beta-D-Galactoside transport in Escherichia coli: substrate recognition.

1. A number of galactosides and other sugar compounds were examined as inhibitors of facilitated or active transport by the lactose permease system of Escherichia coli. Efficient inhibition required an alpha- or beta-anomeric galactopyranosyl ring of D-configuration, a free 6-hydroxyl group, and a certain aglycone size which was reached, for example, by monosaccharide or nitrophenyl substituents. 2. Aromatic alpha-D-galactopyranosides acted as high-affinity inhibitors (Ki, below 50 micrometer). At least two of them were not transported, in contrast to alpha-galactoside disaccharides and to aromatic beta-D-galactopyranosides. 3. beta-D-Galactoside transport was not significantly inhibited by specific inhibitors and transitionstate analogues of beta-galactosidase (D-galactal, D-galactonolascone). 4. The beta-D-galactopyranoside, lactitol, and alpha-D-galactopyranoside, galactinol, were not efficiently bound by the lactose permease system, although the maximal rate of uptake of lacitol was similar to that of lactose. By comparison with several structurally related D-galactopyranosides, the decreased affinity was attributed to an effect of the membrane/water interface. A model for substrate recognition by the lactose permease system is presented.     

Binding of hydrophobic D-galactopyranosides to the lactose permease of Escherichia coli

Binding of alpha- and beta-D-galactopyranosides with different hydrophobic aglycons was compared using substrate protection against N-ethylmaleimide alkylation of single-Cys148 lactose permease. As demonstrated previously, methyl- or allyl-substituted alpha-D-galactopyranosides exhibit a 60-fold increase in binding affinity (K(D) = 0.5 mM), relative to galactose (K(D) = 30 mM), while methyl beta-D-galactopyranoside binds only 3-fold better. In the present study, galactopyranosides with cyclohexyl or phenyl substitutions, both in alpha and beta anomeric configurations, were synthesized. Surprisingly, relative to methyl alpha-D-galactopyranoside, binding of cyclohexyl alpha-D-galactopyranoside to lactose permease is essentially unchanged (K(D) = 0.4 mM), and phenyl alpha-D-galactopyranoside exhibits only a modest increase in binding affinity (K(D) = 0.15 mM). Nitro- or methyl-substituted phenyl alpha-D-galactopyranosides bind with significantly higher affinities (K(D) = 0.014-0.067 mM), and the strongest binding is observed with analogues containing para substituents. In contrast, D-galactopyranosides with a variety of large hydrophobic substituents (isopropyl, cyclohexyl, phenyl, o- or p-nitrophenyl) in beta anomeric configuration exhibit uniformly weak binding (K(D) = 1.0-2.3 mM). The results confirm and extend previous observations that hydrophobic aglycons of D-galactopyranosides increase binding affinity, with a clear predilection toward alpha-substituted sugars. In addition, the data suggest that the primary interaction between the permease and hydrophobic aglycons is directed toward the carbon atom bonded to the anomeric oxygen. The different positioning of this carbon atom in alpha- or beta-D-galactopyranosides thus may provide a rationale for the characteristic binding preference of the permease for alpha anomers.     

Galactose transport in Streptococcus thermophilus

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 10(10) cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 10(10) cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.     

Galactose transport in Streptococcus thermophilus.

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. I. Val177 and Val177/Asn319 permeases facilitate proton uniport and sugar uniport

