Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.

Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms.     

Cytochrome P-450 and the activation of promutagens in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the cellular content of cytochrome P-450 was investigated and shown to be related to the growth phase of aerobic cultures when glucose was the carbon source. When grown on glucose medium the log-phase cells of the diploid strain D5 contained about 9× more cytochrome P-450 than log-phase cells of the diploid strain D4. The D4 cells grown on medium containing glucose contained about 10× more cytochrome P-450 than D4 cell grown on medium containing galactose as carbon source. Cells of strain D4, harvested from log-phase cultures grown on glucose, were capable of metabolizing aflatoxin B₁, dimethylnitrosamine, β-naphthylamine, ethyl carbamate, cyclophosphamide and dimethylsulphoxide to products active genetically in the same cells. The metabolism of the compounds tested was attributed to cyctochrome P-450-dependent mixed-function oxidation since genetic activity was high in log cells grown on medium containing glucose but negligible in log cells grown on medium containing galactose. However, aflatoxin B₁ differed from the other promutagens tested since the genetic activity of this compound in cells grown on galactose medium was similar to the activity in cells grown on glucose medium. This result is discussed in relation to enzyme systems which could metabolize aflatoxin B₁. The results of treating log-phase cells of the strain D5, grown on medium containing glucose, with aflatoxin B₁ and dimethylnitrosamine are presented and compared with the results from the strain D4.     

Reduced Lysis upon Growth of Lactococcus lactis on Galactose Is a Consequence of Decreased Binding of the Autolysin AcmA

When Lactococcus lactis subsp. lactis IL1403 or L. lactis subsp. cremoris MG1363 is grown in a medium with galactose as the carbon source, the culture lyses to a lesser extent in stationary phase than when the bacteria are grown in a medium containing glucose. Expression of AcmA, the major autolysin of L. lactis, is not influenced by the carbon source. Binding studies with a fusion protein consisting of the MSA2 protein of Plasmodium falciparum and the C-terminal peptidoglycan-binding domain of AcmA revealed that cell walls of cells from both subspecies grown on galactose bind less AcmA than cell walls of cells grown on glucose. Cells grown on glucose or galactose and treated with trichloroacetic acid prior to AcmA binding bind similar amounts of AcmA. Analysis of the composition of the lipoteichoic acids (LTAs) of L. lactis IL1403 cells grown on glucose or galactose showed that the LTA composition is influenced by the carbon source: cells grown on galactose contain LTA with less galactose than cells grown on glucose. In conclusion, growth of L. lactis on galactose changes the LTA composition in the cell wall in such a way that less AcmA is able to bind to the peptidoglycan, resulting in a decrease in autolysis.     

The Effect of Ethanol on the Growth of Dipodascus aggregatus

Ethanol (68.2 mM) did not appreciably affect the growth of Dipodascus aggregatus with glucose (55.5 mM] as carbon source. Growth with fructose was inhibited whereas growth with galactose was stimulated by ethanol in this concentration. The fungus could grow with ethanol as the sole carbon source. D. aggregatus did not grown with maltose as the sole carbon source. Growth with maltose + ethanol started much earlier than growth with ethanol alone. The maltose concentration of the medium did not measurably decrease during growth with maltose-n ethanol. D. aggregatus did not grow with sucrose as the sole carbon source     

The Effect of Nonanal on Dipodascus aggregatus IV. Studies with Different Carbon Sources

Nonanal (80 μ) in ethanolic solution stimulated the growth of Dipodascus aggregatus with fructose (55.5 mM) as carbon source (inoculum grown with fructose or glucose). If the inoculum had been grown with galactose, neither growth with galactose nor growth with glucose was affected by nonanal. If the inoculum had been grown with glucose, growth with galactose was weakly. stimulated. —Growth with galactose (galactose-grown inoculum) was strongly stimulated by nonanal if xylose at a low concentration (0.53 mM) was added. — The oxygen uptake of glucose grown cells with glucose as substrate was stimulated by 200 μM nonanal in the absence of ethanol. The respiratory activity of galactose-grown cells was also stimulated with galactose as well as with glucose as substrate. In the absence of exogenous substrate the oxygen uptake of glucose-grown cells was weakly stimulated by nonanal whereas that of galactose-grown cells was strongly stimulated.     

The role of catalases in protection of proteins against oxidation in Saccharomyces cerevisiae utilizing ethanol as a carbon source

The content of protein carbonyls and thiobarbituric acid reactive substances (TBARS) in the wild and catalase-deficient strains of the yeast Saccharomyces cerevisiae grown in glucose and ethanol media are compared. The deficient strain cells reproduced 10.6-fold slower in ethanol-containing medium. Activity of glucose-6-phosphate dehydrogenase in YWT1 cells was 1.7-fold lower when yeast are grown in ethanol, and content of protein carbonyls was 4.7-fold higher, than when they are grown in the medium with glucose. At the same time, reproduction of the wild type cells in ethanol was 2.7-fold slower and carbonyl groups of protein content was 2-fold lower, than under cultivation in glucose. TBARS content in both strains was similar when they were grown in ethanol and in glucose. It has been supposed that catalases play a certain role in the protection of S. cerevisiae proteins against oxidative modification when they are grown on the media with glucose and ethanol.     

