Characterization of Streptococcus mutans strains deficient in EIIAB Man of the sugar phosphotransferase system

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression.     

The glucose-specific carrier of the Escherichia coli phosphotransferase system.

Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate-sugar phosphotransferase system (PTS). 2',3'-Epoxypropyl beta-d-glucopyranoside (3a) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates. They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc-specific but structurally different transporter. 3a is the only analogue that inhibits EIIGlc by binding to the high-affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro, methyl 6,7-anhydro-d,l-glycero-alpha-d-gluco-heptopyranoside (1d), preferentially interacts with the low-affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EIIGlc is rapidly inactivated by the 6-O-bromoacetyl esters of methyl alpha-d-glucopyranoside (1a) and methyl alpha-d-mannopyranoside (1c), methyl 6-deoxy-6-isothiocyanato-alpha-d-glucopyranoside (1e), beta-d-glucopyranosyl isothiocyanate (3c) and beta-d-glucopyranosyl phenyl isothiocyanate (3d). Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421. Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose-induced conformational changes in the dimeric transporter. Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells. This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.     

The phosphoenolpyruvate-dependent glucose-phosphotransferase system from Escherichia coli K-12 as the center of a network regulating carbohydrate flux in the cell

The phosphoenolpyruvate-(PEP)-dependent-carbohydrate:phosphotransferase systems (PTSs) of enteric bacteria constitute a complex transport and sensory system. Such a PTS usually consists of two cytoplasmic energy-coupling proteins, Enzyme I (EI) and HPr, and one of more than 20 different carbohydrate-specific membrane proteins named Enzyme II (EII), which catalyze the uptake and concomitant phosphorylation of numerous carbohydrates. The most prominent representative is the glucose-PTS, which uses a PTS-typical phosphorylation cascade to transport and phosphorylate glucose. All components of the glucose-PTS interact with a large number of non-PTS proteins to regulate the carbohydrate flux in the bacterial cell. Several aspects of the glucose-PTS have been intensively investigated in various research projects of many groups. In this article we will review our recent findings on a Glc-PTS-dependent metalloprotease, on the interaction of EIICB(Glc) with the regulatory peptide SgrT, on the structure of the membrane spanning C-domain of the glucose transporter and on the modeling approaches of ptsG regulation, respectively, and discuss them in context of general PTS research.     

Genetic and biochemical characterization of the phosphoenolpyruvate:glucose/mannose phosphotransferase system of Streptococcus thermophilus

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB(Man), two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB(Man), IIC(Man), IID(Man), and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB(Man) as well as membrane fragments containing IIC(Man) and IID(Man). These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB(Man).     

The phosphotransferase system of Corynebacterium glutamicum: features of sugar transport and carbon regulation

In this review, we describe the phosphotransferase system (PTS) of Corynebacterium glutamicum and discuss genes for putative global carbon regulation associated with the PTS. C. glutamicum ATCC 13032 has PTS genes encoding the general phosphotransferases enzyme I, HPr and four enzyme II permeases, specific for glucose, fructose, sucrose and one yet unknown substrate. C. gluamicum has a peculiar sugar transport system involving fructose efflux after hydrolyzing sucrose transported via sucrose EII. Also, in addition to their primary PTS, fructose and glucose are each transported by a second transporter, glucose EII and a non-PTS permease, respectively. Interestingly, C. glutamicum does not show any preference for glucose, and thus co-metabolizes glucose with other sugars or organic acids. Studies on PTS-mediated sugar uptake and its related regulation in C. glutamicum are important because the production yield of lysine and cell growth are dependent on the PTS sugars used as substrates for fermentation. In many bacteria, the PTS is also involved in several regulatory processes. However, the detailed molecular mechanism of global carbon regulation associated with the PTS in this organism has not yet been revealed.     

Mlc of Thermus thermophilus: a glucose-specific regulator for a glucose/mannose ABC transporter in the absence of the phosphotransferase system

