Proton-linked D-xylose transport in Escherichia coli.

The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.     

Energization of the transport systems for arabinose and comparison with galactose transport in Escherichia coli.

1. Strains of Escherichia coli were obtained containing either the AraE or the AraF transport system for arabinose. AraE+,AraF- strains effected energized accumulation and displayed an arabinose-evoked alkaline pH change indicative of arabinose-H+ symport. In contrast, AraE-,AraF+ strains accumulated arabinose but did not display H+ symport. 2. The ability of different sugars and their derivatives to elicit sugar-H+ symport in AraE+ strains was examined. Only L-arabinose and D-fucose were good substrates, and arabinose was the only inducer. 3. Membrane vesicles prepared from an AraE+,AraF+ strain accumulated the sugar, energized most efficiently by the respiratory substrates ascorbate + phenazine methosulphate. Addition of arabinose or fucose to an anaerobic suspension of membrane vesicles caused an alkaline pH change indicative or sugar-H+ symport on the membrane-bound transport system. 4. Kinetic studies and the effects of arsenate and uncoupling agents in intact cells and membrane vesicles gave further evidence that AraE is a low-affinity membrane-bound sugar-H+ symport system and that AraF is a binding-protein-dependent high-affinity system that does not require a transmembrane protonmotive force for energization. 5. The interpretation of these results is that arabinose transport into E. coli is energized by an electrochemical gradient of protons (AraE system) or by phosphate bond energy (AraF system). 6. In batch cultures the rates of growth and carbon cell yields on arabinose were lower in AraE-,AraF+ strains than in AraE+,AraF- or AraE+,AraF+ strains. The AraF system was more susceptible to catabolite repression than was the AraE system. 7. The properties of the two transport systems for arabinose are compared with those of the genetically and biochemically distinct transport systems for galactose, GalP and MglP. It appears that AraE is analogous to GalP, and AraF to MglP.     

Pyruvate formate lyase and acetate kinase are essential for anaerobic growth of Escherichia coli on xylose

During anaerobic growth of bacteria, organic intermediates of metabolism, such as pyruvate or its derivatives, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate-level phosphorylation. In Escherichia coli, conversion of glucose to pyruvate yields 2 net ATPs, while metabolism of a pentose, such as xylose, to pyruvate only yields 0.67 net ATP per xylose due to the need for one (each) ATP for xylose transport and xylulose phosphorylation. During fermentative growth, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP from two pyruvates (one hexose equivalent) while still maintaining the overall redox balance. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose. An E. coli pfl mutant lacking pyruvate formate lyase cannot convert pyruvate to acetyl coenzyme A, the required precursor for acetate and ethanol production, and could not produce this additional ATP. E. coli pfl mutants failed to grow under anaerobic conditions in xylose minimal medium without any negative effect on their survival or aerobic growth. An ackA mutant, lacking the ability to generate ATP from acetyl phosphate, also failed to grow in xylose minimal medium under anaerobic conditions, confirming the need for the ATP produced by acetate kinase for anaerobic growth on xylose. Since arabinose transport by AraE, the low-affinity, high-capacity, arabinose/H+ symport, conserves the ATP expended in pentose transport by the ABC transporter, both pfl and ackA mutants grew anaerobically with arabinose. AraE-based xylose transport, achieved after constitutively expressing araE, also supported the growth of the pfl mutant in xylose minimal medium. These results suggest that a net ATP yield of 0.67 per pentose is only enough to provide for maintenance energy but not enough to support growth of E. coli in minimal medium. Thus, pyruvate formate lyase and acetate kinase are essential for anaerobic growth of E. coli on xylose due to energetic constraints.     

The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli K12

The gene xylE, coding for xylose-proton symport in Escherichia coli, was cloned and its DNA sequence determined. The cloning strategy utilized lambda placMu insertions and exploited the proximity of xylE to malB. A 2.8-kilobase HincII fragment of cloned DNA restored [14C]xylose transport and xylose-proton symport activities to a xylose transport-negative strain. The xylE gene was identified as a 1473-base pair open reading frame, located 373 base pairs downstream of malG, encoding a hydrophobic protein of Mr 53,607. The amino acid sequence of XylE bore little resemblance to the lactose-proton LacY symporter or melibiose-sodium MelB symporter, but a high degree of homology was found with the arabinose-proton AraE symporter of E. coli and glucose transport proteins of mammals. Structural analyses and comparisons suggest that 12 membrane-spanning segments may occur in the XylE protein.     

