Single‐cell characterization of metabolic switching in the sugar phosphotransferase system of Escherichia coli

The utilization of several sugars in Escherichia coli is regulated by the Phosphotransferase System (PTS), in which diverse sugar utilization modules compete for phosphoryl flux from the general PTS proteins. Existing theoretical work predicts a winner‐take‐all outcome when this flux limits carbon uptake. To date, no experimental work has interrogated competing PTS uptake modules with single‐cell resolution. Using time‐lapse microscopy in perfused microchannels, we analyzed the competition between N‐acetyl‐glucosamine and sorbitol, as representative PTS sugars, by measuring both the expression of their utilization systems and the concomitant impact of sugar utilization on growth rates. We find two distinct regimes: hierarchical usage of the carbohydrates, and co‐expression of the genes for both systems. Simulations of a mathematical model incorporating asymmetric sugar quality reproduce our metabolic phase diagram, indicating that under conditions of nonlimiting phosphate flux, co‐expression is due to uncoupling of both sugar utilization systems. Our model reproduces hierarchical winner‐take‐all behaviour and stochastic co‐expression, and predicts the switching between both strategies as a function of available phosphate flux. Hence, experiments and theory both suggest that PTS sugar utilization involves not only switching between the sugars utilized but also switching of utilization strategies to accommodate prevailing environmental conditions.     

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.     

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.     

A flux-sensing mechanism could regulate the switch between respiration and fermentation

The yeast Saccharomyces cerevisiae can show different metabolic phenotypes (e.g. fermentation and respiration). Based on data from the literature, we argue that the substrate uptake rate is the core variable in the system that controls the global metabolic phenotype. Consequently the metabolic phenotype that the cell expresses is not dependent on the type of the sugar or its concentration, but only on the rate at which the sugar enters the cell. As this requires the cells to 'measure' metabolic flux, we discuss the existing clues toward a flux-sensing mechanism in this organism and also outline several aspects of the involved flux-dependent regulation system. It becomes clear that the sensing and regulation system that divides the taken up carbon flux into the respiratory or fermentative pathways is complex with many molecular components interacting on multiple levels. To obtain a true understanding about how the global metabolic phenotype of S. cerevisiae is controlled by the glucose uptake rate, different tools and approaches from systems biology will be required.     

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.     

Computer‐aided rational design of the phosphotransferase system for enhanced glucose uptake in Escherichia coli

The phosphotransferase system (PTS) is the sugar transportation machinery that is widely distributed in prokaryotes and is critical for enhanced production of useful metabolites. To increase the glucose uptake rate, we propose a rational strategy for designing the molecular architecture of the Escherichia coli glucose PTS by using a computer‐aided design (CAD) system and verified the simulated results with biological experiments. CAD supports construction of a biochemical map, mathematical modeling, simulation, and system analysis. Assuming that the PTS aims at controlling the glucose uptake rate, the PTS was decomposed into hierarchical modules, functional and flux modules, and the effect of changes in gene expression on the glucose uptake rate was simulated to make a rational strategy of how the gene regulatory network is engineered. Such design and analysis predicted that the mlc knockout mutant with ptsI gene overexpression would greatly increase the specific glucose uptake rate. By using biological experiments, we validated the prediction and the presented strategy, thereby enhancing the specific glucose uptake rate.     

The phosphoenolpyruvate:glucose phosphotransferase system of Salmonella typhimurium. The phosphorylated form of IIIGlc

Enzyme IIIGlc of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) of Salmonella typhimurium can occur in two forms: phosphorylated and nonphosphorylated. Phosphorylated IIIGlc (P-IIIGlc) has a slightly lower mobility during sodium dodecyl sulphate/polyacrylamide gel electrophoresis than IIIGlc. In bacterial extracts both phosphoenolpyruvate (the physiological phosphoryl donor of the PTS) as well as ATP can phosphorylate IIIGlc. The ATP-catalyzed reaction is dependent on phosphoenolpyruvate synthase, however, and is due to prior conversion of ATP to phosphoenolpyruvate. The phosphoryl group of phosphorylated IIIGlc is hydrolysed after boiling in sodium dodecyl sulfate but phosphorylated IIIGlc can be discriminated from IIIGlc if treated with this detergent at room temperature. We have used the different mobilities of IIIGlc and P-IIIGlc to estimate the proportion of these two forms in intact cells. Wild-type cells contain predominantly P-IIIGlc in the absence of PTS sugars. In an S. typhimurium mutant containing a leaky ptsI17 mutation (0.1% enzyme I activity remaining) both forms of IIIGlc occur in approximately equal amounts. Addition of PTS sugars such as glucose results, both in wild-type and mutant, in a dephosphorylation of P-IIIGlc. This correlates well with the observed inhibition of non-PTS uptake systems by PTS sugars via nonphosphorylated IIIGlc.     

