Lactose-grown Cells Research Articles

Sugar transport systems in Kluyveromyces marxianus CCT 7735.

The pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H+-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.

Catabolism of Glucose and Lactose in Bifidobacterium animalis subsp. lactis, Studied by 13 C Nuclear Magnetic Resonance

Bifidobacteria are widely used as probiotics in several commercial products; however, to date there is little knowledge about their carbohydrate metabolic pathways. In this work, we studied the metabolism of glucose and lactose in the widely used probiotic strain Bifidobacterium animalis subsp. lactis BB-12 by in vivo (13)C nuclear magnetic resonance (NMR) spectroscopy. The metabolism of [1-(13)C]glucose was characterized in cells grown in glucose as the sole carbon source. Moreover, the metabolism of lactose specifically labeled with (13)C on carbon 1 of the glucose or the galactose moiety was determined in suspensions of cells grown in lactose. These experiments allowed the quantification of some intermediate and end products of the metabolic pathways, as well as determination of the consumption rate of carbon sources. Additionally, the labeling patterns in metabolites derived from the metabolism of glucose specifically labeled with (13)C on carbon 1, 2, or 3 in cells grown in glucose or lactose specifically labeled in carbon 1 of the glucose moiety ([1-(13)Cglucose]lactose), lactose specifically labeled in carbon 1 of the galactose moiety ([1-(13)Cgalactose]lactose), and [1-(13)C]glucose in lactose-grown cells were determined in cell extracts by (13)C NMR. The NMR analysis showed that the recovery of carbon was fully compatible with the fructose 6-phosphate, or bifid, shunt. The activity of lactate dehydrogenase, acetate kinase, fructose 6-phosphate phosphoketolase, and pyruvate formate lyase differed significantly between glucose and lactose cultures. The transcriptional analysis of several putative glucose and lactose transporters showed a significant induction of Balat_0475 in the presence of lactose, suggesting a role for this protein as a lactose permease. This report provides the first in vivo experimental evidence of the metabolic flux distribution in the catabolic pathway of glucose and lactose in bifidobacteria and shows that the bifid shunt is the only pathway involved in energy recruitment from these two sugars. On the basis of our experimental results, a model of sugar metabolism in B. animalis subsp. lactis is proposed.

Restricted sugar uptake by sugar-induced internalization of the yeast lactose/galactose permease Lac12

Kluyveromyces lactis Lac12 permease mediates lactose and low-affinity galactose transports. In this study we investigated the effects of carbon sources on internalization of Lac12 using a LAC12-GFP fusion construct. When galactose- or lactose-grown cells are shifted to a fresh sugar medium, Lac12-GFP is removed from the plasma membrane and is localized intracellularly. Surprisingly, either galactose or lactose in the new media caused the internalization, and cells responded differently to these two sugars. Our results reveal that this process is dependent on sugar species and also sugar concentration. Lac12-GFP internalization causes reduction of [C(14) ]lactose uptake rates and also occurs in a Klsnf1 mutant strain; it is therefore independent of KlSnf1 activity. We suggest that glucose-6-phosphate is the intracellular signal, as internalization was induced by 2-deoxyglucose, and inhibition of phosphoglucomutase by lithium prevented galactose- but not lactose- or glucose-induced internalization. Lac12-GFP internalization was not triggered by 6-deoxyglucose, and was irreversible in the absence of protein synthesis.

A study of Streptococcus thermophilus proteome by integrated analytical procedures and differential expression investigations

Streptococcus thermophilus is a Gram-positive bacterium belonging to the group of lactic acid bacteria, among which several genera play an essential role in manufacture of food products. Recently, a genomic consortium sequenced and annotated its entire genome, which has been demonstrated to contain 1900 coding sequences. In this study, we have revealed the expression products of almost 200 different genes using a proteomic strategy combining 2-DE plus MALDI-TOF PMF and differential 1-DE plus muLC-ESI-IT-MS/MS. Thus, a number of cellular pathways related to important physiological processes were described at the proteomic level. Almost 50 genes were related to multiple electrophoretic species, whose heterogeneity was mainly due to variability in pI values. A 2-DE reference map obtained for lactose-grown cells was compared with those obtained after heat, cold, acid, oxidative and starvation stresses. Protein up/down-regulation measurements demonstrated that adaptation to different environmental challenges may involve the contribution of unique as well as combined physiological mechanisms. Common regulatory sites in the promoter region of genes whose expression was induced after stress were identified. These results provide a better comprehension of biochemical processes related to stress resistance in S. thermophilus, allowing defining the molecular bases of adaptative responses or markers for the identification of strains with potential industrial applications.