Characterization and functional expression in Escherichia coli of the sodium/proton/glutamate symport proteins of Bacillus stearothermophilus and Bacillus caldotenax

The genes encoding the Na+/H+/L-glutamate symport proteins of the thermophilic organisms Bacillus stearothermophilus (gltTBs) and Bacillus caldotenax (gltTBc) were cloned by complementation of Escherichia coli JC5412 for growth on glutamate as sole source of carbon, energy and nitrogen. The nucleotide sequences of the gltTBs and gltTBc genes were determined. In both cases the translated sequences corresponded with proteins of 421 amino acid residues (96.7% amino acid identity between GltTBs and GltTBc). Putative promoter, terminator and ribosome-binding-site sequences were found in the flanking regions. These expression signals were functional in E. coli. The hydropathy profiles indicate that the proteins are hydrophobic and could form 12 membrane-spanning regions. The Na+/H+ coupled L-glutamate symport proteins GltTBs and GltTBc are homologous to the strictly H+ coupled L-glutamate transport protein of E. coli K-12 (overall 57.2% identity). Functional expression of glutamate transport activity was demonstrated by uptake of glutamate in whole cells and membrane vesicles. In accordance with previous observations (de Vrij et al., 1989; Heyne et al., 1991), glutamate uptake was driven by the electrochemical gradients of sodium ions and protons.     

High capacity xylose transport in Candida intermedia PYCC 4715

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (mu(max)=0.5 h-1, 30 degrees C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km = 0.2 mM, Vmax = 7.5 mmol h-1 g-1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K = 50 mM, Vmax = 11 mmol h-1 g-1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.     

Transport of xylose and glucose in the xylose-fermenting yeast Pichia stipitis

Summary A low-affinity and a high-affinity sylose proton symport operated simultaneously in both starved and non-starved cells of Pichia stipitis. Glucose competed with xylose for transport by the low-affinity system and inhibited xylose transport by the high-affinity system non-competitively. The low affinity system was subject to substrate inhibition when glucose but not when xylose was the substrate. The differences between the characteristics of monosaccharide transport by Pichia stipitis and its imperfect state, Candida shehatae, are discussed.     

Analysis of NADPH supply during xylitol production by engineered Escherichia coli

Escherichia coli strain PC09 (DeltaxylB, cAMP-independent CRP (crp*) mutant) expressing an NADPH-dependent xylose reductase from Candida boidinii (CbXR) was previously reported to produce xylitol from xylose while metabolizing glucose [Cirino et al. (2006) Biotechnol Bioeng 95(6): 1167-1176]. This study aims to understand the role of NADPH supply in xylitol yield and the contribution of key central carbon metabolism enzymes toward xylitol production. Studies in which the expression of CbXR or a xylose transporter was increased suggest that enzyme activity and xylose transport are not limiting xylitol production in PC09. A constraints-based stoichiometric metabolic network model was used to understand the roles of central carbon metabolism reactions and xylose transport energetics on the theoretical maximum molar xylitol yield (xylitol produced per glucose consumed), and xylitol yields (Y(RPG)) were measured from resting cell biotransformations with various PC09 derivative strains. For the case of xylose-proton symport, omitting the Zwf (glucose-6-phosphate dehydrogenase) or PntAB (membrane-bound transhydrogenase) reactions or TCA cycle activity from the model reduces the theoretical maximum yield from 9.2 to 8.8, 3.6, and 8.0 mol xylitol (mol glucose)(-1), respectively. Experimentally, deleting pgi (encoding phosphoglucose isomerase) from strain PC09 improves the yield from 3.4 to 4.0 mol xylitol (mol glucose)(-1), while deleting either or both E. coli transhydrogenases (sthA and pntA) has no significant effect on the measured yield. Deleting either zwf or sucC (TCA cycle) significantly reduces the yield from 3.4 to 2.0 and 2.3 mol xylitol (mol glucose)(-1), respectively. Expression of a xylose reductase with relaxed cofactor specificity increases the yield to 4.0. The large discrepancy between theoretical maximum and experimentally determined yield values suggests that biocatalysis is compromised by pathways competing for reducing equivalents and dissipating energy. The metabolic role of transhydrogenases during E. coli biocatalysis has remained largely unspecified. Our results demonstrate the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions, and suggest that the pool of reduced cofactors available for biotransformation is not readily interchangeable via transhydrogenase.     

