Glucose-grown Cells Research Articles

Induction and glucose repression of endo- β-xylanase in the yeast Trichosporon cutaneum SL409

Extracellular endo-1,4- β-xylanase (EC 3.2.1.8) synthesis in the yeast Trichosporon cutaneum SL409 is inducible. The enzyme can be induced in washed glucose-grown cells by xylan or xylose. Methyl β- d-xylopyranoside, a synthetic analogue of xylobiose, however, was not an inducer of xylanase in Trichosporon cutaneum SL409. The induction of xylanase by xylan lasted longer and the final total activities were significantly higher compared to the induction by xylose. Xylanase induction was subject to glucose repression.

Short Communication: Effect of carbon source on exopolysaccharide production by Sphingomonas paucimobilis ATCC 31461

The ability of the bacterium Sphingomonas paucimobilis ATCC 31461 to produce the exopolysaccharide gellan on various carbon sources was examined. The bacterium was found to be capable of synthesizing gellan on glucose, corn syrup, sucrose, mannose, galactose, fructose or maltose as a carbon source after 72 hours of growth. Gellan production was found to double after 72 hours for corn syrup-grown cells relative to glucose-grown cells. Lesser amounts of polysaccharide were produced when sucrose, mannose, galactose, fructose or maltose served as the carbon source instead of glucose or corn syrup. It did not appear that an increase in bacterial cell weight could be correlated with enhanced gellan synthesis.

Pathway of glucose catabolism by strain VeGlc2, an anaerobe belonging to the verrucomicrobiales lineage of bacterial descent

Strain VeGlc2, an anaerobic ultramicrobacterium belonging to the Verrucomicrobiales lineage of bacterial descent, fermented glucose to acetate, propionate, succinate, and CO2. The distribution of radiolabel in the fermentation end products produced from position-labelled glucose and in vitro measurements of enzyme activities in crude cell extracts prepared from glucose-grown cells showed that glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The 6-phosphofructokinase (EC 2.7.1.90) activity required pyrophosphate as the phosphoryl donor, and ATP could not replace pyrophosphate. The other enzyme activities were those of a classical Embden-Meyerhof-Parnas pathway. 14CO2 was incorporated into propionate and succinate, suggesting that a carboxylation reaction rather than a transcarboxylation reaction was involved in the reductive pathway leading to succinate and propionate. Difference spectra showed that a type b cytochrome was present, which could be involved in electron transport in the reductive pathway.

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast.

Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane. Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Their composition, after acid methanolysis, was analysed by gas-liquid chromatography. The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells. The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources. The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells. The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells. The ceramides contained an unusual long-chain base composition. In hexadecane-grown cells more than 60% of the long-chain bases were C19 phytosphingosine. In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine.

Snf1 protein kinase regulates phosphorylation of the Mig1 repressor in Saccharomyces cerevisiae.

In glucose-grown cells, the Mig1 DNA-binding protein recruits the Ssn6-Tup1 corepressor to glucose-repressed promoters in the yeast Saccharomyces cerevisiae. Previous work showed that Mig1 is differentially phosphorylated in response to glucose. Here we examine the role of Mig1 in regulating repression and the role of the Snf1 protein kinase in regulating Mig1 function. Immunoblot analysis of Mig1 protein from a snf1 mutant showed that Snf1 is required for the phosphorylation of Mig1; moreover, hxk2 and reg1 mutations, which relieve glucose inhibition of Snf1, correspondingly affect phosphorylation of Mig1. We show that Snf1 and Mig1 interact in the two-hybrid system and also coimmunoprecipitate from cell extracts, indicating that the two proteins interact in vivo. In immune complex assays of Snf1, coprecipitating Mig1 is phosphorylated in a Snf1-dependent reaction. Mutation of four putative Snf1 recognition sites in Mig1 eliminated most of the differential phosphorylation of Mig1 in response to glucose in vivo and improved the two-hybrid interaction with Snf1. These studies, together with previous genetic findings, indicate that the Snf1 protein kinase regulates phosphorylation of Mig1 in response to glucose.

A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris.