We report the presence of Mlc in a thermophilic bacterium. Mlc is known as a global regulator of sugar metabolism in gram-negative enteric bacteria that is controlled by sequestration to a glucose-transporting EII(Glc) of the phosphotransferase system (PTS). Since thermophilic bacteria do not possess PTS, Mlc in Thermus thermophilus must be differently controlled. DNA sequence alignments between Mlc from T. thermophilus (Mlc(Tth)) and Mlc from E. coli (Mlc(Eco)) revealed that Mlc(Tth) conserved five residues of the glucose-binding motif of glucokinases. Here we show that Mlc(Tth) is not a glucokinase but is indeed able to bind glucose (K(D) = 20 microM), unlike Mlc(Eco). We found that mlc of T. thermophilus is the first gene within an operon encoding an ABC transporter for glucose and mannose, including a glucose/mannose-binding protein and two permeases. malK1, encoding the cognate ATP-hydrolyzing subunit, is located elsewhere on the chromosome. The system transports glucose at 70 degrees C with a K(m) of 0.15 microM and a V(max) of 4.22 nmol per min per ml at an optical density (OD) of 1. Mlc(Tth) negatively regulates itself and the entire glucose/mannose ABC transport system operon but not malK1, with glucose acting as an inducer. MalK1 is shared with the ABC transporter for trehalose, maltose, sucrose, and palatinose (TMSP). Mutants lacking malK1 do not transport either glucose or maltose. The TMSP transporter is also able to transport glucose with a K(m) of 1.4 microM and a V(max) of 7.6 nmol per min per ml at an OD of 1, but it does not transport mannose.     

Signal transduction between a membrane-bound transporter, PtsG, and a soluble transcription factor, Mlc, of Escherichia coli

The global regulator Mlc controls several genes implicated in sugar utilization systems, notably the phosphotransferase system (PTS) genes, ptsG, manXYZ and ptsHI, as well as the malT activator. No specific low molecular weight inducer has been identified that can inactivate Mlc, but its activity appeared to be modulated by transport of glucose via Enzyme IICB(Glc) (PtsG). Here we demonstrate that inactivation of Mlc is achieved by sequestration of Mlc to membranes containing dephosphorylated Enzyme IICB(Glc). We show that Mlc binds specifically to membrane fractions which carry PtsG and that excess Mlc can inhibit Enzyme IICB(Glc) phosphorylation by the general PTS proteins and also Enzyme IICB(Glc)-mediated phosphorylation of alpha-methylglucoside. Binding of Mlc to Enzyme IICB(Glc) in vitro required the IIB domain and the IIC-B junction region. Moreover, we show that these same regions are sufficient for Mlc regulation in vivo, via cross-dephosphorylation of IIB(Glc) during transport of other PTS sugars. The control of Mlc activity by sequestration to a transport protein represents a novel form of signal transduction in gene regulation.     

Substrate‐dependent cluster density dynamics of Corynebacterium glutamicum phosphotransferase system permeases

Many bacteria take up carbohydrates by membrane‐integral sugar specific phosphoenolpyruvate‐dependent carbohydrate:phosphotransferase systems (PTS). Although the PTS is centrally involved in regulation of carbon metabolism in different bacteria, little is known about localization and putative oligomerization of the permease subunits (EII). Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters, as well as the general components EI and HPr from Corynebacterium glutamicum using widefield and single molecule localization microscopy. PtsF and PtsG form membrane embedded clusters that localize in a punctate pattern. Size, number and fluorescence of the membrane clusters change upon presence or absence of the transported substrate, and a direct influence of EI and HPr was not observed. In presence of the transport substrate, EII clusters significantly increased in size. Photo‐activated localization microscopy data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however, the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas.     

Transport and metabolism of glucose and arabinose in Bifidobacterium breve

Glucose was required for the transport of arabinose into Bifidobacterium breve. The non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) did not facilitate assimilation of arabinose. Studies using d-[U-¹⁴C]-labelled arabinose showed that it was fermented to pyruvate, formate, lactate and acetate, whereas the principal metabolic products of d-[U-¹⁴C]-labelled glucose were acetate and formate. In contrast to glucose, arabinose was not incorporated into cellular macromolecules. A variety of metabolic inhibitors and inhibitors of sugar transport (proton ionophores, metal ionophores, compounds associated with electron transport) were used to investigate the mechanisms of sugar uptake. Only NaF, an inhibitor of substrate level phosphorylation, and 2-DG inhibited glucose assimilation. 2-DG had no effect on arabinose uptake, but NaF was stimulatory. High levels of phosphorylation of glucose and 2-DG by PEP and to a lesser degree, ATP were seen in phosphoenolpyruvate: phosphotransferase (PEP:PTS) assays. These data together with strong inhibition of glucose uptake by NaF suggest a role for phosphorylation in the transport process. Arabinose uptake in B. breve was not directly dependent on phosphorylation or any other energy-linked form of transport but may be assimilated by glucose-dependent facilitated diffusion.Abbreviations (2,4-DNP) 2,4-dinitrophenol - (2,4-DNP) carbonylcyanide m-chlorophenylhydrazone - (CCCP) (phosphoenolpyruvate phosphotransferase system) - PEP: PTS trichloroacetic acid - (TCA) 2-deoxy-d-glucose - (2-DG) 2-deoxy-d-glucose     

