Distinct galactose phosphoenolpyruvate-dependent phosphotransferase system in Streptococcus lactis.

Lactose-negative (Lac-) mutants were isolated from a variant of Streptococcus lactis C2 in which the lactose plasmid had become integrated into the chromosome. These mutants retained their parental growth characteristics on galactose (Lac- Gal+). This is in contrast to the Lac- variants obtained when the lactose plasmid is lost from S. lactis, which results in a slower growth rate on galactose (Lac- Gal+). The Lac- Gal+ mutants were defective in [14C]thiomethyl-beta-D-galactopyranoside accumulation, suggesting a defect in the lactose phosphoenolpyruvate-dependent phosphotransferase system, but still possessed the ability to form galactose-1-phosphate and galactose-6-phosphate from galactose in a ratio similar to that observed from the parental strain. The Lac- Gald variant formed only galactose-1-phosphate. The results imply that galactose is not translocated via the lactose phosphoenolpyruvate-dependent phosphotransferase system, but rather by a specific galactose phosphoenolpyruvate-dependent phosphotransferase system for which the genetic locus is also found on the lactose plasmid in S. lactis.     

Characterization of lactose-fermenting revertants from lactose-negative Streptococcus lactis C2 mutants

Partial lactose-fermenting revertants from lactose-negative (lac(-)) mutants of Streptococcus lactis C2 appeared on a lawn of lac(-) cells after 3 to 5 days of incubation at 25 C. The revertants grew slowly on lactose with a growth response similar to that for cryptic cells. In contrast to lac(+)S. lactis C2, the revertants were defective in the accumulation of [(14)C]thiomethyl-beta-d-galactoside, indicating that they were devoid of a transport system. Hydrolysis of o-nitrophenyl-beta-d-galactoside-6-phosphate by toluene-treated cells confirmed the presence of phospho-beta-d-galactosidase (P-beta-gal) in the revertant. However, this enzyme was induced only when the cells were grown in the presence of lactose; galactose was not an inducer. In lac(+)S. lactis C2, enzyme induction occurred in lactose- or galactose-grown cells. The revertants were defective in EII-lactose and FIII-lactose of the phosphoenolpyruvate-dependent phosphotransferase system. Galactokinase activity was detected in cell extracts of lac(+)S. lactis C2, but the activity was 9 to 13 times higher in extracts from the revertant and lac(-), respectively. This suggested that the lac(-) and the revertants use the Leloir pathway for galactose metabolism and that galactose-1-phosphate rather than galactose-6-phosphate was being formed. This may explain why lactose, but not galactose, induced P-beta-gal in the revertants. Because the revertant was unable to form galactose-6-phosphate, induction could not occur. This compound would be formed on hydrolysis of lactose phosphate. The data also indicate that galactose-6-phosphate may serve not only as an inducer of the lactose genes in S. lactis C2, but also as a repressor of the Leloir pathway for galactose metabolism.     

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.     

Regulation of formation of the intracellular beta-galactosidase activity of Aspergillus nidulans

The regulation of formation of the single intracellular beta-galactosidase activity of Aspergillus nidulans was investigated. beta-Galactosidase was not formed during growth on glucose or glycerol, but was rapidly induced during growth on lactose or D-galactose. L-Arabinose, and -- with lower efficacy -- D-xylose also induced beta-galactosidase activity. Addition of glucose to cultures growing on lactose led to a rapid decrease in beta-galactosidase activity. In contrast, in cultures growing on D-galactose, addition of glucose decreased the activity of beta-galactosidase only slightly. Glucose inhibited the uptake of lactose, but not of D-galactose, and required the carbon catabolite repressor CreA for this. In addition, CreA also repressed the formation of basal levels of beta-galactosidase and partially interfered with the induction of beta-galactosidase by D-galactose, L-arabinose, and D-xylose. D-Galactose phosphorylation was not necessary for beta-galactosidase induction, since induction by D-galactose occurred in an A. nidulans mutant defective in galactose kinase, and by the non-metabolizable D-galactose analogue fucose in the wild-type strain. Interestingly, a mutant in galactose-1-phosphate uridylyl transferase produced beta-galactosidase at a low, constitutive level even on glucose and glycerol and was no longer inducible by D-galactose, whereas it was still inducible by L-arabinose. We conclude that biosynthesis of the intracellular beta-galactosidase of A. nidulans is regulated by CreA, partially repressed by galactose-1-phosphate uridylyl transferase, and induced by D-galactose and L-arabinose in independent ways.     