Effect of membrane potential on the kinetic parameters of the Na+ or H+ melibiose symport in Escherichia coli membrane vesicles

Comparison of the transport properties of the melibiose permease of E. coli acting as a H+-symport or a Na+-symport has been performed by measuring initial rates of [3H]-melibiose transport or its accumulation in isolated membrane vesicles. The results show strikingly that although the membrane potential primarily drives melibiose accumulation by both types of symport, it selectively affects the apparent affinity constant Kt of the H+-melibiose symport while it specifically changes the maximal rate of transport (Vmax) of the Na+-melibiose symport. It is suggested that modification(s) of some partial reaction constants of a given transport cycle might lead to important changes in the kinetic properties of this transport system.     

Identification of the AraE transport protein of Escherichia coli.

1. Two arabinose-inducible proteins are detected in membrane preparations from strains of Escherichia coli containing arabinose-H+ (or fucose-H+) transport activity; one protein has an apparent subunit relative molecular mass (Mr) of 36 000-37 000 and the other has Mr 27 000. 2. An araE deletion mutant was isolated and characterized; it has lost arabinose-H+ symport activity and the arabinose-inducible protein of Mr 36 000, but not the protein of Mr 27 000. 3. An araE+ specialized transducing phage was characterized and used to re-introduce the araE+ gene into the deletion strain, a procedure that restores both arabinose-H+ symport activity and the protein of Mr 36,000. 4. N-Ethylmaleimide inhibits arabinose transport and partially inhibits arabinose-H+ symport activity. 5. N-Ethylmaleimide modifies an arabinose-inducible protein of Mr 36 000-38 000, and arabinose protects the protein against the reagent. 6. These observations identify an arabinose-transport protein of Escherichia coli as the product of the araE+ gene. 7. The protein was recognized as a single spot staining with Coomassie Blue after two-dimensional gel electrophoresis.     

Cation-selectivity of the l-glutamate transporters of Escherichia coli, Bacillus stearothermophilus and Bacillus caldotenax: dependence on the environment in which the proteins are expressed

l -Glutamate transport by the H⁺-glutamate and Na⁺-glutamate symport proteins of Escherichia coli K-12 (GltP_Ec and GltS_Ec, respectively) and the Na⁺-H⁺-glutamate symport proteins of Bacillus stearothermophilus (GltT_Bs) and Bacillus caldotenax (GltT_Bc) was studied in membrane vesicles derived from cells in which the proteins were either homologously or heterologously expressed. Substrate and inhibitor specificity studies indicate that GltP_Ec, GltT_Bs and GltT_Bc fall into the same group of transporters, whereas GltS_Ec is distinctly different from the others. Also, the cation specificity of GltS_Ec is different; GltS_Ec transported l -glutamate with (at least) two Na⁺, whereas GltP_Ec, GltT_Bs and GltT_Bc catalysed an electrogenic symport of l -glutamate with ≥two H⁺, i.e. when the proteins were expressed in E. coli Surprisingly studies in membrane vesicles of B. stearothermophilus and B. caldotenax indicated a Na⁺-H⁺- l -glutamate symport for both GltT_Bs and GltT_Bc. The Na⁺ dependency of the GltT transporters in the Bacillus strains increased with temperature. These observations suggest that the conformation of the transport proteins in the E. coli and the Bacillus membranes differs, which influences the coupling ion selectivity.     

Mechanism of Na(+)-dependent citrate transport in Klebsiella pneumoniae.

Citrate transport via CitS of Klebsiella pneumoniae has been shown to depend on the presence of Na+. This transport system has been expressed in Escherichia coli, and uptake of citrate in E. coli membrane vesicles via this uptake system was found to be an electrogenic process, although the pH gradient is the main driving force for citrate uptake (M. E. van der Rest, R. M. Siewe, T. Abee, E. Schwartz, D. Oesterhelt, and W. N. Konings, J. Biol. Chem. 267:8971-8976, 1992). Analysis of the affinity constants for the different citrate species at different pH values of the medium indicates that H-citrate2- is the transported species. Since the electrical potential across the membrane is a driving force for citrate transport, this indicates that transport occurs in symport with at least three monovalent cations. Citrate efflux is stimulated by Na+ concentrations of up to 5 mM but inhibited by higher Na+ concentrations. Citrate exchange, however, is stimulated by all Na+ concentrations, indicating sequential events in which Na+ binds before citrate for translocation followed by a release of Na+ after release of citrate. CitS has, at pH 6.0 and in the presence of 5 mM citrate on both sides of the membrane, an apparent affinity (K(app)) for Na+ of 200 microM. The Na+/citrate stoichiometry was found to be 1. It is postulated that H-citrate2- is transported via CitS in symport with one Na+ and at least two H+ ions.     

