Carbohydrate uptake in the oral pathogen Streptococcus mutans: mechanisms and regulation by protein phosphorylation

Streptococcus mutans is the primary etiological agent of dental caries in man and other animals. This organism and other related oral streptococci use carbohydrates almost exclusively as carbon and energy sources, fermenting them primarily to lactic acid which initiates erosion of tooth surfaces. Investigations over the past decade have shown that the major uptake mechanism for most carbohydrates in S. mutans is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), although non-PTS systems have also been identified for glucose and sucrose. Regulation of sugar uptake occurs by induction/repression and inducer exclusion mechanisms in S. mutans, but apparently not by inducer expulsion as is found in some other streptococci. In addition, ATP-dependent protein kinases have also been identified in S. mutans and other oral streptococci, and a regulatory function for at least one of these has been postulated. Among a number of proteins that are phosphorylated by these enzymes, the predominant soluble protein substrate is the general phospho-carrier protein of the PTS, HPr, as had previously been observed in a variety of Gram-positive bacteria. Recent results have provided evidence for a role for ATP-dependent phosphorylation of HPr in the coordination of sugar uptake and its catabolism in S. mutans. In this review, these results are summarized, and directions for future research in this area are discussed.     

Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria.

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.     

Corynebacterium glutamicum: a dissection of the PTS

The high-GC Gram-positive actinomycete Corynebacterium glutamicum is commercially exploited as a producer of amino acids that are used as animal feed additives and flavor enhancers. Despite its beneficial role, carbon metabolism and its possible influence on amino acid metabolism is poorly understood. We have addressed this issue by analyzing the phosphotransferase system (PTS), which in many bacteria controls the flux of nutrients and therefore regulates carbon metabolism. The general PTS phosphotransferases enzyme I (EI) and HPr were characterized by demonstration of PEP-dependent phosphotransferase activity. An EI mutant exhibited a pleiotropic negative phenotype in carbon utilization. The role of the PTS as a major sugar uptake system was further demonstrated by the finding that glucose and fructose negative mutants were deficient in the respective enzyme II PTS permease activities. These carbon sources also caused repression of glutamate uptake, which suggests an involvement of the PTS in carbon regulation. The observation that no HPr kinase/phosphatase could be detected suggests that the mechanism of carbon regulation in C. glutamicum is different to the one found in low-GC Gram-positive bacteria.     

The ptsI gene encoding enzyme I of the phosphotransferase system of Corynebacterium glutamicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is widespread among bacteria where it mediates carbohydrate uptake and often serves in carbon control. Here we present cloning and analysis of the monocistronic ptsI gene of Corynebacterium glutamicum R, which encodes PTS Enzyme I (EI). EI catalyzes the first reaction of PTS and the reported ptsI was shown to complement the corresponding defect in Escherichia coli. The deduced 59.2-kDa EI of 564 amino acids shares more than 50% homology with EIs from Bacillus stearothermophilus, Bacillus subtilis, and Lactobacillus sake. Chromosomal inactivation of ptsI demonstrated that EI plays an indispensable role in PTS of C. glutamicum R and this system represents a dominant sugar uptake system. Cellobiose was only transported and utilized in adaptive mutants of C. glutamicum R. Cellobiose transport was also found to be PTS-dependent and repressed by PTS sugar glucose.     

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.     

Sugar transport systems of Bifidobacterium longum NCC2705

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.     

Carbohydrate uptake by escherichia coli

In contrast to active transport, the uptake of carbohydrates via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) leads to the appearance in the cell of the sugar initially as a 1- or 6- phosphate ester. The components of the PTS that transfer phosphate to the sugar are not absolutely specific for any one sugar. Both their synthesis and their activity are controlled; in the latter, “fine” control, glucose-6-phosphate appears to play an important role. Studies of growth on, and uptake of, galactose by E.coli mutants devoid of components of the PTS and also devoid of active transport systems for galactose, suggest that proteins effecting facilitated diffusion of hexoses may be part of, or be closely associated with, the sugar-specific components of the PTS.     

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.     

A conserved glutamate residue, Glu-257, is important for substrate binding and transport by the Escherichia coli mannitol permease.