The budding yeast Pichia pastoris is an attractive system for exploring certain questions in cell biology, but experimental use of this organism has been limited by a lack of convenient expression vectors. Here we describe a set of compact vectors that should allow for the expression of a wide range of endogenous or foreign genes in P. pastoris. A gene of interest is inserted into a modified pUC19 polylinker; targeted integration into the genome then results in stable and uniform expression of this gene. The utility of these vectors was illustrated by expressing the bacterial beta-glucuronidase (GUS) gene. Constitutive GUS expression was obtained with the strong GAP promoter or the moderate YPT1 promoter. The regulatable AOX1 promoter yielded very strong GUS expression in methanol-grown cells, negligible expression in glucose-grown cells, and intermediate expression in mannitol-grown cells. GenBank Accession Numbers are: pIB1, AF027958; pIB2, AF0279959; pIB3, AF027960; pIB4, AF027961.

Identification and characterization of MAE1, the Saccharomyces cerevisiae structural gene encoding mitochondrial malic enzyme.

Pyruvate, a precursor for several amino acids, can be synthesized from phosphoenolpyruvate by pyruvate kinase. Nevertheless, pyk1 pyk2 mutants of Saccharomyces cerevisiae devoid of pyruvate kinase activity grew normally on ethanol in defined media, indicating the presence of an alternative route for pyruvate synthesis. A candidate for this role is malic enzyme, which catalyzes the oxidative decarboxylation of malate to pyruvate. Disruption of open reading frame YKL029c, which is homologous to malic enzyme genes from other organisms, abolished malic enzyme activity in extracts of glucose-grown cells. Conversely, overexpression of YKL029c/MAE1 from the MET25 promoter resulted in an up to 33-fold increase of malic enzyme activity. Growth studies with mutants demonstrated that presence of either Pyk1p or Mae1p is required for growth on ethanol. Mutants lacking both enzymes could be rescued by addition of alanine or pyruvate to ethanol cultures. Disruption of MAE1 alone did not result in a clear phenotype. Regulation of MAE1 was studied by determining enzyme activities and MAE1 mRNA levels in wild-type cultures and by measuring beta-galactosidase activities in a strain carrying a MAE1::lacZ fusion. Both in shake flask cultures and in carbon-limited chemostat cultures, MAE1 was constitutively expressed. A three- to fourfold induction was observed during anaerobic growth on glucose. Subcellular fractionation experiments indicated that malic enzyme in S. cerevisiae is a mitochondrial enzyme. Its regulation and localization suggest a role in the provision of intramitochondrial NADPH or pyruvate under anaerobic growth conditions. However, since null mutants could still grow anaerobically, this function is apparently not essential.

Insulin signaling in the yeast Saccharomyces cerevisiae. 1. Stimulation of glucose metabolism and Snf1 kinase by human insulin.

Effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae were studied in this report. Under two conditions of growth limitation (glucose-grown cells during transition to stationary phase or spheroplasts during incubation in synthetic glucose medium), human insulin (10 and 1 microM, respectively) enhanced glycogen accumulation and glycogen synthase activity by 40-60% compared to control cells. Glycogen phosphorylase activity was also increased under the same conditions, but this stimulation was diminished by 35-45% in insulin-treated compared to control cells. Thus, under growth limitation, insulin causes glycogen phosphorylase and glycogen synthase to become more sensitive to inactivation and activation, respectively. In glucose-induced spheroplasts, insulin (1 microM), in addition to glycogen accumulation, led to about 2-fold increases of the rates of ethanol production and glucose oxidation compared to control cells, and the maximal concentration of hexose 6-phosphate was increased by 30-40%. In contrast, glucose transport as well as the levels of the allosteric regulators, fructose 2,6-bisphosphate and cAMP, were not altered at all. Snf1 kinase is assumed to be involved in the regulation of glycogen metabolism in yeast, although it does not seem to be modulated directly by the glucose concentration. Snf1 kinase activity was elevated 5-10-fold in response to insulin both during glucose induction of yeast spheroplasts and during transition to stationary phase of glucose-grown cells. We conclude that Saccharomyces cerevisiae and insulin-sensitive mammalian cells share some parts of the signaling cascades regulating oxidative and nonoxidative glucose metabolism in response to glucose and insulin.