Low Glucose but Not Galactose Enhances Oxidative Mitochondrial Metabolism in C2C12 Myoblasts and Myotubes

Background

Substituting galactose for glucose in cell culture media has been suggested to enhance mitochondrial metabolism in a variety of cell lines. We studied the effects of carbohydrate availability on growth, differentiation and metabolism of C2C12 myoblasts and myotubes.

Methodology/Principal Findings

We measured growth rates, ability to differentiate, citrate synthase and respiratory chain activities and several parameters of mitochondrial respiration in C2C12 cells grown in media with varying carbohydrate availability (5 g/l glucose, 1 g/l glucose, 1 g/l galactose, and no added carbohydrates). C2C12 myoblasts grow more slowly without glucose irrespective of the presence of galactose, which is not consumed by the cells, and they fail to differentiate without glucose in the medium. Cells grown in a no-glucose medium (with or without galactose) have lower maximal respiration and spare respiratory capacity than cells grown in the presence of glucose. However, increasing glucose concentration above physiological levels decreases the achievable maximal respiration. C2C12 myotubes differentiated at a high glucose concentration showed higher dependency on oxidative respiration under basal conditions but had lower maximal and spare respiratory capacity when compared to cells differentiated under low glucose condition. Citrate synthase activity or mitochondrial yield were not significantly affected by changes in the available substrate concentration but a trend towards a higher respiratory chain activity was observed at reduced glucose levels.

Conclusions/Significance

Our results show that using galactose to increase oxidative metabolism may not be applicable to every cell line, and the changes in mitochondrial respiratory parameters associated with treating cells with galactose are mainly due to glucose deprivation. Moderate concentrations of glucose (1 g/l) in a growth medium are optimal for mitochondrial respiration in C2C12 cell line while supraphysiological concentrations of glucose cause mitochondrial dysfunction in C2C12 myoblasts and myotubes.     

Effects of pH and type of sugar in the medium on tyrosinase activity in cultured melanoma cells.

The tyrosinase (EC 1.14.18.1) activity of cultured mouse melanoma cells B16 in the stationary phase of growth, depends greatly on the pH of the medium and the kind of sugar present. The enzyme activity of a homogenate of cells grown at pH 7.2 in Eagles's MEM supplemented with 10% new born calf serum and con taining galactose in place of glucose, was about ten times that of a homogenate of cells cultured at pH 6.3 in the same medium. The tyrosinase activity changed reversibly on changing the pH of the culture medium. When cultured at a constant pH of 7.2, cells grown with 1 mM galactose had about five times higher tyrosinase activity than cells grown with 1 mM glucose. Only a small amount of lactate accumulated in cultures with glucose and it had little effect on the enzyme activity. These two findings explain the very low tyrosinase activity of cells cultured in medium with 5 mM glucose: the low activity is due to the presence of glucose and to the low pH resulting from conversion of glucose to lactic acid.     

The effect of growth medium on the antioxidant defense of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We compared the oxidation of dihydrorhodamine 123, glutathione contents and activities of superoxide dismutase (SOD) and catalase for three wild-type strains of Saccharomyces cerevisiae grown on media with different carbon sources. The rate of oxidation of dihydrorhodamine 123 was much higher in respiring cells grown on ethanol or glycerol media than in fermenting cells grown on glucose medium. The total SOD activity was highest on glycerol medium and lowest on ethanol medium, while the catalase activity was highest on glycerol medium. The sequence of glutathione content values was: glucose > ethanol > glycerol.     

Effect of carbon source during growth on sensitivity of Pseudomonas fluorescens to actinomycin D.

Sensitivity to actinomycin D(AD) varies in Pseudomonas fluorescens cells grown in glucose or succinate minimal salts medium. Growth is inhibited in succinate minimal medium by much lower concentrations of AD than in glucose minimal medium. Uptake of selected radioactive metabolites is inhibited by AD in cells incubated for 2 h in succinate medium containing AD but glucose-grown cells were not sensitive. EDTA treatment promotes increased sensitivity to AD in succinate-grown cells but does not alter sensitivity in glucose-grown cells. Succinate-grown cells bound 2-3 times as much 3H-AD as glucose-grown cells. Glucose-grown cells had much higher lipopolysaccharide levels in the envelope than succinate-grown cells. It is proposed that the lipopolysaccharide masks the binding sites and, therefore, is responsible for the difference in binding of AD by the glucose- and succinate-grown cells. The availability of the binding sites is also reflected in the sensitivity of the cells to the antibiotic.     

