Sugar uptake in a 2-deoxy-d-glucose resistant mutant ofSaccharomyces cerevisiae

Summary The non-metabolizable and toxic glucose analogue 2-deoxy-d-glucose (2-DOG) has been widely employed to screen for regulatory mutants which lack catabolite repression. A number of yeast mutants resistant to 2-DOG have recently been isolated in this laboratory. One such mutant, derived from aSaccharomyces cerevisiae haploid strain, was demonstrated to be derepressed for maltose, galactose and sucrose uptake. Furthermore, kinetic analysis of glucose transport suggested that the high affinity glucose transport system was also derepressed in the mutant strain. In addition, the mutant had an increased intracellular concentration of trehalose relative to the parental strain. These results indicate that the 2-DOG resistant mutant is defective in general glucose repression.     

Isolation of mutants of Penicillium purpurogenum resistant to catabolite repression

 The strain Penicillium purpurogenum P-26 was subjected to UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment and mutants were isolated capable of synthesizing cellulase under the conditions of a high concentration of glucose. Initially mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated on Walseth’s cellulose/agar plates containing 15–45 mM 2-deoxy-D-glucose. These mutants were again screened for resistance to catabolite repression by glycerol or glucose on Walseth’s cellulose/agar plates containing 50 g/l glycerol or 50 g/l glucose respectively. Four mutants with different sizes of clearing zone on Walseth’s cellulose/agar plates containing 50 g/l glucose were selected for flask culture. Among them, the mutant NTUV-45-4 showed better carboxymethylcellulase activity in flask culture containing 1% Avicel plus 3% glucose than did the parental strain. Received: 9 October 1995/Received revision: 27 November 1995/Accepted: 8 January 1996     

Construction of industrial baker's yeast strains able to assimilate maltose under catabolite repression conditions

Spore progeny from an industrial baker's yeast strain were mutagenized with UV and mutants resistant to 2-deoxyglucose isolated. One of these mutants (10a12–13) showed high levels of maltase (-glucosidase) and external invertase, and assimilated maltose when growing under catabolite repression conditions. This mutant was not allelic to any of the catabolite repression mutants tested cat4, cat80, cid1, cyc8, hex2, hxk2 and tup1. Mutant 10a12–13 was crossed with appropriate strains to construct hybrids that were also able to assimilate maltose in the presence of glucose. These hybrids may be useful in fermentation processes where both glucose and maltose are present.     

std1, a Gene Involved in Glucose Transport in Schizosaccharomyces pombe

A wild-type strain, Sp972 h⁻, of Schizosaccharomyces pombe was mutagenized with ethylmethanesulfonate (EMS), and 2-deoxyglucose (2-DOG)-resistant mutants were isolated. Out of 300 independent 2-DOG-resistant mutants, 2 failed to grow on glucose and fructose (mutants 3/8 and 3/23); however, their hexokinase activity was normal. They have been characterized as defective in their sugar transport properties, and the mutations have been designated as std1-8 and std1-23 (sugar transport defective). The mutations are allelic and segregate as part of a single gene when the mutants carrying them are crossed to a wild-type strain. We confirmed the transport deficiency of these mutants by [¹⁴C]glucose uptake. They also fail to grow on other monosaccharides, such as fructose, mannose, and xylulose, as well as disaccharides, such as sucrose and maltose, unlike the wild-type strain. Lack of growth of the glucose transport-deficient mutants on maltose revealed the extracellular breakdown of maltose in S. pombe, unlike in Saccharomyces cerevisiae. Both of the mutants are unable to grow on low concentrations of glucose (10 to 20 mM), while one of them, 3/23, grows on high concentrations (50 to 100 mM) as if altered in its affinity for glucose. This mutant (3/23) shows a lag period of 12 to 18 h when grown on high concentrations of glucose. The lag disappears when the culture is transferred from the log phase of its growth on high concentrations. These mutants complement phenotypically similar sugar transport mutants (YGS4 and YGS5) reported earlier by Milbradt and Hoefer (Microbiology 140:2617–2623, 1994), and the clone complementing YGS4 and YGS5 was identified as the only glucose transporter in fission yeast having 12 transmembrane domains. These mutants also demonstrate two other defects: lack of induction and repression of shunt pathway enzymes and defective mating.     

