPr.

By using both metabolizable and nonmetabolizable sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), we show that PTS sugar uptake into intact cells and membrane vesicles of Lactococcus lactis and Bacillus subtilis is strongly inhibited by high concentrations of any of several metabolizable PTS sugars. Inhibition requires phosphorylation of seryl residue 46 in the phosphocarrier protein of the PTS, HPr, by the metabolite-activated, ATP-dependent protein kinase. Inhibition does not occur when wild-type HPr is replaced by the S46A mutant form of this protein either in vesicles of L. lactis or B. subtilis or in intact cells of B. subtilis. Nonmetabolizable PTS sugar analogs such as 2-deoxyglucose inhibit PTS sugar uptake by a distinct mechanism that is independent of HPr(ser-P) and probably involves cellular phosphoenolpyruvate depletion.     

Evidence of in vivo cross talk between the nitrogen-related and fructose-related branches of the carbohydrate phosphotransferase system of Pseudomonas putida

The genome of Pseudomonas putida KT2440 encodes only five recognizable proteins belonging to the phosphoenolpyruvate (PEP)-carbohydrate phosphotransferase system (PTS). Two of these PTS constituents (FruA and FruB) form a complete system for fructose intake. The other three products, encoded by ptsP (EI(Ntr)), ptsO (NPr), and ptsN (EIIA(Ntr)), comprise a branch of the system unrelated to sugar traffic but thought to have an influence on coordination of N and C metabolism. We used a genetic approach to clarify the course of high-energy phosphate through this reduced set of PTS proteins. To this end, we monitored the phosphorylation state in vivo of the EIIA(Ntr) enzyme in various genetic backgrounds and growth conditions. Our results show that the source of phosphate available to the system is PEP and that the primary flow of phosphate through the N/C-sensing PTS proceeds from PEP to EI(Ntr) to NPr to EIIA(Ntr). We also found that in the presence of fructose, unlike in the presence of succinate, EIIA(Ntr) can be phosphorylated in a ptsP strain but not in a ptsP fruB double mutant. This result revealed that the fructose transport system has the ability to cross talk in vivo with the N-related PTS branch. The data reported here thus document an unexpected connection in vivo between the sugar-dependent and sugar-independent PTSs.