Kinetic analysis of simultaneously occurring proton-sorbose symport and passive sorbose transport in Saccharomyces fragilis

Sorbose transport in Saccharomyces fragilis takes place both via an active sugar-H+ symport system and via facilitated diffusion. To establish whether the two modes of transport proceed via the same transporter or via two different carriers, the kinetic consequences of both models were investigated. The kinetic equations for initial transport were derived for three possible reaction sequences with respect to sugar and H+ binding to the symport carrier: random binding and obligatory ordered binding with either sugar or H+ binding first, yielding six sets of kinetic parameters. Analysis of experimental data of sorbose transport in S. fragilis showed the existence of separate carriers for active, sorbose-H+ symport and facilitated diffusion. Furthermore, it could be concluded that the symport carrier shows random binding of sugar and H+. In recent literature, a similar combination of active and passive sugar transport in Rhodotorula gracilis and Chlorella vulgaris was interpreted as two modes of action of the same carrier, viz., active symport via the protonated, and facilitated diffusion via the unprotonated carrier. Analysis of the experimental data according to the criteria presented in this paper showed, however, that this supposition is untenable and that two different carriers must also be involved in these micro-organisms.     

Evidence for catabolite inhibition in regulation of pentose utilization and transport in the ruminal bacterium Selenomonas ruminantium.

Pentose sugars can be an important energy source for ruminal bacteria, but there has been relatively little study regarding the regulation of pentose utilization and transport by these organisms. Selenomonas ruminantium, a prevalent ruminal bacterium, actively metabolizes xylose and arabinose. When strain D was incubated with a combination of glucose and xylose or arabinose, the hexose was preferentially utilized over pentoses, and similar preferences were observed for sucrose and maltose. However, there was simultaneous utilization of cellobiose and pentoses. Continuous-culture studies indicated that at a low dilution rate (0.10 h-1) the organism was able to co-utilize glucose and xylose. This co-utilization was associated with growth rate-dependent decreases in glucose phosphotransferase activity, and it appeared that inhibition of pentose utilization was due to catabolite inhibition by the glucose phosphotransferase transport system. Xylose transport activity in strain D required induction, while arabinose permease synthesis did not require inducer but was subject to repression by glucose. Since an electrical potential or a chemical gradient of protons drove xylose and arabinose uptake, pentose-proton symport systems apparently contributed to transport.     

Secondary active transport

Secondary active transport is defined as the transport of a solute in the direction of its increasing electrochemical potential coupled to the facilitated diffusion of a second solute (usually an ion) in the direction of its decreasing electrochemical potential. The coupling agents are membrane proteins (carriers), each of which catalyzes simultaneously the facilitated diffusion of the driving ion and the active transport of a given solute. The review starts with some considerations on the energetics followed by a presentation of the kinetics of secondary active transport. Examples of information which may be gained by such studies are discussed. In the second part, some examples of secondary transport are given; we also describe the characteristics of the corresponding carriers. The various transport systems presented are: the D-glucose/Na+ symport in brush-border membranes, the lactose/H+ symport in E. coli, the Na+/H+ antiport, the different transport systems in the inner mitochondrial membrane.     

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes

Separate proteins for proton-linked transport of D-xylose, L-arabinose, D-galactose, L-rhamnose and L-fucose into Escherichia coli are being studied. By cloning and sequencing the appropriate genes, the amino acid sequences of proteins for D-xylose/H+ symport (XylE), L-arabinose/H+ symport (AraE), and part of the protein for D-galactose/H+ symport (GalP) have been determined. These are homologous, with at least 28% identical amino acid residues conserved in the aligned sequences, although their primary sequences are not similar to those of other E. coli transport proteins for lactose, melibiose, or D-glucose. However, they are equally homologous to the passive D-glucose transport proteins from yeast, rat brain, rat adipocytes, human erythrocytes, human liver, and a human hepatoma cell line. The substrate specificity of GalP from E. coli is similar to that of the mammalian glucose transporters. Furthermore, the activities of GalP, AraE and the mammalian glucose transporters are all inhibited by cytochalasin B and N-ethylmaleimide. Conserved residues in the aligned sequences of the bacterial and mammalian transporters are identified, and the possible roles of some in sugar binding, cation binding, cytochalasin binding, and reaction with N-ethylmaleimide are discussed. Each protein is independently predicted to form 12 hydrophobic, membrane-spanning alpha-helices with a central hydrophilic segment, also comprised of alpha-helix. This unifying structural model of the sugar transporters shares features with other ion-linked transport proteins for citrate or tetracycline.     