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.     

Melibiose transport system in Lactobacillus plantarum.

Lactobacillus plantarum ATCC 8014 grew on melibiose at 30 C, but not at 37 C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37 C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30 C, but not at 37 C. The transport activity itself was found to be stable for at least 3 hr at 37 C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form alpha-galactosidase at 37 C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37 C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and alpha-galactosidase were induced by galactose, melibiose and o-nitrophenyl-alpha-D-galactopyranoside when the strain was grown at 30 C. Raffinose induced melibiose transport system only a little, while it was a good inducer for alpha-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.     

Studies on the Fermentative Production and Metabolism of Uridine Coenzymes

Enzyme activities involved in the galactose metabolism of Torulopsis Candida grown on a. lactose medium were investigated with the cell-free extract and ammonium sulfate fraction. Remarkable activities of galactokinase, galactose-1-phosphate uridylyltransferase and UDPG pyrophosphorylase were detected, whereas UDPGal pyrophosphorylase activity was weak. UDPGal formation proceeded by the cell-free extract along a coupling reaction catalyzed by UDPG pyrophosphorylase and galactose-1-phosphate uridylyltransferase where UDPG or glucose-l-phosphate acted as a catalyst.

The mechanism of UDPGal accumulation under the fermentative condition could be explained by a concerted inhibition of UDPGal-4- epimerase activity by 5′-UMP and galactose present as fermentation substrates.     

Selection procedure for mutants defective in the beta-methylgalactoside transport system of Escherichia coli utilizing the compound 2R-glyceryl-beta-D-galactopyranoside

A procedure has been devised that allows selection of mutants defective in the beta-methylgalactoside transport system (mgl) of Escherichia coli. This procedure utilizes the compound 2R-glyceryl-beta-d-galactopyranoside (glycerylgalactoside), which is known to be transported by only two transport system in E. coli, namely, the lactose and the beta-methylgalactoside transport systems. Mutants lacking glycerol-3-phosphate dehydrogenase (glpD) are sensitive to glycerol. Similarly, mutants lacking uridine diphosphate-galactose-4-epimerase (galE) are sensitive to galactose. Glycerylgalactoside is an inducer of the lactose operon and also a substrate for beta-galactosidase. Thus, a mgl(+)glpD galE lacY strain will not grow in the presence of glycerylgalactoside owing to accumulated glycerol-3-phosphate, galactose-1-phosphate, and uridine diphosphate-galactose. We have constructed such a strain and shown that mgl mutants can be obtained by selecting for those that grow in the presence of glycerylgalactoside.     

Overexpression of human UDP-glucose pyrophosphorylase rescues galactose-1-phosphate uridyltransferase-deficient yeast