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.     

Influence of glucose and galactose on the morphology and biological properties of Yersinia pseudotuberculosis

When cultivated in the presence of glucose, irrespective of temperature and the degree of aeration, Y. pseudotuberculosis cells have the ovoid form, constant size and low hydrophobic properties of their surface. Meanwhile the characteristics of the bacteria grown in the medium, carbohydrate-free or with galactose added, essentially depend on the conditions of medium aeration. Under the conditions of intensive stirring at both temperatures these bacteria acquire the coccoid form, not typical for Yersinia, they have a smaller area (approximately 2 times) and more hydrophobic surface in comparison with the cells grown in the presence of glucose. Under stationary conditions the differences between the cells, cultivated in the presence of galactose and glucose, in form and area disappear, but the differences in the hydrophobic properties of the surface are retained. As revealed in this study, the cells grown in the presence of galactose and under the conditions of intensive medium stirring, in contrast to those grown with glucose, have 1.5-fold greater invasive activity, irrespective of aeration conditions, eightfold greater resistance to ampicillin and twofold greater resistance to streptomycin and erythromycin.     

Polygalacturonase production by Kluyveromyces marxianus: effect of medium composition

A strain of Kluyveromyces marxianus (CCT 3172), isolated from a cocoa fermentation in Brazil, secreted an endopolygalacturonase (PG) when grown under self-induced anaerobic conditions; neither polymethylesterase nor pectate lyase appeared in culture filtrates. Replacing glucose in the medium with sucrose had no effect on PG secretion or ethanol production. Growth in fructose-containing medium retarded secretion of PG and ethanol, but had no effect on growth. Growth and ethanol production in media containing galactose resembled those in fructose-containing medium, although PG secretion was lowered. Growth and PG secretion were considerably retarded in xylose-containing medium, and were similarly affected in media containing different concentrations of glucose. Varying the concentration of ammonium sulphate in media had no effect on growth or PG secretion.     

Growth responses of cell cultures in a galactose medium

Under specific conditions utilized, Eagle's minimum essential medium containing “substantially glucose-free” galactose instead of glucose supported the growth of every cell culture tested with the exception of embryonic cells. Growth of various primary kidney cultures in galactose-EMEM was greater or, at least, equal to that obtained with the same medium containing glucose. Cell lines of non-human origin showed extensive growth in galactose-EMEM and were further stimulated by supplemental pyruvate. Only limited, if any, growth of human cell lines occurred in galactose-EMEM under routine conditions of culture. The growth response of these cells was greatly increased and approached that with glucose if the initial pH of the galactose medium was adjusted in the acid pH range or, with WISH cells, if the galactose medium was supplemented with pyruvate.     

Galactose repression of beta-galactosidase induction in Escherichia coli

Beggs, William H. (University of Minnesota, Minneapolis), and Palmer Rogers. Galactose repression of beta-galactosidase induction in Escherichia coli. J. Bacteriol. 91:1869-1874. 1966.-Galactose repression of beta-galactosidase induction in Escherichia coli was investigated to determine whether the galactose molecule itself is the catabolite repressor of this enzyme system. Without exception, beta-galactosidase induction by cells grown in a synthetic salts medium with lactate or glycerol as the carbon source was more strongly repressed by glucose than by galactose. This relationship existed even when the organism was previously grown in the synthetic medium containing galactose as the source of carbon. Two observations suggested that the ability of galactose to repress beta-galactosidase formation by Escherichia coli depends directly upon the cells' capacity to catabolize galactose. First, galactose repression of beta-galactosidase synthesis was markedly enhanced in bacteria tested subsequent to gratuitous induction of the galactose-degrading enzymes with d-fucose. Second, galactose failed to exert a repressive effect on beta-galactosidase in a galactose-negative mutant lacking the first two enzymes involved in galactose catabolism. Glucose completely repressed enzyme formation in this mutant. This same mutant, into which the genes for inducible galactose utilization had been introduced previously by transduction, again exhibited galactose repression. Pyruvate was found to be at least as effective as galactose in repressing beta-galactosidase induction by cells grown in synthetic salts medium plus glycerol. It is concluded that the galactose molecule itself is not the catabolite repressor of beta-galactosidase, but that repression is exerted through some intermediate in galactose catabolism.     

Gluconeogenesis in Saccharomyces cerevisiae: determination of fructose-1,6-bisphosphatase activity in cells grown in the presence of glycolytic carbon sources.

