Metabolic control of lactose entry in Escherichia coli.

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain beta-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by beta-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of omicron-nitrophenyl-beta-D-galactopyranoside (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H+/sugar cotransport. Entry of omicron-nitrophenyl-beta-D-galactopyranoside is less dependent on the need for proton reextrusion, probably because the stoichiometry of H+/substrate cotransport is greater for lactose than for ONPG.     

Involvement of phosphoenolpyruvate in lactose utilization by group N streptococci

The effect of sodium fluoride on lactose metabolism and o-nitrophenyl-beta-d-galactopyranoside (ONPG) hydrolysis by Streptococcus lactis strains 7962 and C(2)F suggested that different mechanisms of lactose utilization existed in the two strains. Sodium fluoride prevented lactose utilization and ONPG hydrolysis by whole cells of S. lactis C(2)F but had no effect on S. lactis 7962. Although hydrolysis of ONPG by toluene-treated cells of S. lactis 7962 occurred without addition of phospho-enolpyruvate (PEP), toluene-treated cells of S. lactis C(2)F required the presence of this cofactor. Concentrated cell extracts of S. lactis C(2)F hydrolyzed ONPG; this hydrolysis was inhibited by NaF, but the addition of PEP, in the presence of NaF, restored maximal activity. Addition of acetyl-phosphate, carbamyl-phosphate, adenosine-5'-triphosphate, guanosine-5'-triphosphate, or uridine-5'-triphosphate did not stimulate activity. The presence of cofactors did not stimulate and NaF did not inhibit the hydrolysis in extracts of S. lactis 7962. To confirm the operation of two mechanisms, S. lactis 7962 was shown to hydrolyze lactose to glucose and galactose, whereas S. lactis C(2)F was unable to split the disaccharide. In addition, whole cells of S. lactis C(2)F rapidly accumulated a phosphorylated derivative of thiomethyl-beta-d-galactoside (TMG) which behaved chromatographically and electrophoretically like TMG-PO(4). Unexpectedly, S. lactis 7962 also accumulated a TMG derivative, although the rate was extremely low. These data indicate that different mechanisms of lactose utilization exist in the two strains, with a phosphorylation step dependent on PEP involved in S. lactis C(2)F.     

Effects of Dimethylsulfoxide on the Lactose Operon in Escherichia coli

Fowler, Audree V. (University of California, Los Angeles), and Irving Zabin. Effects of dimethylsulfoxide on the lactose operon in Escherichia coli. J. Bacteriol. 92:353-357. 1966.-Dimethylsulfoxide (DMSO) at a concentration of 5% (v/v) in the culture medium inhibits the growth of Escherichia coli to only a slight extent, and does not affect the differential rate of synthesis of beta-galactosidase. Resting cells remain viable after shaking in the presence of 20% DMSO for 3 hr at 37 C. Both beta-galactosidase and thiogalactoside transacetylase retain almost all activity after incubation in even higher concentrations of the solvent for many hours. DMSO decreases the permeability barrier. The rate of hydrolysis of o-nitrophenyl-beta-d-galactoside (ONPG) in whole cells containing beta-galactosidase but lacking permease is increased in cells treated with 5% DMSO. Several permeaseless strains preinduced for beta-galactosidase will grow on lactose in the presence, but not in the absence, of 5% DMSO. When permeaseless strains are grown on tetrazolium-lactose-agar, the presence of 5% DMSO causes a definite but not marked shift toward the lactose-positive character.     