Proteomic analysis of Lactococcus lactis, a lactic acid bacterium.

Lactococcus lactis is a Gram-positive bacteria, which belongs to the group of lactic acid bacteria among which several genera play an essential role in the manufacture of food products. Cytosolic proteins of L. lactis IL1403 cultivated in M17 broth have been resolved by two-dimensional gel electrophoresis using two pH gradients (pH 4-7, 4.5-5.5). More than 230 spots were identified by peptide mass fingerprints, corresponding to 25% of the predicted acid proteome. The present study made it possible to describe at the proteome level a significant number of cellular pathways (glycolysis, fermentation, nucleotide metabolism, proteolysis, fatty acid and peptidoglycan synthesis) related to important physiological processes and technological properties. It also indicated that the fermentative metabolism, which characterizes L. lactis is associated with a high expression of glycolytic enzymes. Thirty-four proteins were matched to open reading frames for which there is no assigned function. The comparison at the proteome level of two strains of L. lactis showed an important protein polymorphism. The comparison of the proteomes of glucose- and lactose-grown cells revealed an unexpected link between the nature of the carbon source and the metabolism of pyrimidine nucleotides.

Differential gene expression in Thermotoga neapolitana in response to growth substrate

We have previously shown that β-galactosidase activity expressed in Thermotoga neapolitana cells grown on lactose is subject to repression by glucose when they are grown on both substrates whereas β-galactosidase and β-glucosidase activities observed in cells grown on cellobiose are not repressed by growth on both glucose and cellobiose. To examine the differential expression of bgalA, bgalB, bglA and bglB in T. neapolitana, total RNA was isolated from cells growing on either glucose, lactose or cellobiose as the sole source of carbon and transcripts encoding these genes were quantitated by Northern blot analyses. BglA expression was induced by cellobiose while bglB was expressed under all three conditions at a lower level. Expression of the β-galactosidase genes, bgalA and bgalB, was detected only in lactose-grown cells. β-Glucosidase enzyme activity was only found in cell extracts of cellobiose-grown cells while β-galactosidase activity was found in both lactose- and cellobiose-grown cell extracts. Our results show that in cellobiose-grown cells, the high β-glucosidase activity is likely due to expression of bglA and that neither bgalA nor bgalB is responsible for the β-galactosidase activity.

Differential gene expression in Thermotoga neapolitana in response to growth substrate.

We have previously shown that beta-galactosidase activity expressed in Thermotoga neapolitana cells grown on lactose is subject to repression by glucose when they are grown on both substrates whereas beta-galactosidase and beta-glucosidase activities observed in cells grown on cellobiose are not repressed by growth on both glucose and cellobiose. To examine the differential expression of bgalA, bgalB, bglA and bglB in T. neapolitana, total RNA was isolated from cells growing on either glucose, lactose or cellobiose as the sole source of carbon and transcripts encoding these genes were quantitated by Northern blot analyses. BglA expression was induced by cellobiose while bglB was expressed under all three conditions at a lower level. Expression of the beta-galactosidase genes, bgalA and bgalB, was detected only in lactose-grown cells. beta-Glucosidase enzyme activity was only found in cell extracts of cellobiose-grown cells while beta-galactosidase activity was found in both lactose- and cellobiose-grown cell extracts. Our results show that in cellobiose-grown cells, the high beta-glucosidase activity is likely due to expression of bglA and that neither bgalA nor bgalB is responsible for the beta-galactosidase activity.

Properties of two sugar phosphate phosphatases from Streptococcus bovis and their potential involvement in inducer expulsion.

Streptococcus bovis possesses two sugar phosphate phosphatases (Pases). Pase I is a soluble enzyme that is inhibited by the membrane fractions from lactose-grown cells and is insensitive to activation by S46D HPr, an analog of HPr(ser-P) of the sugar phosphotransferase system. Pase II is a membrane-associated enzyme that can be activated 10-fold by S46D HPr, and it appears to play a role in inducer expulsion.

Fermentation of lactose by yeast cells secreting recombinant fungal lactase.