Isolation and characterization of thiodigalactoside-resistant mutants of the lactose permease which possess an enhanced recognition for maltose

In the current study, lactose permease mutants were isolated which exhibited an enhanced recognition for maltose (an alpha-glucoside) but a diminished recognition for thiodigalactoside, TDG (a beta-galactoside). Maltose/TDGR mutants were obtained from four different parental strains encoding either a wild-type permease (pTE18), a mutant lactose permease which recognizes maltose (pB15) or mutant lactose permeases which recognize maltose but are resistant to inhibition by cellobiose (pTG and pBA). A total of 27 independent mutants were isolated: 12 from pTE18, 10 from pB15, 3 from pTG, and 2 from pBA. DNA sequencing of the 27 mutants revealed that the mutants contain single base pair substitutions within the lac Y gene which result in single amino acid substitutions within the lactose permease. All of the mutants obtained from pTE18, pTG, and pBA involved a change of Tyr-236 to histidine, phenylalanine, or asparagine. From pB15, three different types of mutants were obtained: Tyr-236 to histidine, Ile-303 to phenylalanine, or His-322 to asparagine. When assayed for [14C]maltose transport, the maltose/TDGR mutants were seen to transport maltose significantly faster than the wild type. Furthermore, although TDG was shown to inhibit the uptake of maltose in the four parental strains, all of the mutant strains exhibited a dramatic resistance to TDG inhibition. Most of the maltose/TDGR mutants were also shown to be very defective in the transport of lactose. However, certain mutants (i.e., Asn-322) exhibited moderate lactose transport activity. Finally, it was observed that all of the mutant strains were unable to facilitate the uphill accumulation of beta-methylthiogalactopyranoside. The locations of the amino acid substitutions are discussed with regard to their possible role in sugar recognition.     

Genetic control of lysine permeases in Saccharomycopsis lipolytica

In order to obtain strains of Saccharomycopsis lipolytica impaired in the active transport of l-lysine, mutants resistant to a mixture of l-canavanine, l-4-5-transdehydrolysine and l-S-amino ethylcysteine, taken either all three or two by two, were isolated. These compounds were shown previously to be competitive inhibitors of l-lysine uptake.The resistance patterns and excretion capacity of the mutants were established. All mutants behaved as monogenic. Recombination tests indicated that four genes at least were involved. All mutants were impaired in both high and low affinity l-lysine transport systems.Several hypotheses on the functions of these genes are put forward and discussed.     

Transport of maltose by Pseudomonas fluorescens W

The system for uptake of maltose in Pseudomonas fluorescens W was inducible. Using a mutant strain unable to hydrolyze maltose, it was shown that maltose was taken up unaltered against a concentration gradient. Uptake of ¹⁴C maltose was only significantly inhibited by nonradioactive maltose or maltotriose. These were the only sugars that could displace accumulated radioactive maltose in the strain unable to hydrolyze maltose. Uptake exhibited saturation kinetics and was inhibited by energy poisons, indicating that this system was one of active transport. Sulfhydryl-binding reagents reversibly inhibited maltose uptake. No transport ability was lost when cells were subjected to osmotic shock. Using the protein-binding dye 7-diazonium-1,3-naphthalene disulfonate a protein or proteins located in or external to the cell membrane was implicated in maltose transport. The hydrolysis of p-nitrophenyl--D-glucoside (PNPG) was used as an indirect measure of transport ability since penetration of PNPG, not its hydrolysis, was the rate-limiting step.Abbreviations PNPG paranitrophenyl--D-glucoside - NDS 7-diazonium-1,3-naphthalene disulfonic acid - PMB p-hydroxymercuribenzoate - MBS p-chloromercuriphenylsulfonic acid - PCMB p-chloromercuribenzoate - CCCP carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide     