Galactosidase activity of lactose-positive Neisseria

The chromogenic substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG) was hydrolyzed by lactose-positive Neisseria. Eight strains of pharyngeal origin were examined. In culture reactions, seven strains resembled Neisseria meningitidis with the exception that they produced acid from 1% (w/v) lactose. An eighth strain (V8) differed in that it did not form acid from maltose or from 1% lactose. However, acid formation was observed in 10% lactose cultures of strain V8, suggesting that entry of lactose occurred by passive diffusion, rather than as a result of permease activity. The enzymes which hydrolyzed ONPG were produced constitutively by the cells of all eight strains. Thus, specific activity in these strains was not increased by prior exposure to lactose, or to two other possible inducers, isopropyl-beta-d-thiogalactoside or methyl-beta-d-thiogalactoside. Study of cell-free extracts of one strain showed that the enzyme was heat-labile, having a half-life of 10 min at 45 C. The enzyme was unstable at low protein concentrations, but it was protected completely or partially when albumin or manganous ions were added. The enzyme appeared to be a typical beta-galactosidase: alpha-galactosides (melibiose and p-nitrophenyl-alpha-d-galactopyranoside) were not hydrolyzed, activity against ONPG was not dependent upon inorganic phosphate, and galactose was released by cleavage of ONPG. ONPG hydrolysis provided a simple and rapid method for detecting lactose-positive Neisseria.     

Diffusion and chemical reaction rates with nonuniform enzyme distribution: an experimental approach

The study of the effects of nonuniform distributions of immobilized beta-galactosidase on the overall reaction rate of the hydrolysis of lactose are presented. Diffusion inside the particles has been characterized by measuring the diffusion rates of two beta-galactosidase substrates: lactose and ONPG in a commercial silica-alumina support. Effective diffusivities have been determined by the chromatographic method under inert conditions. The results obtained for tortuosity can be explained assuming that the transport only takes place in the macropores. The distribution of the immobilized enzyme has been measured by means of confocal microscopy technique. The enzyme has been tagged with FITC and immobilized in particles of different diameters, the internal local concentrations of the enzyme have been determined with the aid of an image computer program. As expected, a more nonuniform internal profile of the enzyme was found when the particle diameter was bigger. Experiments under reaction conditions were carried out in batch reactors using lactose and ONPG as substrates and particles of the immobilized beta-galactosidase of different diameter (1 x 10(-4) to 5 x 10(-3) m) as catalyst, employing a temperature of 40 degrees C for lactose and 25 and 40 degrees C for ONPG, respectively. The mass balance inside the particle for the substrates has been solved for the internal profiles of the immobilized enzyme inside particles of different size and the enzymatic reactions considered. The calculated and the experimental effectiveness factor values were similar when particles under 2.75 x 10(-3) m in diameter were employed. For the same Thiele modulus, a particle with nonuniform distribution of enzyme showed a higher effectiveness as a catalyst than particles with a more uniform distribution.     

Activation and regeneration of whole cell biocatalysts: initial and periodic induction behavior in starved Escherichia coli after immobilization in thin synthetic films

Activation and regeneration of whole cell biocatalytic activity via initial and subsequent induction of the lacZ gene was investigated in starved Escherichia coli using a novel synthetic biofilm. Stationary-phase bacteria were entrapped in 10-80 mum thick multi-layer films, where a copolymer of acrylic and vinyl acetate was the immobilization matrix. The E. coli were placed in a defined starvation medium containing essentially no nitrogen or carbon source and induced initially using lactose or isopropylthiogalactoside (IPTG). Subsequent inductions were performed with IPTG. Comparison studies with suspended bacteria showed that when IPTG was the initial inducing agent, induction kinetics are linear for both immobilized and suspended cells. After induction with lactose, however, a lag time is noted for suspended cells, but not for E. coli in the biofilm. Biocatalytic activity was successfully regenerated by re-inducing starved suspended cells 1-3 days after an initial induction with lactose. This regeneration was demonstrated in the synthesis of additional active beta-galactosidase. However, immobilized cells could be re-induced for at least 17 days after the initial induction, and viability in the synthetic biofilms remained greater than 90%, demonstrating that periodic induction is a valuable method for extending the life of whole cell biocatalysts. (c) 1996 John Wiley & Sons, Inc.     

Co-induction of beta-galactosidase and the lactose-P-enolpyruvate phosphotransferase system in Streptococcus salivarius and Streptococcus mutans.