Role of secondary transporters and phosphotransferase systems in glucose transport by Oenococcus oeni

Glucose uptake by the heterofermentative lactic acid bacterium Oenococcus oeni B1 was studied at the physiological and gene expression levels. Glucose- or fructose-grown bacteria catalyzed uptake of [(14)C]glucose over a pH range from pH 4 to 9, with maxima at pHs 5.5 and 7. Uptake occurred in two-step kinetics in a high- and low-affinity reaction. The high-affinity uptake followed Michaelis-Menten kinetics and required energization. It accumulated the radioactivity of glucose by a factor of 55 within the bacteria. A large portion (about 80%) of the uptake of glucose was inhibited by protonophores and ionophores. Uptake of the glucose at neutral pH was not sensitive to degradation of the proton potential, Δp. Expression of the genes OEOE_0819 and OEOE_1574 (here referred to as 0819 and 1574), coding for secondary transporters, was induced by glucose as identified by quantitative real-time (RT)-PCR. The genes 1574 and 0819 were able to complement growth of a Bacillus subtilis hexose transport-deficient mutant on glucose but not on fructose. The genes 1574 and 0819 therefore encode secondary transporters for glucose, and the transports are presumably Δp dependent. O. oeni codes, in addition, for a phosphotransferase transport system (PTS) (gene OEOE_0464 [0464] for the permease) with similarity to the fructose- and mannose-specific PTS of lactic acid bacteria. Quantitative RT-PCR showed induction of the gene 0464 by glucose and by fructose. The data suggest that the PTS is responsible for Δp-independent hexose transport at neutral pH and for the residual Δp-independent transport of hexoses at acidic pH.     

Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 has four enzyme II (EII) genes of the phosphotransferase system in its genome encoding transporters for sucrose, glucose, fructose, and an unidentified EII. To analyze the function of these EII genes, they were inactivated via homologous recombination and the resulting mutants characterized for sugar utilization. Whereas the sucrose EII was the only transport system for sucrose in C. glutamicum, fructose and glucose were each transported by a second transporter in addition to their corresponding EII. In addition, the ptsF ptsG double mutant carrying deletions in the EII genes for fructose and glucose accumulated fructose in the culture broth when growing on sucrose. As no fructokinase gene exists in the C. glutamicum genome, the fructokinase gene from Clostridium acetobutylicum was expressed in C. glutamicum and resulted in the direct phosphorylation of fructose without any fructose efflux. Accordingly, since fructokinase could direct fructose flux to the pentose phosphate pathway for the supply of NADPH, fructokinase expression may be a potential strategy for enhancing amino acid production.     

Mutational analysis provides molecular insight into the carbohydrate-binding region of calreticulin: pivotal roles of tyrosine-109 and aspartate-135 in carbohydrate recognition

Calreticulin (CRT) is a lectin chaperone present in the lumen of the endoplasmic reticulum. It interacts with various glycoproteins by binding via their attached Glc(1)Man(9)GlcNAc(2) moiety. To provide further insight into these lectin-glycan interactions, we are investigating the interaction of CRT with various sugars. We have earlier modeled the complex between CRT and the Glc(1)Man(3) tetrasaccharide, a derivative of the native Glc(1)Man(9)GlcNAc(2) sugar moiety. Here, we have systematically mutated the residues implicated by the model in the interaction of CRT to its sugar substrates and categorized the role played by each of the subsites of calreticulin toward the glycan binding. The CRT mutants Y109F and D135L did not show any binding to the sugar substrates interacting with the wild-type protein, demonstrating the great importance of these residues in the carbohydrate-binding site of CRT. Also, D317L and M131A showed weak affinity toward the trisaccharide. The mutation of residues from the primary binding site of CRT, i.e., those interacting with glucose, appears to be far less tolerated as compared to mutations in residues that interact with the mannose residues of the glycan. Also, methyl-2-deoxy-glucopyranosyl-alpha(1-->3)-mannopyranoside failed to bind, asserting to the significance of the interactions between the primary binding site of CRT and the 2'-OH of the glucose residue of the oligosaccharide substrate in generating specificity for this recognition. These studies provide detailed molecular insight into the sugar binding specificity of CRT.     