Strains of Saccharomyces cerevisiae transformed with a yeast multicopy expression vector carrying the cDNA for Aspergillus niger secretory beta-galactosidase under the control of ADH1 promoter and terminator were studied for their fermentation properties on lactose (V. Kumar, S. Ramakrishnan, T. T. Teeri, J. K. C. Knowles, and B. S. Hartley, Biotechnology 10:82-85, 1992). Lactose was hydrolyzed extracellularly into glucose and galactose, and both sugars were utilized simultaneously. Diauxic growth patterns were not observed. However, a typical biphasic growth was observed on a mixture of glucose and galactose under aerobic and anaerobic conditions with transformants of a haploid S. cerevisiae strain, GRF167. Polyploid distiller's yeast (Mauri) transformants were selected simply on the basis of the cloned gene expression on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates. Rapid and complete lactose hydrolysis and higher ethanol (0.31 g/g of sugar) and biomass (0.24 g/g of sugar) production were observed with distiller's yeast grown under aerobic conditions. A constant proportion (10%) of the population retained the plasmid throughout the fermentation period (48 h). Nearly theoretical yields of ethanol were obtained under anaerobic conditions on lactose, glucose, galactose, and whey permeate media. However, the rate and the amount of lactose hydrolysis were lower under anaerobic than aerobic conditions. All lactose-grown cells expressed partial galactokinase activity.

Regulation of polysaccharide formation by Streptococcus thermophilus in batch and fed-batch cultures

A commercial strain of Streptococcus thermophilus possessing galactokinase activity was examined for the effect of lactose flow rate on the production and composition of polysaccharides through the early steps of biosynthesis. In all cases, lactose-grown cells did not release free galactose into the medium and produced polysaccharides containing galactose, glucose, mannose, uronic acids and minor amounts of hexosamines. In batch cultures with excess lactose present the cells converted nearly 80% of the carbon source to L-lactate and produced 2.4 g 1−1 (eq. glucose) polysaccharides. However, when the carbon flow was set at 1.5 mM h−1, only 47% of the fermented sugar was converted to L-lactate by the strain, which synthesized 22% more polysaccharides. As lactose became limiting, the level of some glycolytic enzymes and nucleotidyltransferases markedly decreased while phosphoglucomutase, phosphomannose isomerase and galactokinase activities were stimulated. The shift in the key enzyme ratios was reflected by major changes in polysaccharide distribution, which definitely altered in favour of galactose. Data suggested a diversion of lactose flow towards polysaccharide production at the expense of lactic acid and biomass formation, as well as a fine regulation of polymer distribution when the cell growth of S. thermophilus was limited by the carbon source feed rate.

Effect of Sodium Chloride on Uptake of Substrate by Staphylococcus aureus 196E

Sodium chloride inhibited a number of biochemical parameters in Staphylococcus aureus 196E. Induction of phospho-β-galactosidase, synthesis of staphylococcal enterotoxin A, enzyme activity (phospho-β-galactosidase) and glucose utilization were approximately four times more sensitive to the inhibitory effects of salt than was growth. Uptake of 14C-2-deoxyglucose and respiratory activity with a number of substrates were inhibited also. The breakdown of o-nitrophenyl-β-galactoside (ONPG) by lactose-grown S. aureus 196E was inhibited by NaCl as well as by other solutes (salts, carbohydrates, amino acids) which suggested that the inhibitory effect is a general one of solutes and not restricted to NaCl. Various ionophores (gramicidin, valinomycin, monensin, lasalocid, m-chlorophenylhydrazone), the H+-ATPase inhibitor (N,N′,-dicyclohexylcarbodiimide), and ion channel blockers (quinine, quinidine, chlorpromazinc, tetracaine, verapamil) reversed the inhibitory action of salt on ONPG breakdown by lactose-grown cells; however, these compounds did not reverse NaCl inhibition of glucose utilization. The effects observed here suggest that NaCl (and probably other solutes) exerts an inhibitory effect on transport of substrates into the cells of S. aureus 196E.