The addition of lactose, galactose, or isopropyl-beta-D-thiogalactoside (IPTG) to glucose-grown cells of Streptococcus salivarius 25975 resulted in the co-induction of both the lactose-P-enolpyruvate phosphotransferase system (lactose-PTS) and beta-galactosidase, with the latter the predominant metabolic system. With various strains of Streptococcus mutans and Streptococcus sanguis 10556, on the other hand, the lactose-PTS was the major metabolic pathway with beta-galactosidase induced either to low or negligible levels. In all cases, induction of the lactose-PTS resulted in the concomitant induction of 6-P-beta-galactosidase. The induction by lactose of both the lactose-PTS and beta-galactosidase in all strains was repressed by glucose and other catabolites, notably, fructose. Induction of beta-galactosidase in S. salivarius 25975 by IPTG was, however, relatively resistant to glucose repression. Induction experiments with IPTG and lactose suggested that a cellular metabolite of lactose metabolism was a repressor of enzyme activity. Exogenous cAMP was shown to reverse the transient repression by glucose of beta-galactosidase induction in cells of S. salivarius 25975 receiving lactose, provided the cells were grown with small amounts of toluene to overcome the permeability barrier to this nucleotide, cAMP, was however, unable to overcome the permanent repression of beta-galactosidase activity to a significant extent under these conditions.     

Mechanisms of lactose utilization by lactic acid streptococci: enzymatic and genetic analyses

The apparent instability of beta-galactosidase in toluene-treated cells or cell-free extracts of lactic streptococci is explained by the fact that these organisms do not contain the expected enzyme. Instead, various strains of Streptococcus lactis, S. cremoris, and S. diacetilactis were shown to hydrolyze o-nitrophenyl-beta-d-galactoside-6-phosphate (ONPG-6-P), indicating the presence of a different enzyme. In addition, lactose metabolism in S. lactis C(2)F was found to involve enzyme I (EI), enzyme II (EII), factor III (FIII), and a heat-stable protein (HPr) of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system analogous to that of Staphylococcus aureus. Mutants of S. lactis C(2)F, defective in lactose metabolism, possessed the phenotype lac(-) gal(-). These strains were unable to accumulate (14)C-thiomethyl-beta-d-galactoside, to hydrolyze ONPG, or to utilize lactose when grown in lactose or galactose broth. In addition, these mutants contained EI and HPr, but lacked EII, FIII, and the ability to hydrolyze ONPG-6-P. This suggested that the defect was in the phosphorylation step. Lactose-negative mutants of S. lactis 7962, a strain containing beta-galactosidase, could be separated into several classes, which indicated that this organism is not dependent upon the PEP-phosphotransferase system for lactose metabolism.     

Induction of beta-galactosidase in Streptomyces violaceus.

Synthesis of beta-galactosidase by Streptomyces violaceus was induced by D-galactose and L-arabinose, and to a lesser extent by lactose, D-arabinose, and methyl-beta-D-galactopyranoside. The synthesis of the enzyme was linear and started to increase 2--3 h after induction by galactose, reaching a maximum after 5--7 h. The highest level of specific activity was observed in 2% galactose, with an increase of 45 times over the basal level in glycerol. Isopropyl-beta-D-thiogalactopyranoside (IPTG) and methyl-beta-D-thiogalactopyranoside (TMG) inhibited induction by D-galactose, but did not influence enzymatic activity. Cellular extracts hydrolyzed O-nitrophenyl-beta-D-galactopyranoside, but did not significantly hydrolyze lactose, melibiose, p-nitrophenyl-alpha-D-galactopyranoside, p-nitrophenyl-beta-D-fucoside, or p-nitrophenyl-beta-D-glucopyranoside. Rifampicin and chloramphenicol inhibited beta-galactosidase synthesis in non-preinduced and in preinduced cells. The inhibition by chloramphenicol was reversible.     