To better understand the pathophysiology of galactose-1-phosphate uridyltransferase (GALT) deficiency in humans, we studied the mechanisms by which a GALT-deficient yeast survived on galactose medium. Under normal conditions, GALT-deficient yeast cannot grow in medium that contains 0.2% galactose as the sole carbohydrate, a phenotype of Gal(-). We isolated revertants from a GALT-deficient yeast by direct selection for growth in galactose, a phenotype of Gal(+). Comparison of gene expression profiles among wild-type and revertant strains on galactose medium revealed that the revertant down-regulated genes encoding enzymes including galactokinase, galactose permease, and UDP-galactose-4-epimerase (the GAL regulon). By contrast, the revertant strain up-regulated the gene for UDP-glucose pyrophosphorylase, UGP1. There was reduced accumulation of galactose-1-phosphate in the galactose-grown revertant cells when compared to the GALT-deficient parent cells. In vitro biochemical analysis showed that UDP-glucose pyrophosphorylase had bifunctional properties and could catalyze the conversion of galactose-1-phosphate to UDP-galactose in the presence of UTP. To test if augmented expression of this gene could produce a Gal(+) phenotype in the GALT-deficient parent cells, we overexpressed the yeast UGP1 and the human homolog, hUGP2 in the mutant strain. The Gal(-) yeast transformed with either UGP1 or hUGP2 regained their ability to grow on galactose. We conclude that revertant can grow on galactose medium by reducing the accumulation of toxic precursors through down-regulation of the GAL regulon and up-regulation of the UGP1 gene. We speculate that increased expression of hUGP2 in humans could alleviate poor outcomes in humans with classic galactosemia.     

Phosphoenolpyruvate:carbohydrate phosphotransferase systems in Enterococcus faecalis

A wild-type strain of Enterococcus faecalis and its mutants resistant to 2-deoxy-D-glucose (2DG) were examined for the presence of phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs) with 12 carbohydrates, which were utilized by the organism, as the substrates. The wild-type strain possessed a constitutive mannose-PTS, which was reactive with glucose, mannose, glucosamine, 2DG and fructose. This activity was absent in the mutants. No independent glucose- or fructose-PTS was found in the mannose-PTS-defective mutants. The mutants, however, showed a low level of a constitutive PTS activity with maltose, suggesting the existence of an independent maltose-PTS in the organism. Both wild-type and mutant strains possessed inducible lactose-, mannitol-, and trehalose-PTSs. Lactose-PTS was induced by either lactose or galactose in the parent, but only by lactose in the mutants. The lactose-PTS was not reactive with galactose, and no separate galactose-PTS was present. These observations suggest that the inducer for lactose-PTS, probably being galactose 6-phosphate, may not be formed from galactose in the organism when the constitutive mannose-PTS is lost by mutation.     

Plasmid linkage of the D-tagatose 6-phosphate pathway in Streptococcus lactis: effect on lactose and galactose metabolism

The three enzymes of the D-tagatose 6-phosphate pathway (galactose 6-phosphate isomerase, D-tagatose 6-phosphate kinase, and tagatose 1,6-diphosphate aldolase) were absent in lactose-negative (Lac-) derivatives of Streptococcus lactis C10, H1, and 133 grown on galactose. The lactose phosphoenolpyruvate-dependent phosphotransferase system and phospho-beta-galactosidase activities were also absent in Lac- derivatives of strains H1 and 133 and were low (possibly absent) in C10 Lac-. In all three Lac- derivatives, low galactose phosphotransferase system activity was found. On galactose, Lac- derivatives grew more slowly (presumably using the Leloir pathway) than the wild-type strains and accumulated high intracellular concentrations of galactose 6-phosphate (up to 49 mM); no intracellular tagatose 1,6-diphosphate was detected. The data suggest that the Lac phenotype is plasmid linked in the three strains studied, with the evidence being more substantial for strain H1. A Lac- derivative of H1 contained a single plasmid (33 megadaltons) which was absent from the Lac- mutant. We suggest that the genes linked to the lactose plasmid in S. lactis are more numerous than previously envisaged, coding for all of the enzymes involved in lactose metabolism from initial transport to the formation of triose phosphates via the D-tagatose 6-phosphate pathway.     