The sugar specificity mutants of the lactose permease containing Val177 or Val177/Asn319 were analyzed with regard to their ability to couple H+ and sugar co-transport. Both mutants were able to transport lactose downhill to a significant degree. The Val177 mutant was partially defective in the active accumulation of galactosides, whereas the Val177/Asn319 mutant was completely defective in the uphill accumulation of sugars. With regard to coupling, the Val177 mutant was shown to catalyze the uncoupled transport of H+ to a substantial degree. This led to a decrease in the H+ electrochemical gradient under aerobic conditions and also resulted in faster H+ uptake when a transient H+ electrochemical gradient was generated under anaerobic conditions. Interestingly, galactosides were shown to diminish the rate of uncoupled H+ transport in the Val177 strain. The Val177/Asn319 strain also catalyzed uncoupled H+ transport, but to a lesser degree than the single Val177 mutant. In addition, the Val177/Asn319 mutant was shown to transport galactosides with or without H+. The observed H+/lactose stoichiometry was 0.30 in the double mutant compared to 0.98 in the wild-type strain. When an H+ electrochemical gradient was generated across the membrane, the Val177/Asn319 mutant permease was shown to facilitate an extremely rapid net H+ leak if nonmetabolizable galactosides had been equilibrated across the membrane. The mechanism of this leak is consistent with a circular pathway involving H+/galactoside influx and uncoupled galactoside efflux. The magnitude of the H+ leak in the presence of nonmetabolizable galactosides was so great in the double mutant that low concentrations of certain galactosides (i.e. 0.5 mM thiodigalactoside) resulted in a complete inhibition of growth. These results are discussed with regard to the possibility that cation and sugar binding to the lactose permease may involve a direct physical coupling at a common recognition site.     

The transient kinetics of uptake of galactosides into Escherichia coli.

The uptake of galactosides into Escherichia coli via the lactose permease was studied in the time range 0.01-10s by rapid mixing and quenched flow. An initial transient was observed under two conditions. Firstly, a lag in the approach to the steady state was observed at low galactoside concentrations (less than Km). Secondly, a burst of uptake was observed when anaerobic cell suspensions were mixed with aerobic substrate solutions. However, the cause of the burst of uptake appears to be a burst in the rate of respiration. The rate of galactoside uptake during this phase is 10-fold greater than during the steady state.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

A Molecular Investigation of Genotype by Environment Interactions

The fitnesses conferred by seven lactose operons, which had been transduced into a common genetic background from natural isolates of Escherichia coli, were determined during competition for growth rate-limiting quantities of galactosyl-glycerol, a naturally occurring galactoside. The fitnesses of these same operons have been previously determined on lactose and three artificial galactosides, lactulose, methyl-galactoside and galactosyl-arabinose. Analysis suggests that although marked genotype by environment interactions occur, changes in the fitness rankings are rare. The relative activities of the β-galactosidases and the permeases were determined on galactosyl-glycerol, lactose, lactulose and methyl-galactoside. Both enzymes display considerable kinetic variation. The β-galactosidase alleles provide no evidence for genotype by environment interactions at the level of enzyme activity. The permease alleles display genotype by environment interactions with a few causing changes in activity rankings. The contributions to fitness made by the permeases and the β-galactosidases were partitioned using metabolic control analysis. Most of the genotype by environment interaction at the level of fitness is generated by changes in the distribution of control among steps in the pathway, particularly at the permease where large control coefficients ensure that its kinetic variation has marked fitness effects. Indeed, changes in activity rankings at the permease account for the few changes in fitness rankings. In contrast, the control coefficients of the β-galactosidase are sufficiently small that its kinetic variation is in, or close to, the neutral limit. The selection coefficients are larger on the artificial galactosides because the control coefficients of the permease and β-galactosidase are larger. The flux summation theorem requires that control coefficients associated with other steps in the pathway must be reduced, implying that the selection at these steps will be less intense on the artificial galactosides. This suggests that selection intensities need not be greater in novel environments.     