Mechanism of proline transport in Escherichia coli K12. III. Inhibition of membrane potential-driven proline transport by syn-coupled ions and evidence for symmetrical transition states of the 2H+/proline symport carrier

Specific inhibition of 2H+/proline symport by syn-coupled ions (Na+, Li+, and H+) was investigated using cytoplasmic membrane vesicles prepared from the proline carrier-overproducing strain MinS/ pLC4 -45 of Escherichia coli K12. The 2H+/proline symport driven by the membrane potential generated via respiration with 20 mM ascorbate/Tris, 0.1 mM phenazine methosulfate was specifically inhibited by Na+. The inhibition by Na+ was described by a fully noncompetitive mechanism, and the apparent Ki for Na+ was 15 mM. A linear correlation between the apparent Vmax and the apparent Kd was observed. Li+ stimulated the transport activity 2-fold at 10 mM and inhibited it at concentrations above 50 mM. H+ caused fully noncompetitive inhibition of 2H+/proline symport, and its apparent Ki was 0.6 microM. These results indicate that the concentrations of Na+ and H+ strictly and independently regulate the amount of the active C state carrier responsible for 2H+/proline symport driven by the membrane potential by inhibiting the transition from the C* state carrier which exhibits Na+- and H+-dependent binding of proline and is predominant in nonenergized conditions.     

Proton-linked L-fucose transport in Escherichia coli.

1. Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.     

The lactose/H+ carrier of Escherichia coli: lac YUN mutation decreases the rate of active transport and mimics an energy-uncoupled phenotype.

The Escherichia coli K12 strain X71-54 carries the lac YUN allele, coding for a lactose/H+ carrier defective in the accumulation of a number of galactosides [Wilson, Kusch & Kashket (1970) Biochem. Biophys. Res. Commun. 40, 1409-1414]. Previous studies proposed that the lower accumulation in the mutant be due to a faulty coupling of H+ and galactoside fluxes via the carrier. Immunochemical characterization of the carriers in membranes from mutant and parent strains with an antibody directed against the C-terminal decapeptide of the wild-type carrier leads to the conclusion that the mutant carrier is similar to the wild-type in terms of apparent Mr, C-terminal sequence, and level of incorporation into the membrane. The pH-dependence of galactoside transport was compared in the mutant and the parent. At pH 8.0-9.0, mutant and parent behave similarly with respect to the accumulation of beta-D-galactosyl 1-thio-beta-D-galactoside and to the ability to grow on the carrier substrate melibiose. At pH 6.0, both the maximal velocity for active transport and the level of accumulation of beta-D-galactosyl-1-thio-beta-D-galactoside are lower in the mutant. The mutant also is unable to grow on melibiose at pH 5.5. However, at pH 6.0 and low galactoside concentrations, the symport stoichiometry is 0.90 H+ per galactoside in the mutant as compared with 1.07 in the parent. These observations suggest that symport is normal in the mutant and that the lower rate of transport in the mutant is responsible for the phenotype. At higher galactoside concentrations, accumulation is determined not only thermodynamically but also kinetically, contrary to a simple interpretation of the chemiosmotic theory. Therefore lower rates of active transport can mimic the effect of uncoupling H+ and galactoside symport. Examination of countertransport in poisoned cells at pH 6.0 reveals that the rate constants for the reorientation of the loaded and unloaded carrier are altered in the mutant. The reorientation of the unloaded carrier is slower in the mutant. However, the reorientation of the galactoside-H+-carrier complex is slower for substrates like melibiose, but faster for substrates like lactose. These findings suggest that lactose-like and melibiose-like substrates interact with the carrier in slightly different ways.     

Variable H+/substrate stoicheiometries in Rhodotorula gracilis are caused by a pH-dependent protonation of the carrier(s).

Two carrier-mediated systems transport sugars in the yeast Rhodotorula gracilis depending on the pH. One system, with higher affinity for sugars, catalyses a symport of protons with sugar, whereas the other system, having lower affinity, is independent of protons. This was shown in three different ways. (1) At low pH, where only the high-affinity system works, a H+/sugar stoicheiometry of 1 was found. An increase of the pH and of the sugar concentration, which allowed the low-affinity system to operate, brought about a drop of the stoicheiometry to values below 1. (2) During H+ symport the influx of positive charge was electrically compensated by an equivalent efflux of K+ from the cells. At high pH and high sugar concentrations this stoicheiometry of K+ and sugar decreased concomitant with the H+/sugar stoicheiometry. (3) At pH 7.5 both transport systems were operating, as shown by biphasic saturation kinetics. Under these conditions only the high-affinity transport was found to be electrogenic. These results agree with the theory of an electrogenic H+/sugar symport where changes in the affinity for substrate are brought about by reversible protonation and deprotonation of the carrier.