In vivo 13C-NMR studies of polymer synthesis in rhizobium meliloti M5N1 strain

The use of in vivo 13C-NMR approach for the monitoring of the synthesis of various polymers within cells of Rhizobium meliloti (M5N1 strain) is reported. Significant differences in polymer biosynthesis have been shown as a function of the metabolic state of the cells and the labeled carbon source used. Consumption of carbon source and produced glycogen was complete with mid-exponential phase harvested cells. This was not the case with stationary phase harvested cells, for which polyhydroxybutyrate synthesis was higher and gluconate synthesis was lower than the former. [1-13C]fructose-grown cells produced more exopolysaccharide and polyhydroxybutyrate, but less beta-(1,2) glucan and gluconate than [1-13C]glucose-grown cells. This approach offers a suitable tool to examine the kinetics of polymer biosynthesis by Rhizobia. Copyright 1998 John Wiley & Sons, Inc.

Identification and Characterization of Epoxide Carboxylase Activity in Cell Extracts of Nocardia corallina B276

The metabolism of aliphatic epoxides (epoxyalkanes) by the alkene-utilizing actinomycete Nocardia corallina B276 was investigated. Suspensions of N. corallina cells grown with propylene as the carbon source readily degraded propylene and epoxypropane, while suspensions of glucose-grown cells did not. The addition of propylene and epoxypropane to glucose-grown cells resulted in a time-dependent increase in propylene- and epoxypropane-degrading activities that was prevented by the addition of rifampin and chloramphenicol. The expression of alkene- and epoxide-degrading activities was correlated with the high-level expression of several polypeptides not present in extracts of glucose-grown cells. Epoxypropane and epoxybutane degradation by propylene-grown cell suspensions of N. corallina was stimulated by the addition of CO2 and inhibited by the depletion of CO2. Cell extracts catalyzed the carboxylation of epoxypropane to form acetoacetate in a reaction that was dependent on the addition of CO2, NAD+, and a reductant (NADPH or dithiothreitol). In the absence of CO2, epoxypropane was isomerized by cell extracts to form acetone at a rate approximately 10-fold lower than the rate of epoxypropane carboxylation. Methylepoxypropane was found to be a time-dependent, irreversible inactivator of epoxyalkane-degrading activity. These properties demonstrate that epoxyalkane metabolism in N. corallina occurs by a carboxylation reaction forming beta-keto acids as products and provide evidence for the involvement in this reaction of an epoxide carboxylase with properties and cofactor requirements similar to those of the four-component epoxide carboxylase enzyme system of the gram-negative bacterium Xanthobacter strain Py2 (J. R. Allen and S. A. Ensign, J. Biol. Chem. 272:32121-32128, 1997). The addition of epoxide carboxylase component I from Xanthobacter strain Py2 to methylepoxypropane-inactivated N. corallina extracts restored epoxide carboxylase activity, and the addition of epoxide carboxylase component II from Xanthobacter Py2 to active N. corallina extracts stimulated epoxide isomerase rates to the same levels observed with the purified Xanthobacter system. Antibodies raised against Xanthobacter strain Py2 epoxide carboxylase component I cross-reacted with a polypeptide in propylene-grown N. corallina extracts with the same molecular weight as component I but did not cross-react with glucose-grown extracts. Together, these results suggest a common pathway of epoxyalkane metabolism for phylogenetically distinct bacteria that involves CO2 fixation and the activity of a multicomponent epoxide carboxylase enzyme system.

Mechanisms underlying the transport and intracellular metabolism of acetic acid in the presence of glucose in the yeast Zygosaccharomyces bailii.

Zygosaccharomyces bailii ISA 1307 displays biphasic growth in a medium containing a mixture of glucose (0.5%, w/v) and acetic acid (0.5%, w/v), pH 5.0 and 3.0. In cells harvested during the first growth phase, no activity of a mediated acetic acid transport system was found. Incubation of these cells in phosphate buffer with cycloheximide for 1 h restored activity of an acetic acid carrier which behaved as the one present in glucose-grown cells. These results indicated that the acetic acid carrier is probably present in cells from the first growth phase of the mixed medium but its activity was affected by the presence of acetic acid in the culture medium. In glucose-grown cells, after incubation in phosphate buffer with glucose and acetic acid, the activity of the acetic acid carrier decreased significantly with increased acid concentration in the incubation buffer. At acid concentrations above 16.7 mM, no significant carrier activity was detectable. Furthermore, the intracellular acid concentration increased with the extracellular one and was inversely correlated with the activity of the acetic acid carrier, suggesting the involvement of a feedback inhibition mechanism in the regulation of the carrier. During biphasic growth, the first phase corresponded to a simultaneous consumption of glucose and acetic acid, and the second to the utilization of the remaining acid. The enzyme acetyl-CoA synthetase was active in both growth phases, even in the presence of glucose. Activity of isocitrate lyase and phosphoenolpyruvate carboxykinase was found only in acetic-acid-grown cells. Thus it appears that both membrane transport and acetyl-CoA synthetase and their regulation are important for Z. bailii to metabolize acetic acid in the presence of glucose. This fact correlates with the high resistance of this yeast to environments with mixtures of sugars and acetic acid such as those often present during wine fermentation.