Sugar transport systems in Corynebacterium glutamicum: features and applications to strain development

Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to take up and phosphorylate glucose, fructose, and sucrose, the major sugars from agricultural crops that are used as the primary feedstocks for industrial amino acid fermentation. This means that worldwide amino acid production using this organism has depended exclusively on the PTS. Recently, a better understanding not only of PTS-mediated sugar uptake but also of global regulation associated with the PTS has permitted the correction of certain negative aspects of this sugar transport system for amino acid production. In addition, the recent identification of different glucose uptake systems in this organism has led to a strategy for the generation of C. glutamicum strains that express non-PTS routes instead of the original PTS. The potential practical advantages of the development of such strains are discussed.     

Characteristic phenotypes associated with ptsN-null mutants in Escherichia coli K-12 are absent in strains with functional ilvG

The phosphotransferase system (PTS), encompassing EI, HPr, and assorted EII proteins, uses phosphoenolpyruvate to import and phosphorylate sugars. A paralog of EIIA of the sugar PTS system known as ptsN has been purported to regulate organic nitrogen source utilization in Escherichia coli K-12. Its known biochemical function, however, relates to potassium homeostasis. The evidence for regulation of organic nitrogen source utilization by ptsN is based primarily on the defective growth of ΔptsN mutants on amino acid nitrogen sources and other nutrient combinations. These observations were made with E. coli strains MG1655 and W3110, which carry a nonfunctional version of ilvG. There are three isozymes that effectively catalyze the first committed step of branched-chain amino acid biosynthesis, but ilvG is unique for doing so effectively across a range of potassium concentrations. Here we show that all of the nutrient utilization phenotypes attributed to ptsN are manifested selectively in strains lacking functional ilvG. We conclude that the ptsN gene product does not regulate organic nitrogen source utilization as previously proposed.     

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.     

Fructose transport by Escherichia coli

The utilization of fructose by Escherichia coli involves, as first step, the uptake of the sugar, normally via the phosphoenolpyruvate-dependent phosphotransferase system (PTS). This fructose-specific PTS differs in several ways from that effecting the uptake of other sugars that also possess the 3,4,5-D-arabino-hexose configuration: these differences are discussed. Mutants that lack the genes ptsI and ptsH, which specify components of the PTS common to most PT-sugars, can mutate further to regain the ability to utilize fructose when this is present in relatively high concentration (i.e. greater than 2 mM) in the medium. Some of the properties of this unusual uptake system is discussed.     

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.     

Sugar Preferences in Nectar‐ and Fruit‐Eating Birds: Behavioral Patterns and Physiological Causes1

Sucrose, glucose, and fructose are the three sugars that commonly occur in floral nectar and fruit pulp. The relative proportions of these three sugars in nectar and fruit in relation to the sugar preferences of pollinators and seed dispersers have received considerable attention. Based on the research of Herbert and Irene Baker and their collaborators, a dichotomy between sucrose‐dominant hummingbird‐pollinated flowers and hexose‐dominant passerine flowers and fruits was proposed. Data on sugar preferences of several hummingbird species (which prefer sucrose) vs. a smaller sample of passerines (which prefer hexoses) neatly fitted this apparent dichotomy. This hummingbird–passerine dichotomy was strongly emphasized until the discovery of South African plants with sucrose‐dominant nectars, which are pollinated by passerines that are able to digest, and prefer sucrose. Now we know that, with the exception of two clades, most passerines are able to assimilate sucrose. Most sugar preference studies have been conducted using a single, relatively high, sugar concentration in the nectar (ca 20%). Thus, we lack information about the role that sugar concentration might play in sugar selection. Because many digestive traits are strongly affected not only by sugar composition, but also by sugar concentration, we suggest that preferences for different sugar compositions are concentration‐dependent. Indeed, recent studies on several unrelated nectar‐feeding birds have found a distinct switch from hexose preference at low concentrations to sucrose preference at higher concentrations. Finally, we present some hypotheses about the role that birds could have played in molding the sugar composition of plant rewards.     

The Uptake of Sugars by Chara corallina

The influx of several monosaccharides into Chara corallina wasstudied under varying conditions of temperature, in the presenceof inhibitors, and by the use of competition experiments. Itwas found that the mechanism of transport was stereospecific,and the complex interactions between the sugars were characteristicof a carrier-mediated system. There is reason to believe thatthe carrier involved in this system has a narrower range ofaffinities than is usually attributed to a sugar carrier, forglucose and fructose appear to have different sites of entry,though each can influence the uptake of the other. In additionthe carrier system is situated in the plasmalemma and maintainedby metabolic energy.     