The indirect nature of interaction of glucose transport with the system transporting galactosides

The membrane transport systems for galactosides and glucose derivatives interact in enteric microorganisms. Stop-flow experiments with a double wavelength spectrophotometer and a flow-through cuvette (designed to minimize light-scattering effects) were used to measure the speed of interaction in Escherichia coli. The in vivo hydrolysis of ortho-nitrophenol-beta-D-galactopyranoside was measured by comparing the light transmitted by cell suspensions at 420 nm with that at 500 nm. Measurements at the latter wavelength corrected for residual scattering effects. The stop-flow experiment allowed the study of the early kinetics of transport and hydrolysis. It was found with strain ML308 that there was a significant lag in the achievement of steady-state inhibition by glucose and its derivative methyl-alpha-D-glucopyranoside (alpha MG). This strain constitutively produces high levels of permease and beta-galactosidase. The absorbancy increases at 420 nm are limited by transport because the beta-galactosidase is present inside the cells in excess. From earlier results, it was not surprising that inhibition is delayed with low concentrations of the glucose compounds, but the new double wavelength technique showed no kinetic component of rapid inhibition. This result therefore excludes competition for some membrane-bound component and is consistent with the production of the dephosphorylated form of the soluble Enzyme IIIglu that binds and inhibits the permease system in the membrane.     

Induction and characterization of -galactosidase in an extreme thermophile

A thermostable beta-galactosidase (EC 3.2.1.23; beta-dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the alpha-galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic beta-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K(m) of 2.0 x 10(-3)m for the chromogenic substrate o-nitrophenyl beta-d-galactopyranoside (ONPG) and a K(1) of 7.5 x 10(-3)m for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10(5) daltons was estimated by elution of the enzyme from Sephadex 4B.     

Metabolic control of lactose entry in Escherichia coli

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H⁺/sugar cotransport. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ is less dependent on the need for proton reextrusion, probably because the stoichiometry of H⁺/substrate cotransport is greater for lactose than for ONPG.     

Energy cost of galactoside transport to Escherichia coli.

Energy reserves of Escherichia coli can be depleted by our previously reported procedure to a level such that even the "downhill" transport of o-nitrophenyl-beta-D-galactopyranoside (ONPG) is completely dependent upon the exogenous energy supply. The ONPG concentration is high externally to the cells and is low intracellular because of the action of cytoplasmic beta-galactosidase. In the present work, depleted cell suspensions have been infused at low, steady rates with glucose and other energy sources while measurements of transport were being made. Comparing the rate of ONPG transport with the rate of introduction of glucose under conditions where the chosen glucose infusion rate limits transport, we find that 89 molecules of ONPG are transported per molecule of fully oxidized glucose. This transport yield is constant over a 6.5-fold range in rate of glucose addition. This constancy over a range of infusion rates implies that transport is the major cellular function under these special conditions. The yield value if 89 is in the agreement with the predicitions of 76 from Mitchell's chemiosmotic theory and constitutes an independent proff of its validity, since all the other proposed mechanisms of engery coupling predict much smaller yields. The lag from the start of glucose infusion into the reaction cuvette, to the extrapolated time at which a steady rate of transport and concomitant hydrolysis are achieved, is short (approximately 1 min). Similarly, the time after the infusion is stopped until the rate of transport returns to the background rate is also short. The latter implies that the energy metabolism is directed almost entirely to transport and/or other ongoing cellular processes and not to repair or renewal of an energy-independent, facilitated diffusion system.     

Immobilization of enzymes on spongy polyvinyl alcohol cryogels: the example of beta-galactosidase from Aspergillus oryzae.

The catalytic properties of a beta-galactosidase from Aspergillus oryzae, entrapped into a spongy polyvinyl alcohol cryogel, were studied. This polymeric matrix was selected because of its mild conditions of preparation and its stability, biocompatibility, structural strength and diffusive properties. The enzyme was entrapped, in high percentage, into cryogel sponges and its activity and kinetic parameters were determined and compared with those of the free enzyme, using as substrates o-nitrophenyl-beta-galactopyranoside (ONPG) or lactose. The immobilized enzyme showed a reduced activity with ONPG and lactose, probably because of substrate diffusion limitations through the matrix, but it was more stable to temperature, pH and ionic strength than the free enzyme. Lactose hydrolysis under continuous experimental conditions was performed using the matrix-enzyme cited above.     