Three-dimensional structures of protein-protein complexes in the E. coli PTS

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) includes a collection of proteins that accomplish phosphoryl transfer from phosphoenolpyruvate (PEP) to a sugar in the course of transport. The soluble proteins of the glucose transport pathway also function as regulators of diverse systems. The mechanism of interaction of the phosphoryl carrier proteins with each other as well as with their regulation targets has been amenable to study by nuclear magnetic resonance (NMR) spectroscopy. The three-dimensional solution structures of the complexes between the N-terminal domain of enzyme I and HPr and between HPr and enzyme IIA(Glc) have been elucidated. An analysis of the binding interfaces of HPr with enzyme I, IIA(Glc) and glycogen phosphorylase revealed that a common surface on HPr is involved in all these interactions. Similarly, a common surface on IIA(Glc) interacts with HPr, IIB(Glc) and glycerol kinase. Thus, there is a common motif for the protein-protein interactions characteristic of the PTS.     

Defective enzyme II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system leading to uncoupling of transport and phosphorylation in Salmonella typhimurium.

Transport and phosphorylation of glucose via enzymes II-A/II-B and II-BGlc of the phosphoenolpyruvate:sugar phosphotransferase system are tightly coupled in Salmonella typhimurium. Mutant strains (pts) that lack the phosphorylating proteins of this system, enzyme I and HPr, are unable to transport or to grow on glucose. From ptsHI deletion strains of S. typhimurium, mutants were isolated that regained growth on and transport of glucose. Several lines of evidence suggest that these Glc+ mutants have an altered enzyme II-BGlc as follows. (i) Insertion of a ptsG::Tn10 mutation (resulting in a defective II-BGlc) abolished growth on and transport of glucose in these Glc+ strains. Introduction of a ptsM mutation, on the other hand, which abolishes II-A/II-B activity, had no effect. (ii) Methyl alpha-glucoside transport and phosphorylation (specific for II-BGlc) was lowered or absent in ptsH+,I+ transductants of these Glc+ strains. Transport and phosphorylation of other phosphoenolpyurate:sugar phosphotransferase system sugars were normal. (iii) Membranes isolated from these Glc+ mutants were unable to catalyze transphosphorylation of methyl alpha-glucoside by glucose 6-phosphate, but transphosphorylation of mannose by glucose 6-phosphate was normal. (iv) The mutation was in the ptsG gene or closely linked to it. We conclude that the altered enzyme II-BGlc has acquired the capacity to transport glucose in the absence of phosphoenolpyruvate:sugar phosphotransferase system-mediated phosphorylation. However, the affinity for glucose decreased at least 1,000-fold as compared to the wild-type strain. At the same time the mutated enzyme II-BGlc lost the ability to catalyze the phosphorylation of its substrates via IIIGlc.     

Cooperative binding of the sugar substrates and allosteric regulatory protein (enzyme IIIGlc of the phosphotransferase system) to the lactose and melibiose permeases in Escherichia coli and Salmonella typhimurium

An Escherichia coli strain which overproduces the lactose permease was used to investigate the mechanism of allosteric regulation of this permease and those specific for melibiose, glycerol, and maltose by the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Thio-beta-digalactoside, a high affinity substrate of the lactose permease, released the glycerol and maltose permeases from inhibition by methyl-alpha-d-glucoside. Resumption of glycerol uptake occurred immediately upon addition of the galactoside. The effect was not observed in a strain which lacked or contained normal levels of the lactose permease, but growth of wild-type E. coli in the presence of isopropyl-beta-thiogalactoside plus cyclic AMP resulted in enhanced synthesis of the lactose permease so that galactosides relieved inhibition of glycerol uptake. Thiodigalactoside also relieved the inhibition of glycerol uptake caused by the presence of other PTS substrates such as fructose, mannitol, glucose, 2-deoxyglucose, and 5-thioglucose. Inhibition of adenylate cyclase activity by methyl-alpha-glucoside was also relieved by thiodigalactoside in E. coli T52RT provided that the lactose permease protein was induced to high levels. Cooperative binding of sugar and enzyme III(Glc) to the melibiose permease in Salmonella typhimurium was demonstrated, but no cooperativity was noted with the glycerol and maltose permeases. These results are consistent with a mechanism of PTS-mediated regulation of the lactose and melibiose permeases involving a fixed number of allosteric regulatory proteins (enzyme III(Glc)) which may be titrated by the increased number of substrate-activated permease proteins. This work suggests that the cooperativity in the binding of sugar substrate and enzyme III(Glc) to the permease, demonstrated previously in in vitro experiments, has mechanistic significance in vivo. It substantiates the conclusion that PTS-mediated regulation of non-PTS permease activities involves direct allosteric interaction between the permeases and enzyme III(Glc), the postulated regulatory protein of the PTS.