Characterization of low- and high-affinity glucose transports in the yeast Kluyveromyces marxianus

Glucose transport in the yeast Kluyveromyces marxianus proceeds by two functionally and presumably structurally distinct transporters depending on the carbon source of the culture medium. In lactose-grown cells, glucose was taken up through a high-affinity H +-sugar symporter ( K m = 0.09 mM), whereas a low-affinity transporter ( K m = 3.5 mM) was utilized in glucose-grown cells. The two transporters exhibited different substrate specificities. Galactose was demonstrated to be a selective substrate of the H +-glucose symporter ( K m = 0.14 mM) and did not significantly enter glucose-grown cells. Fructose was a preferential substrate of the low-affinity carrier ( K m = 3.5 mM), but it entered lactose-grown cells through a high-affinity H +-fructose symporter distinct from the H +-glucose one. Other putative substrates of the two glucose transporters were identified by competition experiments. 2-Deoxyglucose recognized both carriers with a similar affinity, while the non-phosphorylatable analogues 6-deoxyglucose, 3- O-methylglucose and d-fucose exhibited a 10–30 fold preference for the high-affinity transporter.

Influence of Carbon Substrates on Lactic Acid, Cell Mass and Diacetyl-Acetoin Production in Lactobacillus plantarum

Production of diacetyl-acetoin, lactic acid and cell mass by L. plantarum strains ATCC 8014, ATCC 14431, ATCC 4008 and ATCC 8041 were examined in the growth medium of Craig and Snell (J. Bacteriol. 61:283,1951) containing glucose, lactose, citrate or pyruvate as substrates. The yield coefficient, μM lactate produced per mg cell dry weight, averaged 73.4 for glucose-grown cells and 64.9 for lactose-grown cells with no significant inter-strain difference. Strains that produced higher lactic acid concentrations did so because they produced more cell mass. Glucose and lactose reduced diacetyl-acetoin synthesis in all strains except 8041. Diacetyl-acetoin synthesis doubled when strains 14431 and 8014 were grown in medium containing 20mM citrate, but was not markedly affected in strains 4008 and 8041. Pyruvate stimulated diacetyl-acetoin synthesis in all four strains 10- to 20-fold. Conversion of pyruvate to diacetyl and acetoin was ⩾30% of the theoretical molar yield for strains 8014, 8041 and 14431 grown in the presence of 20mM pyruvate.

Lactose transport in Streptococcus mutans: isolation and characterization of factor IIIlac, a specific protein component of the phosphoenolpyruvate-lactose phosphotransferase system.

The transport of lactose in Streptococcus mutans is mediated via an inducible phosphoenolpyruvate-lactose phosphotransferase system. This system requires for catalytic activity a membrane fraction (enzyme II), two general proteins called enzyme I and HPr, and a soluble specific protein termed factor IIIlac. This protein factor was purified from S. mutans ATCC 27352 by chromatographies on DEAE-cellulose, hydroxylapatite, Ultrogel AcA 34, and phosphocellulose. The purified protein migrated as a single band with a molecular weight of 10,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea. The molecular weight calculated from the amino acid composition was 10,541. Gel filtration of the native protein gave a molecular weight of 41,500. Its isoelectric point was ca. 4.70. A specific antiserum was prepared against purified factor IIIlac. Immunodiffusion experiments revealed that only cellular extracts from lactose-grown cells contained factor IIIlac. A cross-reaction was observed with all of the S. mutans strains tested as well as with Streptococcus sanguis 10556, Streptococcus lactis 11454, and Staphylococcus aureus 6538. No precipitin band was observed with extracts of Streptococcus salivarius, Streptococcus faecalis, Lactobacillus casei, and Bacillus subtilis.

Arginine metabolism in lactic streptococci.

Streptococcus lactis metabolizes arginine via the arginine deiminase pathway producing ornithine, ammonia, carbon dioxide, and ATP. In the four strains of S. lactis examined, the specific activities of arginine deiminase and ornithine transcarbamylase were 5- to 10-fold higher in galactose-grown cells compared with glucose- or lactose-grown cells. The addition of arginine increased the specific activities of these two enzymes with all growth sugars. The specific activity of the third enzyme involved in arginine metabolism (carbamate kinase) was not altered by the composition of the growth medium. In continuous cultures arginine deiminase was not induced, and arginine was not metabolized, until glucose limitation occurred. In batch cultures the metabolism of glucose and arginine was sequential, whereas galactose and arginine were metabolized concurrently, and the energy derived from arginine metabolism was efficiently coupled to growth. No arginine deiminase activity was detected in the nine Streptococcus cremoris strains examined, thus accounting for their inability to metabolize arginine. All nine strains of S. cremoris had specific activities of carbamate kinase similar to those found in S. lactis, but only five S. cremoris strains had ornithine transcarbamylase activity.

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.