Galactose transport systems in Streptococcus lactis

Galactose-grown cells of Streptococcus lactis ML3 have the capacity to transport the growth sugar by two separate systems: (i) the phosphoenolpyruvate-dependent phosphotransferase system and (ii) an adenosine 5'-triphosphate-energized permease system. Proton-conducting uncouplers (tetrachlorosalicylanilide and carbonyl cyanide-m-chlorophenyl hydrazone) inhibited galactose uptake by the permease system, but had no effect on phosphotransferase activity. Inhibition and efflux experiments conducted using beta-galactoside analogs showed that the galactose permease had a high affinity for galactose, methyl-beta-D-thiogalactopyranoside, and methyl-beta-D-galactopyranoside, but possessed little or no affinity for glucose and lactose. The spatial configurations of hydroxyl groups at C-2, C-4, and C-6 were structurally important in facilitating interaction between the carrier and the sugar analog. Iodoacetate had no inhibitory effect on accumulation of galactose, methyl-beta-D-thiogalactopyranoside, or lactose via the phosphotransferase system. However, after exposure of the cells to p-chloromercuribenzoate, phosphoenolpyruvate-dependent uptake of lactose and methyl-beta-D-thiogalactopyranoside were reduced by 75 and 100%, respectively, whereas galactose phosphotransferase activity remained unchanged. The independent kinetic analysis of each transport system was achieved by the selective generation of the appropriate energy source (adenosine 5'-triphosphate or phosphoenolpyruvate) in vivo. The maximum rates of galactose transport by the two systems were similar, but the permease system exhibited a 10-fold greater affinity for sugar than did the phosphotransferase system.     

Characterization and sequencing of the lac Y54-41 "uncoupled" mutant of the lactose permease

The Escherichia coli strain carrying the lac Y54-41 gene encodes a mutant lactose permease which carries out normal downhill transport of galactosides but is defective in uphill accumulation. In this study, the mutant lac Y54-41 gene was cloned onto the multicopy vector pUR270. As expected, the cloned gene was shown to express normal downhill transport activity but was markedly defective in the uphill transport of methyl-beta-D-thiogalactopyranoside. Direct measurements of H+ transport revealed that the mutant permease can transport H+ with methyl-beta-D-thiogalactopyranoside but at a significantly reduced capacity compared to the wild-type strain. However, under conditions where the mutant and wild-type strains both transport lactose at similar rates, no detectable H+ transport was observed in the mutant strain. The entire cloned lac Y54-41 gene was subjected to DNA sequencing, and a single base substitution was found which replaces glycine 262 in the protein with a cysteine residue. Inhibition experiments showed that the mutant permease is dramatically more sensitive to three different sulfhydryl reagents: N-ethylmaleimide, p-hydroxymericuribenzoate, and p-hydroxymercuriphenylsulfonic acid. However, the lactose analogue, thiodigalactoside, was only marginally effective at protecting against inhibition in the mutant strain. The results are consistent with the idea that the sulfhydryl reagents are inhibiting the mutant permease activity by reacting with cysteine 262.     

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose)

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.     

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.     

Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site.

A galactose-binding lectin (galaptin) from human spleen has been purified to homogeneity by affinity chromatography on asialofetuin-Sepharose. The carbohydrate-binding specificity of galaptin has been investigated by analyzing the binding of galaptin to asialofetuin in the presence of putative inhibitors. An enzyme-linked immunosorbent assay (ELISA) was developed that involved adsorption of asialofetuin to microtiter plates. Galaptin bound to asialofetuin was detected with polyclonal rabbit anti-galaptin serum followed by goat anti-rabbit IgG-peroxidase conjugate. The concentrations of inhibitors giving 50% inhibition of galaptin binding relative to controls were graphically determined and normalized relative to galactose or lactose. These analyses revealed that galaptin has a combining site at least as large as a disaccharide. The disaccharides having non-reducing-terminal beta-galactosyl residues linked (1,3), (1,4), and (1,6) to Glc or GlcNAc are better inhibitors than free Gal. GalNAc, either free or glycosidically linked, appears to have no affinity for the lectin. The nitrophenyl galactosides are better inhibitors than methyl galactosides, indicating the occurrence of hydrophobic interactions. The data indicate that OH groups at C-4 and C-6 of Gal and the OH at C-3 of GlcNAc in Gal beta(1,4)GlcNAc are important for lectin sugar interaction. Our data support the hypothesis that endogenous receptors for galaptin are most likely lactosaminoglycan moieties.     

Physiological function of periplasmic hexose phosphatase in Salmonella typhimurium.