Role of trehalose in survival of Saccharomyces cerevisiae under osmotic stress.

Trehalose is an enigmatic compound that accumulates in Saccharomyces cerevisiae and has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate that may be mobilized during stress. In this study, specific mutants in trehalose metabolism were used to evaluate whether trehalose contributes to survival under severe osmotic stress and generates the compatible solute glycerol under moderate osmotic stress. The survival under severe osmotic stress (0.866 aw' NaCl or sorbitol) of mutants was compared to that of the wild-type strain when cultivated to either the mid-exponential or the stationary growth phase on glucose, galactose or ethanol. Stationary-phase cells survived better than exponential-phase cells. The death rates of ethanol-grown cells were lower than those of galactose-grown cells, which in turn survived better than glucose-grown cells. There was a strong relationship between intracellular trehalose levels and resistance to osmotic stress. The mutant strains unable to produce trehalose (tps1 delta tps2 delta and tps1 delta hxk2 delta) were more sensitive to severe osmotic stress (0.866 aw) than the isogenic wild-type strain, confirming a role for trehalose in survival. Hyperaccumulation of trehalose found in the nth1 delta and the nth1 delta gpd1 delta mutant strains, however, did not improve survival rates compared to the wild-type strain. When wild-type, nth1 delta and nth1 delta gpd1 delta cells were exposed to moderate osmotic stress (0.98 and 0.97 aw' NaCl), which permits growth, glycerol production did not appear to be related to the intracellular trehalose levels although glycerol levels increased more rapidly in nth1 delta cells than in wild-type cells during the initial response to osmotic stress. These data indicate that trehalose does not act as a reserve compound for glycerol synthesis under these conditions. No evidence was found for solutes other than glycerol and trehalose being significant for the survival of or growth by S. cerevisiae under osmotic stress conditions.

Effects of ethanol and other alkanols on transport of acetic acid in Saccharomyces cerevisiae.

In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of Vmax versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.

Effects of oral antihyperglycemic agents on extracellular matrix synthesis by mesangial cells

Increased expression of the glucose transporter GLUT1 in mesangial cells (MCs) markedly stimulates glucose transport and the formation of extracellular matrix (ECM), even when ambient glucose concentrations are low. Certain antihyperglycemic agents cause GLUT1 overexpression and increase glucose transport in various tissues. However, their effects on the kidney are unknown. Because diabetic glomerulosclerosis is characterized by the accumulation of mesangial matrix, was studied the effects of antihyperglycemic agents on matrix metabolism in MCs cultured either in 8 or 20 mM glucose. Membrane-associated GLUT1 was measured by immunoblotting. The initial rate of glucose transport was determined according to the 2-deoxy-D[14C(U)]glucose uptake. Collagen metabolism was studied by metabolic radiolabeling with [14C]-proline. Fibronectin in the medium was measured by ELISA. GLUT1 mRNA was estimated by Northern analysis. The sulfonylurea tolazamide increased GLUT1 protein expression by 107 and 69% in 8 and 20 mM glucose-grown cells, respectively. However, GLUT1 mRNA levels remained unchanged. Transporter-dependent deoxyglucose uptake was increased by tolazamide up to 184% in a dose-dependent fashion and was evident at both glucose concentrations after three or five days of exposure to the drug. Tolazamide significantly stimulated transforming growth factor-beta 1 (TGF-beta 1) secretion and the total synthesis of collagen and collagen and fibronectin accumulation in the medium of MCs maintained in high or low glucose concentrations. The biguanide metformin did not alter GLUT1 expression, glucose transport, fibronectin formation, or collagen metabolism, except at high concentrations. Tolazamide markedly enhances ECM synthesis and accumulation in MCs probably by stimulating GLUT1 expression, glucose transport and TGF-beta 1 secretion, irrespective of the ambient glucose concentration. This effect was dose-dependent and minimally inducible by metformin.