Regulation of competence development and sugar utilization in Haemophilus influenzae Rd by a phosphoenolpyruvate:fructose phosphotransferase system

Changes in intracellular cAMP concentration play important roles in Haemophilus influenzae , regulating both sugar utilization and competence for natural transformation. In enteric bacteria, cAMP levels are controlled by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) in response to changes in availability of the preferred sugars it transports. We have demonstrated the existence of a simple PTS in H. influenzae by several methods. We have cloned the H. influenzae ptsI gene, encoding PTS Enzyme I; genome analysis locates it in a pts operon structurally homologous to those of enteric bacteria. In vitro phosphorylation assays confirmed the presence of functional PTS components. A ptsI null mutation reduced fructose uptake to 1% of the wild-type rate, and abolished fructose fermentation even when exogenous cAMP was provided. The ptsI mutation also prevented fermentation of ribose and galactose, but utilization of these cAMP-dependent sugars was restored by addition of cAMP. In wild-type cells the non-metabolizable fructose analogue xylitol prevented fermentation of these sugars, confirming that the fructose PTS regulates cAMP levels. Development of competence under standard inducing conditions was reduced 250-fold by the ptsI mutation, unless cells were provided with exogenous cAMP. Competence is thus shown to be under direct nutritional control by a fructose-specific PTS.     

Genomic analysis of the phosphotransferase system in Clostridium botulinum

Clostridium botulinum is capable of fermenting carbohydrates, but there have been no detailed studies of the uptake of sugars and related substrates. In bacteria, a common and often predominant system of carbohydrate uptake is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). This multi-protein complex catalyses a group translocation involving both uptake and phosphorylation of carbohydrates, and is also known to play an important role in environmental sensing and metabolic regulation. The genome of C. botulinum encodes 15 PTSs which have a similar domain structure to the PTS in other bacteria. Based on phylogenetic relationships and analysis of gene clusters, the C. botulinum PTS appears to be involved in the uptake of hexoses, hexose derivatives and disaccharides. C. botulinum also contains the components of PTS-associated regulatory mechanisms which have been characterised in other bacteria. It therefore seems likely that the PTS plays a significant, and previously unrecognised, role in the physiology of this bacterium.     

Improvement of <Emphasis Type="Italic">Escherichia coli</Emphasis> production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system

The application of metabolic engineering in Escherichia coli has resulted in the generation of strains with the capacity to produce metabolites of commercial interest. Biotechnological processes with these engineered strains frequently employ culture media containing glucose as the carbon and energy source. In E. coli, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports glucose when this sugar is present at concentrations like those used in production fermentations. This protein system is involved in phosphoenolpyruvate-dependent sugar transport, therefore, its activity has an important impact on carbon flux distribution in the phosphoenolpyruvate and pyruvate nodes. Furthermore, PTS has a very important role in carbon catabolite repression. The properties of PTS impose metabolic and regulatory constraints that can hinder strain productivity. For this reason, PTS has been a target for modification with the purpose of strain improvement. In this review, PTS characteristics most relevant to strain performance and the different strategies of PTS modification for strain improvement are discussed. Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities has resulted in significant improvements in product yield from glucose and productivity for several classes of metabolites. In addition, inactivation of PTS components has been applied successfully as a strategy to abolish carbon catabolite repression, resulting in E. coli strains that use more efficiently sugar mixtures, such as those obtained from lignocellulosic hydrolysates.     

Comparative studies of Escherichia coli strains using different glucose uptake systems: Metabolism and energetics

Modifying substrate uptake systems is a potentially powerful tool in metabolic engineering. This research investigates energetic and metabolic changes brought about by the genetic modification of the glucose uptake and phosphorylation system of Escherichia coli. The engineered strain PPA316, which lacks the E. coli phosphotransferase system (PTS) and uses instead the galactose-proton symport system for glucose uptake, exhibited significantly altered metabolic patterns relative to the parent strain PPA305 which retains PTS activity. Replacement of a PTS uptake system by the galactose-proton symport system is expected to lower the carbon flux to pyruvate in both aerobic and anaerobic cultivations. The extra energy cost in substrate uptake for the non-PTS strain PPA 316 had a greater effect on anaerobic specific growth rate, which was reduced by a factor of five relative to PPA 305, while PPA 316 reached a specific growth rate of 60% of that of the PTS strain under aerobic conditions. The maximal cell densities obtained with PPA 316 were approximately 8% higher than those of the PTS strain under aerobic conditions and 14% lower under anaerobic conditions. In vivo NMR results showed that the non-PTS strain possesses a dramatically different intracellular environment, as evidenced by lower levels of total sugar phosphate, NAD(H), nucleoside triphosphates and phosphoenolpyruvate, and higher levels of nucleoside diphosphates. The sugar phosphate compositions, as measured by extract NMR, were considerably different between these two strains. Data suggest that limitations in the rates of steps catalyzed by glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and pyruvate kinase may be responsible for the low overall rate of glucose metabolism in PPA316. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 583-590, 1997.