Effects of pH and Sugar on Acetoin Production from Citrate by Leuconostoc lactis

The relationship between acetoin production and citrate utilization in Leuconostoc lactis NCW1 was studied. In a complex medium the organism utilized citrate at neutral pH (initial pH, 6.3) and at acid pH (initial pH, 4.5) but produced nine times more acetoin at the latter pH. In resting cells the utilization of citrate was optimum at pH 5.3. Production of acetoin as a function of citrate utilization increased as the pH decreased, and at pH 4.3 all of the citrate utilized was recovered as acetoin. Glucose (10 mM) and lactose (10 mM) markedly stimulated citrate utilization but totally inhibited acetoin production in glucose- and lactose-grown cells. Addition of glucose to cells actively metabolizing citrate caused an immediate increase in citrate uptake and a reduction in the level of acetoin. The apparent K(m) values of lactic dehydrogenase for pyruvate were 1.05, 0.25, and 0.15 mM at pH 7.5, 6.5, and 5.0, respectively. Several heterofermentation intermediates inhibited alpha-acetolactate synthetase and decarboxylase activities. The implications of these results in regulating acetoin formatin are discussed.

Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation.

All of the lactic streptococci examined except Streptococcus lactis ML8 fermented galactose to lactate, formate, acetate, and ethanol. The levels of pyruvate-formate lyase and lactate dehydrogenase were elevated and reduced, respectively, in galactose-grown cells compared with glucose- or lactose-grown cells. Reduced intracellular levels of both the lactate dehydrogenase activator (fructose, 1,6-diphosphate) and pyruvate-formate lyase inhibitors (triose phosphates) appeared to be the main factors involved in the diversion of lactate to the other products. S. lactis ML8 produced only lactate from galactose, apparently due to the maintenance of high intracellular levels of fructose 1,6-diphosphate and triose phosphates. The growth rates of all 10 Streptococcus cremoris strains examined decreased markedly with galactose concentrations below about 30 mM. This effect appeared to be correlated with uptake predominantly by the low-affinity galactose phosphotransferase system and initial metabolism via the D-tagatose 6-phosphate pathway. In contrast, with four of the five S. lactis strains examined, galactose uptake and initial metabolism involved more extensive use of the high-affinity galactose permease and Leloir pathway. With these strains the relative flux of galactose through the alternate pathways would depend on the exogenous galactose concentration.

Branched-chain amino acid transport in Streptococcus agalactiae.

The transport of the branched-chain amino acids in Streptococcus agalactiae was characterized. Glucose-grown cells were able to utilize only glucose as an energy source for transport of L-leucine, whereas lactose-grown cells could utilize both glucose and lactose. It was determined from metabolic inhibitor studies that energy from glycolysis and substrate level phosphorylation was required for active transport. Energy was found to be coupled to transport by the action of adenosine triphosphatase and the generation of a proton motive force. The branched-chain amino acids were found to share a common transport system that may consist of multiple components.

A requirement for ATP for beta-galactoside transport by Bacillus alcalophilus.

Lactose-grown cells of Bacillus alcalophilus actively transported methylthio-beta, D-galactoside (TMG) in a range of pH values from 7.5 to 10.5 with a pH optimum at 8.5. The TMG was accumulated in a chemically unmodified form, and cell extracts failed to catalyze either ATP or P-enolpyruvate-dependent phosphorylation of TMG. At pH 8.5, the lactose-grown cells exhibited a transmembrane proton gradient (deltapH) of 1.38 units, interior acid, and a transmembrane electrical potential (delta psi) of -132 mV. Accordingly, the total protonmotive force at this pH was very low, -51mV. Several lines of evidence indicate that the protonmotive force or delta psi did not directly energize TMG transport but, rather, that ATP was directly required: (a) in cells treated with arsenate so that the delta psi was unaffected and cellular ATP levels were markedly lowered, TMG transport was inhibited in proportion to the reduction of cellular ATP, while electrogenic alpha-aminoisobutyric acid transport was not; (b) when a valinomycin-induced potassium diffusion potential was established in starved cells, alpha-aminoisobutyric acid transport, but not TMG transport, was stimulated; and (c) in a series of experiments in which the delta psi was rapidly abolished by treatment with gramicidin, ATP levels declined slowly and the rate of TMG transport correlated directly with ATP levels rather than with the delta psi. Consumption of cellular ATP concomitant with TMG transport could be demonstrated.