Regulation of N -acetylglucosaminidase production in Candida albicans

The N-acetylglucosaminidase of Candida albicans is a secreted hydrolytic enzyme that contributes to the yeast's virulence. There was a significant increase in the N-acetylglucosaminidase activity of C. albicans cells released from carbon starvation in medium containing N-acetylglucosamine. The increased enzyme activity in N-acetylglucosamine-grown cells correlated with increased transcription of the HEX1 gene, which encodes C. albicans N-acetylglucosaminidase. In contrast, glucose repressed HEX1 transcription, and glucose-grown cells had on average 94-fold lower N-acetylglucosaminidase activities than did N-acetylglucosamine-grown cells. N-acetylglucosaminidase induction in cells grown on N-acetylglucosamine was also repressed by fructose, mannose or galactose, although to a lesser extent than by glucose, and sucrose repressed enzyme production by only 10%. Eighty-eight percent of the enzyme in N-acetylglucosamine-grown cells was localised in the periplasm, and after incubation for 5 h, 30 or 70% of the total enzyme activity was secreted into the medium by yeast or mycelial cells, respectively. The cellular location of the enzyme and the regulation of production by the carbon source indicate a scavenging role for C. albicans N-acetylglucosaminidase.

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H+-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H+-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H+-extrusion. Comparison of the rates of H+-flux with the relatedin vitroH+-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H+-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.

Characteristics of Fps1-dependent and -independent glycerol transport in Saccharomyces cerevisiae.

Eadie-Hofstee plots of glycerol uptake in wild-type Saccharomyces cerevisiae W303-1A grown on glucose showed the presence of both saturable transport and simple diffusion, whereas an fps1delta mutant displayed only simple diffusion. Transformation of the fps1delta mutant with the glpF gene, which encodes glycerol transport in Escherichia coli, restored biphasic transport kinetics. Yeast extract-peptone-dextrose-grown wild-type cells had a higher passive diffusion constant than the fps1delta mutant, and ethanol enhanced the rate of proton diffusion to a greater extent in the wild type than in the fps1delta mutant. In addition, the lipid fraction of the fps1delta mutant contained a lower percentage of phospholipids and a higher percentage of glycolipids than that of the wild type. Fps1p, therefore, may be involved in the regulation of lipid metabolism in S. cerevisiae, affecting membrane permeability in addition to fulfilling its specific role in glycerol transport. Simultaneous uptake of glycerol and protons occurred in both glycerol- and ethanol-grown wild-type and fps1delta cells and resulted in the accumulation of glycerol at an inside-to-outside ratio of 12:1 to 15:1. Carbonyl cyanide m-chlorophenylhydrazone prevented glycerol accumulation in both strains and abolished transport in the fps1delta mutant grown on ethanol. Likewise, 2,4-dinitrophenol inhibited transport in glycerol-grown wild-type cells. These results indicate the presence of an Fps1p-dependent facilitated diffusion system in glucose-grown cells and an Fps1p-independent proton symport system in derepressed cells.

Deviant Pex3p levels affect normal peroxisome formation in Hansenula polymorpha: high steady-state levels of the protein fully abolish matrix protein import.

PEX3 encodes at 52 kDa peroxisomal membrane protein (PMP), essential for peroxisome biogenesis in the yeast Hansenula polymorpha. The relation between Pex3p levels and peroxisome formation was studied in wild type (WT) and delta pex3 strains expressing additional copies of PEX3 under control of a substrate-inducible promoter, namely the strong alcohol oxidase (PAOX) or the weaker amine oxidase (PAMO) promoter. In glucose-grown delta pex3 cells, containing PAOX.PEX3, Pex3p was undetectable and peroxisomes were absent. After induction of these cells on methanol, peroxisomes were rapidly formed. At Pex3p levels up to 7-10 times the values observed in WT controls normal peroxisomes were present. However, at further enhanced Pex3p levels a general matrix protein import defect was observed. This phenomenon was paralleled by aberrant peroxisome assembly and the formation of numerous small vesicles. These vesicles contained Pex3p, together with other H. polymorpha PMPs, but lacked the major matrix proteins which has accumulated in the cytosol. The implications of our results on PEX3 gene regulation and functioning of the peroxisomal matrix protein import machinery in H. polymorpha are discussed.

Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate.

Xyloglucan from the walls of Rosa cells that had been cultured on [12C]- or [13C]-glucose formed bands in caesium trifluoroacetate with mean buoyant densities of 1.575 or 1.616 g/ml respectively. Incubation of a mixture of [13C,3H]xyloglucan and [12C,1H]xyloglucan in the presence of xyloglucan endotransglycosylase (XET) activity caused the mean buoyant density of the radioactive material to decrease, indicating that interpolymeric transglycosylation could be detected in vitro. We used two 13C/3H-dual-labelling protocols to look for interpolymeric transglycosylation in vivo. In protocol A, [13C]glucose-grown Rosa cells were transferred into [12C]glucose medium 6 h after a approximately 2 h pulse of l-[1-3H]arabinose (which radiolabels the xylose residues of xyloglucan). The mean buoyant density of the wall-bound [3H]xyloglucan decreased during the following 7 days in culture. This indicates that, during or after the wall-binding of newly synthesized [12C,1H]xyloglucan, it became covalently attached to previously wall-bound [13C, 3H]xyloglucan. In protocol B, [12C]glycerol- or [12C]glucose-grown Rosa cells were transferred into [13C]glucose medium, 20 or 60 min before a approximately 2 h pulse of [3H]arabinose. The buoyant density of the earliest wall-bound [3H]xyloglucan showed that it had a 12C/13C ratio of approximately 1:1. This indicates that, during (or, implausibly, before) wall-binding, the newly synthesized [13C, 3H]xyloglucan became covalently attached to previously synthesized [12C]xyloglucan. During the following 7 days in culture, the mean buoyant density of the [3H]xyloglucan increased, showing that later-synthesized [13C,1H]xyloglucan can be covalently attached to previously wall-bound [12C,13C,3H]xyloglucan. The only known mechanism by which segments of xyloglucans could become covalently attached to each other in the cell wall is by interpolymeric transglycosylation catalysed by XET. We conclude that XET-catalysed interpolymeric transglycosylation accompanies, and probably causes, the integration of newly secreted xyloglucan into the cell-wall architecture.

The lag phase rather than the exponential-growth phase on glucose is associated with a higher cAMP level in wild-type and cAPK-attenuated strains of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae several phenotypic properties controlled by cAMP-dependent protein kinase (cAPK) are indicative of high cAPK activity during growth on glucose and low activity during growth on non-fermentable carbon sources and in stationary phase. It has been a matter of debate whether the apparently higher activity of cAPK in cells growing on glucose is due to a higher cAMP level or to an alternative mechanism activating cAPK. The cAMP level during diauxic growth of yeast cells in cultures with different initial glucose levels and different initial cell densities has been reinvestigated and the previously reported twofold increase in cAMP during growth initiation has been confirmed. However, this increase was transient and entirely associated with the lag phase of growth. The initiation of exponential growth on glucose was associated with a decrease in the cAMP level and there was no correlation between this decrease in cAMP and the depletion of glucose in the medium. In mutants defective in feedback inhibition of cAMP synthesis, resuspension of exponential-phase glucose-grown cells in glucose medium caused an extended lag phase during which a huge, transient accumulation of cAMP occurred. The latter required the presence of glucose and nitrogen, but not phosphate or sulfate, and was not due to intracellular acidification, as shown by in vivo 31P-NMR spectroscopy. The initiation of exponential growth on glucose was also associated in this case with a decrease in cAMP rather than an increase. This behaviour was also observed in strains with attenuated catalytic subunit activity and lacking the regulatory subunit and even in strains without catalytic subunits of cAPK. This might indicate that other mechanisms are able to cause down-regulation of cAMP synthesis in a way mimicking feedback inhibition. Transfer of glucose-growing cells of wild-type or cAPK-attenuated strains to a nitrogen starvation medium resulted in an increase in the cAMP level rather than a decrease. The results indicate that the apparent changes in cAPK activity in vivo during diauxic growth on glucose and during nitrogen starvation cannot be explained on the basis of changes in the cAMP level.