The mannitol permease, or D-mannitol-specific enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, both transports and phosphorylates its substrate. Previous analyses of the amino acid sequences of PTS permeases specific for various carbohydrates in different species of bacteria revealed several regions of similarity. The most highly conserved region includes a GIXE motif, in which the glutamate residue is completely conserved among the permeases that contain this motif. The corresponding residue in the E. coli mannitol permease is Glu-257, which is located in a large putative cytoplasmic loop of the transmembrane domain of the protein. We used site-directed mutagenesis to investigate the role of Glu-257. The properties of proteins with mutations at position 257 suggest that a carboxylate side chain at this position is essential for mannitol binding. E257A and E257Q mutant proteins did not bind mannitol detectably, while the E257D mutant could still bind this substrate. Kinetic studies with the E257D mutant protein also showed that a glutamate residue at position 257 of this permease is specifically required for efficient mannitol transport. While the E257D permease phosphorylated mannitol with kinetic parameters similar to those of the wild-type protein, the Vmax for mannitol uptake by this mutant protein is less than 5% that of the wild type. These results suggest that Glu-257 of the mannitol permease and the corresponding glutamate residues of other PTS permeases play important roles both in binding the substrate and in transporting it through the membrane.     

Unraveling the evolutionary history of the phosphoryl-transfer chain of the phosphoenolpyruvate:phosphotransferase system through phylogenetic analyses and genome context

Background  

The phosphoenolpyruvate phosphotransferase system (PTS) plays a major role in sugar transport and in the regulation of essential physiological processes in many bacteria. The PTS couples solute transport to its phosphorylation at the expense of phosphoenolpyruvate (PEP) and it consists of general cytoplasmic phosphoryl transfer proteins and specific enzyme II complexes which catalyze the uptake and phosphorylation of solutes. Previous studies have suggested that the evolution of the constituents of the enzyme II complexes has been driven largely by horizontal gene transfer whereas vertical inheritance has been prevalent in the general phosphoryl transfer proteins in some bacterial groups. The aim of this work is to test this hypothesis by studying the evolution of the phosphoryl transfer proteins of the PTS.     

Inducer exclusion in Escherichia coli by non-PTS substrates: the role of the PEP to pyruvate ratio in determining the phosphorylation state of enzyme IIAGlc

The main mechanism causing catabolite repression in Escherichia coli is the dephosphorylation of enzyme IIA^Glc, one of the enzymes of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS is involved in the uptake of a large number of carbohydrates that are phosphorylated during transport, phosphoenolpyruvate (PEP) being the phosphoryl donor. Dephosphorylation of enzyme IIA^Glc causes inhibition of uptake of a number of non-PTS carbon sources, a process called inducer exclusion. In this paper, we show that dephosphorylation of enzyme IIA^Glc is not only caused by the transport of PTS carbohydrates, as has always been thought, and that an additional mechanism causing dephosphorylation exists. Direct monitoring of the phosphorylation state of enzyme IIA^Glc also showed that many carbohydrates that are not transported by the PTS caused dephosphorylation during growth. In the case of glucose 6-phosphate, it was shown that transport and the first metabolic step are not involved in the dephosphorylation of enzyme IIA^Glc, but that later steps in the glycolysis are essential. Evidence is provided that the [PEP]–[pyruvate] ratio, the driving force for the phosphorylation of the PTS proteins, determines the phosphorylation state of enzyme IIA^Glc. The implications of these new findings for our view on catabolite repression and inducer exclusion are discussed.     

Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a beta-glucoside-specific EII and a 6-P-beta-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methyl beta-D-thiogalactoside (TMG) and 2-deoxy-D-glucose (2-DG). In vivo experiments with the ptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.     

Regulation of E. coli glycogen phosphorylase activity by HPr

Bacteria sense continuous changes in their environment and adapt metabolically to effectively compete with other organisms for limiting nutrients. One system which plays an important part in this adaptation response is the phosphoenol-pyruvate:sugar phosphotransferase system (PTS). Many proteins interact with and are regulated by PTS components in bacteria. Here we review the interaction with and allosteric regulation of Escherichia coli glycogen phosphorylase (GP) activity by the histidine phosphocarrier protein HPr, which acts as part of a phosphoryl shuttle between enzyme I and sugar-specific proteins of the PTS. HPr mediates crosstalk between PTS sugar uptake and glycogen breakdown. The evolution of the allosteric regulation of E. coli GP by HPr is compared to that of other phosphorylases.     

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.     

Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems

In Escherichia coli, several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit have not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as glucose-limited continuous cultivations. Deletion of EIIBC disturbs glucose transport severely in batch cultures; cyclic AMP (cAMP)-cAMP receptor protein (CRP) levels rise, and induction of the mgl operon occurs. Nevertheless, Mgl activity is not essential for growth of these mutants, since deletion of this transporter did not affect the growth rate; the activities of the remaining transporters seem to be sufficient. Under conditions of glucose limitation, mgl is upregulated 23-fold compared to levels for growth under glucose excess. Despite the strong induction of mgl upon glucose limitation, deletion of this transport system did not lead to further changes. Although the galactose transporters are often regarded as important for glucose uptake at micromolar concentrations, the glucose as well as mannose PTS might be sufficient for growth at this relatively low dilution rate.     

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.