Hydrolysis of sugar phosphates by crude and purified preparations of periplasmic hexose phosphatase from Salmonella typhimurium followed Michaelis-Menten kinetics. The enzyme bound glucose 1-phosphate with high affinity (Km = 10 microM) but bound glucose 6-phosphate with low affinity (Km = 2,000 microM). The order of substrate affinities was glucose 1-phosphate greater than mannose 1-phosphate = galactose 1-phosphate greater than fructose 1-phosphate greater than glucose 6-phosphate. These results and others suggest that the physiological function of the enzyme is the periplasmic hydrolysis of hexose 1-phosphates.     

Galactose metabolism by Streptococcus mutans

The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-beta-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.     

Functional conservation in the putative substrate binding site of the sucrose permease from Escherichia coli

The sucrose (CscB) permease is the only member of the oligosaccharide:H(+) symporter family in the Major Facilitator Superfamily that transports sucrose but not lactose or other galactosides. In lactose permease (lac permease), the most studied member of the family, three residues have been shown to participate in galactoside binding: Cys148 hydrophobically interacts with the galactosyl ring, while Glu126 and Arg144 are charge paired and form H-bonds with specific galactosyl OH groups. In the present study, the role of the corresponding residues in sucrose permease, Asp126, Arg144, and Ser148, is investigated using a functional Cys-less mutant (see preceding paper). Replacement of Ser148 with Cys has no significant effect on transport activity or expression, but transport becomes highly sensitive to the sulfhydryl reagent N-ethylmaleimide (NEM) in a manner similar to that of lac permease. However, in contrast to lac permease, substrate affords no protection whatsoever against NEM inactivation of transport or alkylation with [(14)C]NEM. Neutral (Ala, Cys) mutations of Asp126 and Arg144 abolish sucrose transport, while membrane expression is not affected. Similarly, combination of two Ala mutations within the same molecule (Asp126-->Ala/Arg144-->Ala) yields normally expressed, but completely inactive permease. Conservative replacements result in highly active molecules: Asp126-->Glu permease catalyzes sucrose transport comparable to Cys-less permease, while mutant Arg144-->Lys exhibits decreased but significant activity. The observations demonstrate that charge pair Asp126-Arg144 plays an essential role in sucrose transport and suggest that the overall architecture of the substrate binding sites is conserved between sucrose and lac permeases.     

2-deoxygalactose, a specific substrate of the Salmonella typhiimurium galactose permease: its use for the isolation of galP mutants.

2-Deoxygalactose is a specific substrate of the galactose permease. The apparent Km is about 500 micron, compared to 45 micron for galactose, whereas the maximal rate of uptake is one-half to one-third of that of galactose. None of the other galactose transport systems, including methyl beta-D-thiogalactosides I and II, the beta-methyl-galactoside permease, and both arabinose systems, is able to catalyze transport of 2-deoxygalactose to a significant extent. 2-Deoxygalactose can also be used to isolate mutants defective in galactose permease, since it is bacteriostatic. Colonies that grow with lactate, malate, or succinate as a carbon source in the presence of 0.5 to 2 mM 2-doexygalactose were found to be mostly galP mutants, lacking galactose permease. Spontaneous 2-deoxygalactose-resistant strains arose with a frequency of about 2 X 10(-6). galP mutants have also been derived from pts deletion mutants that require galactose permease for growth on glucose. Revertants have been obtained that have acquired the parental phenotype.     

Lactose inhibits the growth of Rhizobium meliloti cells that contain an actively expressed Escherichia coli lactose operon.

Expression of the Escherichia coli lactose operon in Rhizobium meliloti 104A14 made the cells sensitive to the addition of the beta-galactosides lactose, phenyl-beta-D-galactoside, and lactobionic acid. Growth stopped when the beta-galactoside was added and viability decreased modestly during the next few hours, but little cell lysis was observed and the cells appeared normal. Protein synthesis was not inhibited. Growth was inhibited only when beta-galactosidase expression was greater than 160 U. Lactose-resistant mutants had defects in the plasmid-carried E. coli beta-galactosidase or beta-galactoside permease and in the R. meliloti genome. We speculate that uncontrolled production of galactose by the action of the lactose operon proteins was responsible for growth inhibition.