The Glucose Transport System in Leishmania tropica Promastigotes*

Transport of glucose by Leishmania tropica promastigotes was measured by the uptake of the nonutilizable glucose analog, 2-deoxy-D-glucose (2-DOG), using the rapid filtration method. Both D-glucose and 2-DOG show identical rates of initial uptake. Intracellular 2-DOG readily exchanges with extracellular D-glucose and 2-DOG uptake is competitively inhibited by D-glucose. These observations suggest that both sugars are taken up by the same system. Neither the glucose analog α-methyl-D-glucoside (α-MG) nor 3-0-methyl glucose (3-0-MG) is taken up to any appreciable extent. Transport of 2-DOG shows saturation kinetics with a V_max of 3.2 nmoles/mg cells/min and a K_m of 0.16 mM. There is thus a stereospecific, carrier-mediated transport system for glucose uptake in L. tropica. About 2/3 of the intracellular pool following transport consists of 2-deoxy-D-glucose phosphate (2-DOG-P) and the remainder is free, unaltered 2-DOG.     

Mutations in the L-arabinose operon of Escherichia coli B-r that result in hypersensitivity to catabolite repression

Two independent mutants resistant to l-arabinose inhibition only in the presence of d-glucose were isolated from an l-arabinose-sensitive strain containing the araD139 mutation. Preliminary mapping studies indicate that these mutations are closely linked to the araIOC region. Addition of d-glucose to growing cultures of these mutants results in a 95 to 98% repression of ara operon expression, as compared to a 50% repression of the parental control. Since cultures of both mutant and parental strains undergo a 50% repression of lac operon expression upon addition of glucose, the hypersensitivity to catabolite repression exhibited by these mutants is specific for the ara operon. Addition of cyclic adenosine monophosphate reverses the catabolite repression of the ara operon in both mutant and parent strains to 70 to 80% of the control. It is suggested that in these mutants the affinity of the ara operon initiator region for the cAMP-catabolite-activator protein complex may have been altered.     

Molecular basis for the differential anti-metabolite action of d-tyrosine in strains 23 and 168 of Bacillus subtilis

Summary The basis for the difference between strains 168 (d-tyrosine-sensitive) and 23 (d-tyrosine-resistant) of Bacillus subtilis at the molecular level is that of transport of d-tyrosine into the cell. Strain 23 does not incorporate significant amounts of d-tyrosine into whole cells. A mutant derivative was isolated from strain 23 which had an altered transport system permitting d-tyrosine uptake, a change which also led to inhibition of growth by d-tyrosine. Strain 168 is extremely sensitive to growth inhibition caused by low concentrations of the d-isomer of tyrosine. A mutant derivative of strain 168 selected for its d-tyrosine resistant phenotype had an altered transport system which no longer recognized the d-isomer of tyrosine. These mutants define at least one element of the tyrosine transport system in B. subtilis and provide a convenient phenotype for the eventual location of the chromosal map position.     

Aspergillus niger mutants with increased glucose oxidase production

Summary Aspergillus niger NRRL-3, an organism used for the industrial scale production of d-gluconic acid and glucose oxidase (EC 1.1.3.4), was subjected to mutagenesis and selection for acid production on diagnostic media containing methyl red. The plates contained 0.1 M d-glucose, a concentration that does not produce a color change in the medium surrounding mycelia of the parental strain under the conditions employed. Mutagenized spores yielded occasional colonies which were able to grow rapidly and were surrounded by a reddish zone. A number of such presumptive mutants were selected and isolated. Twenty-six such strains were grown in shaken cultures with liquid media containing 0.01, 0.1 or 0.5 M d-glucose, harvested, disrupted and the specific activity of d-glucose oxidase determined. Seven of the mutant strains had glucose oxidase specific activities markedly higher than the parental strain.Paper No. 8393, Nebraska Agricultural Research Division.     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

An Escherichia coli Mutant Having Altered D-Xylose Uptake Activity and Cloning of a Gene for D-Xylose Uptake