Galactose Expulsion during Lactose Metabolism in Lactococcus lactis subsp. cremoris FD1 Due to Dephosphorylation of Intracellular Galactose 6-Phosphate

In Lactococcus lactis subsp. cremoris FD1, galactose and lactose are both transported and phosphorylated by phosphotransferase systems. Lactose 6-phosphate (lactose-6P) is hydrolyzed intracellularly to galactose-6P and glucose. Glucose enters glycolysis as glucose-6P, whereas galactose-6P is metabolized via the tagatose-6P pathway and enters glycolysis at the tagatose diphosphate and fructose diphosphate pool. Galactose would therefore be a gluconeogenic sugar in L. lactis subsp. cremoris FD1, but since fructose 1,6-diphosphatase is not present in this strain, galactose cannot serve as an essential biomass precursor (glucose-6P or fructose-6P) but only as an energy (ATP) source. Analysis of the growth energetics shows that transition from N limitation to limitation by glucose-6P or fructose-6P gives rise to a very high growth-related ATP consumption (152 mmol of ATP per g of biomass) compared with the value in cultures which are not limited by glucose-6P or fructose-6P (15 to 50 mmol of ATP per g of biomass). During lactose metabolism, the galactose flux through the tagatose-6P pathway (r(max) = 1.2 h) is lower than the glucose flux through glycolysis (r(max) = 1.5 h) and intracellular galactose-6P is dephosphorylated; this is followed by expulsion of galactose. Expulsion of a metabolizable sugar has not been reported previously, and the specific rate of galactose expulsion is up to 0.61 g of galactose g of biomass h depending on the lactose flux and the metabolic state of the bacteria. Galactose excreted during batch fermentation on lactose is reabsorbed and metabolized when lactose is depleted from the medium. In vitro incubation of galactose-6P (50 mM) and permeabilized cells (8 g/liter) gives a supernatant containing free galactose (50 mM) but no P(i) (less than 0.5 mM). No organic compound except the liberated galactose is present in sufficient concentration to bind the phosphate. Phosphate is quantitatively recovered in the supernatant as P(i) by hydrolysis with alkaline phosphatase (EC 3.1.3.1), whereas inorganic pyrophosphatase (EC 3.6.1.1) cannot hydrolyze the compound. The results indicate that the unknown phosphate-containing compound might be polyphosphate.     

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-β-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.     

The roles of galactitol, galactose-1-phosphate, and phosphoglucomutase in galactose-induced toxicity in Saccharomyces cerevisiae

The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combinations of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furthermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.     

Biochemical analysis of galactose induced bacteriostasis ingalT mutants ofescherichia coli K 12

Escherichia coli mutants completely defective in galactose-1-phosphate uridyl transferase (EC 2.7.7.10) and growing in glycerol medium undergo rapid cessation of growth when exposed to galactose. Toxicity due to galactose is equally pronounced when glycerol is replaced by other carbon sources, like succinate and proline. Gas chromatographic analysis failed to detect even trace amounts of galactitol. Moreover, galactose-1-phosphate had no inhibitory role on some of the critical enzymes of cellular metabolism. General loss of energy (ATP) due to futile phosphorylation of galactose is probably the cause of bacteriostasis. ThegalT mutants can serve as models of human transferaseless galactosemia only to a limited extent     

Galactose Transport in Saccharomyces cerevisiae III. Characteristics of Galactose Uptake in Transferaseless Cells: Evidence Against Transport-Associated Phosphorylation

The characteristics of the inducible galactose transport system in bakers' yeast were studied in uridine diphosphate, galactose-1-phosphate uridylyl-transferaseless cells. Transferaseless cells transport galactose at the same initial rate as wild-type cells and accumulate a mixture of free galactose and galactose-1-phosphate. The addition of ¹⁴C-labeled galactose to cells preloaded with unlabeled galactose and galactose-1-phosphate results in a higher rate of labeling of the free-sugar pool than of the galactose-1-phosphate pool. These results support other evidence that galactose uptake in bakers' yeast is a carrier-mediated, facilitated diffusion and that phosphorylation is an intracellular event after uptake of the free sugar.     

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.     

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.