Lactose/whey utilization and ethanol production by transformed Saccharomyces cerevisiae cells

Strains of Saccharomyces cerevisiae transformed with a multicopy expression vector bearing both the Escherichia coli beta-galactosidase gene under the control of the upstream activating sequence of the GAL1-10 genes and the GAL4 activator gene release part of beta-galactosidase in the growth medium. This release is due to cell lysis of the older mother cells; the enzyme maintains its activity in buffered growth media. Fermentation studies with transformed yeast strains showed that the release of beta-galactosidase allowed an efficient growth on buffered media containing lactose as carbon source as well as on whey-based media. The transformed strains utilized up to 95% of the lactose and a high growth yield was obtained in rich media. High productions of ethanol were also observed in stationary phase after growth in lactose minimal media.     

Lactose utilization in a cryptic strainEscherichia coli ML 35

The permease-negative strainE. coli ML 35 utilized lactose at 30°C 5–6 times more slowly than glucose or permease-positive strains. The rate of utilization could be raised by cultivating the cells in the presence of streptomycin, acriflavine or actinomycin C. Phenethyl alcohol and the basic proteins ribonuclease, protamine or histone stimulated lactose utilization by washed cells of the same strain not precultivated in the presence of these substances. Ribonuclease did not influence lactose utilization by the permease-positive strain ML 308. Under the given experimental conditions, the presence of basic proteins did not cause β-galactosidase to be released into the medium as a result of lysis of the cells. Streptomycin and ribonuclease did not influence TMG transport into the cells. Basic proteins increased the rate of ONPG hydrolysis by intact cells almost to the level observed in the permease-positive strain. Guanidine, spermine, spermidine, putrescine and cadaverine did not influence lactose utilization by strain ML 35. Spermine antagonized the stimulant effect of ribonuclease or histone on lactose utilization. The experiments were supplemented by comparing the effect of temperature on lactose and glucose utilization by a permease-positive and-negative strain ofE. coli.     

Hydrolysis of whey by whole cells of Kluyveromyces bulgaricus immobilized in calcium alginate gels and in hen egg white

Summary Whey hydrolysis was compared in column reactors containing whole yeast cells immobilized in Ca-alginate or in hen egg white in relation to cell -galactosidase activity, flow rates, temperature and time. With cells of 1.3 U/mg dry weight (ONPG method) immobilized in Ca-alginate, 80% hydrolysis was obtained at 4° and 20° C with, respectively 0.50 and 1.65 bed volume/H; the values were 0.2 and 0.74 with cells entrapped in hen egg white. When the flow rate was expressed as ml/H/g wet yeast, no significant difference was observed between both matrices and 80% hydrolysis was reached with a flow rate 1.7 and 5 according to the temperature. The best performance was achieved by the yeast egg white reactor. At 4°C, hydrolysis decreased by 10% after 13 days; by 20% after 17 days. The presence of lactose transport inhibitors in whey did not significantly influence lactose hydrolysis.M. Decleire et al.: Hydrolysis of whey by immobilized whole cells of Kluyveromyces bulgaricus     

Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system.

Several carbohydrate permease systems in Salmonella typhimurium and Escherichia coli are sensitive to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. Mutant Salmonella strains were isolated in which individual transport systems had been rendered insensitive to regulation by sugar substrates of the phosphotransferase system. In one such strain, glycerol uptake was insensitive to regulation; in another, the maltose transport system was resistant to inhibition; and in a third, the regulatory mutation specifically rendered the melibiose permease insensitive to regulation. An analogous mutation in E. coli abolished inhibition of the transport of beta-galactosides via the lactose permease system. The mutations were mapped near the genes which code for the affected transport proteins. The regulatory mutations rendered utilization of the particular carbohydrates resistant to inhibition and synthesis of the corresponding catabolic enzymes partially insensitive to repressive control by sugar substrates of the phosphotransferase system. Studies of repression of beta-galactosidase synthesis in E. coli were conducted with both lactose and isopropyl beta-thiogalactoside as exogenous sources of inducer. Employing high concentrations of isopropyl beta-thiogalactoside, repression of beta-galactosidase synthesis was not altered by the lactose-specific transport regulation-resistant mutation. By contrast, the more severe repression observed with lactose as the exogenous source of inducer was partially abolished by this regulatory mutation. The results support the conclusions that several transport systems, including the lactose permease system, are subject to allosteric regulation and that inhibition of inducer uptake is a primary cause of the repression of catabolic enzyme synthesis.