An Escherichia coli C600 mutant having an altered D-xylose uptake activity was isolated. The growth rate and D-xylose uptake activity of the mutant grown on the minimal medium with D-xylose at 25°C were much lower than those of the parental strain grown under the same conditions, although the activities of D-xylose-binding proteins and the enzymes involved in D-xylose metabolism were almost the same for the two strains. An uptake study on sugars at the low temperature (25°C) indicated that the mutant was deficient in D-xylose uptake activity. A gene responsible for the D-xylose uptake activity at the low temperature was isolated and cloned onto vector plasmid pBR322. The gene specifically improved the D-xylose uptake activity of the mutant at the low temperature when it was introduced into the mutant cells. Based on these results, it was suggested that another D-xylose transport system other than the D-xylose-binding protein mediated system might be functioning in E. coli cells.     

Derepression of a baker's yeast strain for maltose utilization is associated with severe deregulation of HXT gene expression

Aims: We undertook to improve an industrial Saccharomyces cerevisiae strain by derepressing it for maltose utilization in the presence of high glucose concentrations. Methods and Results: A mutant was obtained from an industrial S. cerevisiae strain following random UV mutagenesis and selection on maltose/5‐thioglucose medium. The mutant acquired the ability to utilize glucose simultaneously with maltose and possibly also sucrose and galactose. Aerobic sugar metabolism was still largely fermentative, but an enhanced respirative metabolism resulted in a 31% higher biomass yield on glucose. Kinetic characterization of glucose transport in the mutant revealed the predominance of the high‐affinity component. Northern blot analysis showed that the mutant strain expresses only the HXT6/7 gene irrespective of the glucose concentration in the medium, indicating a severe deregulation in the induction/repression pathways modulating HXT gene expression. Interestingly, maltose‐grown cells of the mutant display inverse diauxy in a glucose/maltose mixture, preferring maltose to glucose. Conclusion: In the mutant here reported, the glucose transport step seems to be uncoupled from downstream regulation, because it seems to be unable to sense abundant glucose, via both repression and induction pathways. Significance and Impact of the Study: We report here the isolation of a S. cerevisiae mutant with a novel derepressed phenotype, potentially interesting for the industrial fermentation of mixed sugar substrates.     

How hexoses and inhibitors influence the malate transport system in Zygosaccharomyces bailii

When grown in fructose or glucose the cells of Zygosaccharomyces bailii were physiologically different. Only the glucose grown cells (glucose cells) possessed an additional transport system for glucose and malate. Experiments with transport mutants had lead to the assumption that malate and glucose were transported by one carrier, but further experiments proved the existence of two separate carrier systems. Glucose was taken up by carriers with high and low affinity. Malate was only transported by an uptake system and it was not liberated by starved malate-loaded cells, probably due to the low affinity of the intracellular anion to the carrier. The uptake of malate was inhibited by fructose, glucose, mannose, and 2-DOG but not by non metabolisable analogues of glucose. The interference of malate transport by glucose, mannose or 2-DOG was prevented by 2,4-dinitrophenol, probably by inhibiting the sugar phosphorylation by hexokinase. Preincubation of glucose-cells with metabolisable hexoses promoted the subsequent malate transport in a sugar free environment. Preincubation of glucose-cells with 2-DOG, but not with 2-DOG/2,4-DNP, decreased the subsequent malate transport. The existence of two separate transport systems for glucose and malate was demonstrated with specific inhibitors: malate transport was inhibited by sodium fluoride and glucose transport by uranylnitrate. A model has been discussed that might explain the interference of hexoses with malate uptake in Z. bailii.Abbreviations 2,4-DNP 2,4-dinitrophenol - 2-DOG 2-deoxyglucose - 6-DOG 6-deoxyglucose - pCMB para-hydroxymercuribenzoate     

Evidence for a specific fructose carrier inRhodotorula glutinis based on kinetic studies with a mutant defective in glucose transport