The activity of fructose-1,6-bisphosphatase (FBP), a gluconeogenic enzyme, was determined in wild-type Saccharomyces cerevisiae X2180 grown in the presence of the glycolytic carbon sources, glucose, fructose, and galactose. The activities of phosphofructokinase (PFK), a glycolytic enzyme, and phosphoglucose isomerase (PGI), an enzyme functioning both in glycolysis and gluconeogenesis, were determined for purposes of comparison. A measurable amount of FBP activity was present in 20-h-old cells grown with moderate shaking in 1% glucose-nutrient or minimal medium. This activity increased significantly in 40 and 60-h-old cells. Similar levels of FBP activity were also present in 20-, 40-, and 60-h-old cells grown in 1% fructose-nutrient medium. A higher level of FBP activity was present in 20-h-old cells grown in 1% galactose-nutrient medium than in 20-h-old cells grown in 1% glucose- or fructose-nutrient medium. The FBP activity in glucose- or fructose-grown cells was higher than the corresponding activity in cells grown under similar conditions for 40 and 60 h in the presence of ethanol, a gluconeogenic carbon source. The PFK activity was significantly less in galactose- and ethanol-grown cells. The PGI activity was relatively constant in 20-, 40-, and 60-h-old cells grown in the presence of glucose, fructose, and galactose, but this activity was reduced approximately 50% in ethanol-grown cells. It is concluded from these results that, depending upon the concentration of carbon source and the time of incubation, FBP, a strictly gloconeogenic enzyme, is synthesized by S. cerevisiae grown in the presence of glycolytic carbon sources.     

Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: Implications for use of alternate sugars

Alternate sugars such as galactose and fructose are metabolized at a slower rate than glucose and result in lower accumulation of lactate. While low lactate accumulation is desirable, we report that complete substitution of glucose with these sugars results in an increase in M5 high mannose glycans. Surprisingly, this increase is much higher when the culture is supplemented with manganese: for example, when cells are cultured with galactose, M5 high mannose glycan content increased from 5% at 1 nM Mn²⁺ in the basal medium to 32% with 16 µM Mn²⁺ supplementation. When galactose is supplemented with glucose maintained at low concentrations, a small reduction in high mannose glycans is seen. In control cultures with glucose, the high mannose content was however <2% in this range of Mn²⁺ concentration. By varying Mn²⁺ and glucose supplementation levels, with or without galactose, we systematically demonstrate that Mn²⁺ concentration and glucose availability, together, significantly affect the high mannose glycan content. To our knowledge, this is the first report that shows that the effect of Mn²⁺ on high mannose glycan content depends on glucose availability. At each Mn²⁺ supplementation level evaluated, galactosylation percentages were highest for cultures where galactose was supplemented with glucose at non‐limiting concentration. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:460–467, 2015     

Modification of membrane protein expression and protein secretion in LLC-PK1 cultures grown on different carbohydrates

Membrane glycoproteins and glycolipids play an important role in epithelial organization, transport and function. To study the effects of exogenous carbohydrates on the expression of glycoproteins, cells of the renal epithelial line LLC-PK1 were cultured on different nutritive carbohydrate sources and on uridine, which is, despite striking differences, known to substitute all essential nutritive functions of glucose. LLC-PK1 cultures were long-term adapted to growth in culture medium containing 0.5, 5, 10 and 25 mM glucose, and 5 mM fructose, galactose and uridine, respectively, as the sole carbohydrate source. These growth conditions elicited adaptive changes in the expression of enzyme activities of alkaline phosphatase and gamma-glutamyltranspeptidase, integral membrane glycoproteins exclusively localized in the apical membrane of LLC-PK1 cells. SDS-PAGE of membrane preparations of adapted LLC-PK1 cells revealed a strong induction of several protein bands between 13.5 and 47 kD in fructose-grown cells, while in plasma membranes of cells grown in galactose several protein bands between 62 and 70 kD decreased. Changes in the secretion pattern of proteins into the culture medium were most prominent in uridine-grown cells compared to controls grown on 25 mM glucose.     

Remodeling of Oxidative Energy Metabolism by Galactose Improves Glucose Handling and Metabolic Switching in Human Skeletal Muscle Cells

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [¹⁴C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [¹⁴C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments.     

Diauxic Growth of Azotobacter vinelandii on Galactose and Glucose: Regulation of Glucose Transport by Another Hexose

The growth curve of Azotobacter vinelandii was biphasic when the organism was grown in a medium containing a mixture of galactose and glucose. Galactose was the primary carbon source; glucose was also consumed, but the rate at which it was consumed was lower than the rate at which galactose was consumed during the first phase of growth. Metabolic pathways for both sugars were induced. Cell cultures exhibited a second lag period as galactose was depleted. The length of this lag phase varied from 2 to 10 h depending on the pregrowth history of the cells. The second log growth phase occurred at the expense of the remaining glucose in the medium and was accompanied by induction of the high-maximum rate of metabolism glucose-induced glucose permease and increases in the levels of glucose metabolic enzymes. The second lag phase of diauxie may have been due to the time required for induction of the glucose-induced glucose permease.