The mutant R₃₃ of the obligatory aerobic yeastRhodotorula glutinis exhibited a defect ind-glucose uptake. Detailed kinetic studies ofd-glucose andd-fructose transport in wild-type and mutant strains provided evidence for the existence in the plasma membrane of a carrier specific for fructose. The transport ofd-fructose in the mutant exhibited saturation kinetics up to 1 mmol/Ld-fructose; at higher concentrations the rate ofd-fructose uptake decreased. In the wild-type strain biphasicd-fructose uptake kinetics were observed; the low-affinity component was not found in the mutant, but the high-affinity transport system persisted. During the exponential phase of growth (ond-glucose) the high-affinityd-fructose system was repressed in the wild-type strain. Mutual competition betweend-fructose andd-glucose as well as the pH dependence of transport of the two hexoses further supported the following conclusion: In the wild-type strain,d-fructose is taken up both by the specific fructose carrier (K _T=0.22 mmol/L) and the glucose carrier (K _T=9.13 mmol/L). The former does not translocated-glucose, the latter is damaged by the mutation. Finally H⁺ co-transport and plasma membrane depolarization induced by the onset ofd-fructose transport indicated that the fructose carrier is an H⁺ symporter.     

Identification of new genes involved in disaccharide fermentation in yeast

Summary Maltose non-fermenting mutants were obtained from strains carrying a MAL4 allele which permits constitutive synthesis of maltase. Cells carrying this allele are able to utilize sucrose in the absence of the classical sucrose genes. All maltose non-fermenting mutants were also sucrose non-fermenters. Eight mutants had become maltase negative; 19 mutants could still form maltase constitutively.In crosses with segregational maltose and sucrose non-fermenting strains, enzyme negative mutants gave diploids unable to ferment maltose and sucrose. Enzyme positive, non-fermenting mutants gave diploids which readily fermented maltose and sucrose. This latter type of mutants was designated dsf (disaccharide fermentation) mutants.The diploids derived from crossing non-fermenting mutants with segregational non-fermenters were subjected to tetrad analysis. Enzyme negative non-fermenters gave only non-fermenting progeny. The dsf mutants segregated both fermenting and non-fermenting progeny, some of which showed the dsf phenotype. This indicated that none of the dsf mutants had a defect in a gene closely linked to MAL4. Crosses between dsf mutants and strains carrying the maltose genes MAL2 and MAL3 showed that the mutations affected maltose fermentation in general. Sucrose fermentation in the presence of the classical sucrose gene SUC3 was not affected, nor were fermentation of glucose, fructose and galactose.The uptake of radioactivity from uniformly labeled maltose appeared to be blocked in mutants of at least four of the dsf genes. Only one non-leaky and a leaky mutant showed a significant uptake.These results suggest that there is an extremely complex transport system for maltose and sucrose or that the utilization of these disaccharides requires a complex series of metabolic reactions.     

Protein phosphatase type-1 regulatory subunits Reg1p and Reg2p act as signal transducers in the glucose-induced inactivation of maltose permease in Saccharomyces cerevisiae

The REG1 gene encodes a regulatory subunit of the type-1 protein phosphatase (PP1) Glc7 in Saccharomyces cerevisiae, which directs the catalytic subunit to substrates involved in glucose repression. Loss of REG1 relieves glucose repression of many genes, including the MAL structural genes that encode the maltose fermentation enzymes. In this report, we explore the role of Reg1p and its homolog Reg2p in glucose-induced inactivation of maltose permease. Glucose stimulates the proteolysis of maltose permease and very rapid loss of maltose transport activity – more rapid than can be explained by loss of the permease protein alone. In a reg1Δ strain we observe a significantly reduced rate of glucose-induced proteolysis of maltose permease, and the rapid loss of maltose transport activity does not occur. Instead, surprisingly, the slow rate of proteolysis of maltose permease is accompanied by an increase in maltose transport activity. Loss of Reg2p modestly reduces the rates of both glucose-induced proteolysis of maltose permease and inactivation of maltose transport activity. Overexpression of Reg2p in a reg1Δ strain suppresses the effect on maltose permease proteolysis and partially restores the inactivation of maltose transport activity, but does not affect the insensitivity of MAL gene expression to repression by glucose observed in this strain. Thus, protein phosphatase type-1 (Glc7p-Reg1p and Glc7p-Reg2p) plays a role in transduction of the glucose signal during glucose-induced proteolysis of maltose permease, but only Glc7p-Reg1p is involved in glucose-induced inactivation of maltose transport activity and glucose repression of MAL gene expression. Overexpression of REG1 partially restores proteolysis of maltose permease in a grr1Δ strain, which lacks glucose signaling, but does not rescue rapid inactivation of maltose transport activity or sensitivity to glucose repression. A model for the role of Reg1p and Reg2p in glucose signaling pathways is discussed. We also uncovered a previously unrecognized G2/M delay in the grr1Δ but not the reg1Δ strains, and this delay is suppressed by REG1 overexpression. The G1/S delay seen in grr1Δ mutants is slightly suppressed as well, but REG1 overexpression does not suppress other grr1Δ phenotypes such as insensitivity to glucose repression. Received: 21 October 1999 / Accepted: 28 December 1999     

Regulation and genetic enhancement of beta-amylase production in Clostridium thermosulfurogenes.

We studied the general mechanism for regulation of beta-amylase synthesis in Clostridium thermosulfurogenes. beta-Amylase was expressed at high levels only when the organism was grown on maltose or other carbohydrates containing maltose units. Three kinds of mutants altered in beta-amylase production were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained starch-glucose agar plates. beta-Amylase was produced only when maltose was added to cells growing on sucrose in wild-type and catabolite repression-resistant mutant strains, but the differential rate of enzyme synthesis in constitutive mutants was constant regardless of the presence of maltose. In carbon-limited chemostats of wild-type and catabolite repression-resistant mutant stains, beta-amylase was expressed on maltose but not on glucose or sucrose. beta-Amylase synthesis was immediately repressed by the addition of glucose. Therefore, we concluded that beta-amylase synthesis in C. thermosulfurogenes was inducible and subject to catabolite repression. The addition of cAMP did not eliminate the repressive effect of glucose. The mutants were generally characterized in terms of beta-amylase production, growth properties, fermentation product formation, and alterations in glucose isomerase and glucoamylase activities. A hyperproductive mutant produced eightfold more beta-amylase on starch medium than the wild type and more rapidly fermented starch to ethanol.     

Maltose and maltotriose can be formed endogenously in Escherichia coli from glucose and glucose-1-phosphate independently of enzymes of the maltose system.

The maltose system in Escherichia coli consists of cell envelope-associated proteins and enzymes that catalyze the uptake and utilization of maltose and alpha,1-4-linked maltodextrins. The presence of these sugars in the growth medium induces the maltose system (exogenous induction), even though only maltotriose has been identified in vitro as an inducer (O. Raibaud and E. Richet, J. Bacteriol., 169:3059-3061, 1987). Induction is dependent on MalT, the positive regulator protein of the system. In the presence of exogenous glucose, the maltose system is normally repressed because of catabolite repression and inducer exclusion brought about by the phosphotransferase-mediated vectorial phosphorylation of glucose. In contrast, the increase of free, unphosphorylated glucose in the cell induces the maltose system. A ptsG ptsM glk mutant which cannot grow on glucose can accumulate [14C]glucose via galactose permeases. In this strain, internal glucose is polymerized to maltose, maltotriose, and maltodextrins in which only the reducing glucose residue is labeled. This polymerization does not require maltose enzymes, since it still occurs in malT mutants. Formation of maltodextrins from external glucose as well as induction of the maltose system is absent in a mutant lacking phosphoglucomutase, and induction by external glucose could be regained by the addition of glucose-1-phosphate entering the cells via a constitutive glucose phosphate transport system. malQ mutants, which lack amylomaltase, are constitutive for the expression of the maltose genes. This constitutive nature is due to the formation of maltose and maltodextrins from the degradation of glycogen.