Proteomics analysis of Bifidobacterium longum NCC2705 growing on glucose, fructose, mannose, xylose, ribose, and galactose

To investigate the molecular mechanisms underlying carbohydrate uptake and connected metabolic pathways of Bifidobacterium longum NCC2705, the proteomic profiles of bacteria grown on different carbon sources including glucose, fructose, mannose, xylose, ribose, and galactose were analyzed. Our results show that all sugars tested were catabolized via the bifid shunt. Sixty-eight proteins that exhibited changes in abundance of threefold or greater were identified by MS. A striking observation was the differential expression of proteins related to the pyruvate metabolism. Further analysis of acetic acid and lactic acid in the culture supernatants by HPLC at the end of fermentation showed that more lactic acid was produced during growth on fructose, ribose, xylose, galactose and more acetic acid was produced during the fermentation of glucose and mannose. Growth experiments revealed that B. longum NCC2705 preferentially used fructose, ribose, xylose, and galactose with higher growth rates over glucose and mannose. Furthermore, five proteins (GroEL, Eno, Tal, Pgm, and BL0033) exhibited clear phosphorylation modifications at serine and/or tyrosine residues. BL0033, a component of an ATP-binding cassette (ABC) transporter, was significantly more abundant in bacteria grown on fructose and, to a lesser extent, ribose and xylose. RT-PCR analysis revealed that all genes of the ABC transporter are induced in the presence of these sugars suggesting that BL0033, BL0034, BL0035, and BL0036 constitute an ABC transporter with fructose as preferred substrate.     

"GIGANTISM" OF CHLORELLA VULGARIS I. RELATION OF GIGANTISM TO CELL GROWTH AND DIVISION

1. The sugars which induced gigantism of Chlorella cells wereglucose,fructose, galactose, mannose, xylose and arabinose.These sugarswere utilized as respiratory substrates by thealgal cells.
2. The cellular division of Chlorella was stimulatedby glucoseand galactose, but suppressed by fructose, mannose,xylose andarabinose, while all these sugars evoked gigantism.No correlationwas found between cellular division and gigantism,
3. The photosynthetic activity of giant Chlorella varied withthesorts of sugars added. It was decreased by glucose, fructoseand mannose, but was unaffected by other sugars such as galactose,xylose and arabinose.
4. The respiratory activity of giant Chlorellacells as much higherthan that of control cells.
5. The amountsof protein-N and dry weight per unit volume of giantChlorellawere much less than those of control cells.

¹ Present address: Department of Chemistry, College of GeneralEducation, Osaka University, Toyonaka, Osaka.     

Synthesis and application of Fmoc-hydrazine for the quantitative determination of saccharides by reversed-phase high-performance liquid chromatography in the low and subpicomole range

A new derivatization reagent, Fmoc-hydrazine, has been synthesized from the reaction of Fmoc-chloroformate with hydrazine as a precolumn fluorometric labeling reagent for reducing sugars such as glucose, galactose, mannose, fructose, fucose, ribose, xylose, arabinose, lactose, and maltose. The optimization of derivatization conditions was examined in detail. Using a reversed-phase high-performance C-8 column and a mobile phase consisting of acetonitrile-aqueous acetic acid, seven sugar derivatives were separated under either isocratic or gradient conditions within 20 min. The Fmoc-hydrazine and sugar Fmoc-hydrazone derivatives exhibit excellent stability. The extent of the hydrazone formation was 77 and 82% for mannose and fucose as assessed by Dionex high-performance anion-exchange chromatography with pulsed amperometric detection. Linear calibration graphs were established in the range from 0.5 to 2 pmol and 12 to 110 pmol for individual sugar derivatives. The determination limits were 0.05-0.09 pmol for mannose, galactose, and ribose; 0.1 pmol for maltose, xylose, and glucose; 0.2 pmol for fucose and lactose; 0.3 pmol for arabinose; and 0.4 pmol for fructose. The component monosaccharides of ultramicroquantities of two glycoproteins (e.g., from 7 ng fetuin and ovalbumin) were determined in the subpicomole range.     

Improved method of analysis of biomass sugars using high-performance liquid chromatography

The precise quantitative analysis of biomass derived sugars is a very important step in the conversion of biomass feedstocks to fuels and chemicals. However, the most accurate method of biomass sugar analysis is based on the gas chromatography analysis of derivatized sugars either as alditol acetates or trimethylsilanes. The derivatization method is time-consuming but the alternative HPLC method cannot resolve most sugars found in biomass hydrolysates. We have demonstrated for the first time that by careful manipulation of the HPLC mobile phase, biomass monomeric sugars (arabinose, xylose, fructose, glucose, mannose, and galactose) can be analyzed quantitatively and there is excellent baseline resolution of all the sugars. This was demonstrated for both standard sugars and corn stover hydrolysates. Our method can also be used to analyze dimmeric sugars (cellobiose and sucrose).     

Different effects of galactose and mannose on cell proliferation and intracellular soluble sugar levels in <Emphasis Type="Italic">Vigna angularis</Emphasis> suspension cultures

Plant cells utilize various sugars as carbon sources for growth, respiration and biosynthesis of cellular components. Suspension-cultured cells of azuki bean (Vigna angularis) proliferated actively in liquid growth medium containing 1% (w/v) sucrose, glucose, fructose, arabinose or xylose, but did not proliferate in medium containing galactose or mannose. These two latter sugars thus appeared distinct from other sugars used as growth substrates. Galactose strongly inhibited cell growth even in the presence of sucrose but mannose did not, suggesting a substantial difference in their effects on cell metabolism. Analysis of intracellular soluble-sugar fractions revealed that galactose, but not mannose, caused a conspicuous decrease in the cellular level of sucrose with no apparent effects on the levels of glucose or fructose. Such a galactose-specific decrease in sucrose levels also occurred in cells that had been cultured together with glucose in place of sucrose, suggesting that galactose inhibits the biosynthesis, rather than uptake, of sucrose in the cells. By contrast, mannose seemed to be metabolically inert in the presence of sucrose. From these results, we conclude that sucrose metabolism is important for the heterotrophic growth of cells in plant suspension-cultures.     

Influence of cosubstrate concentration on xylose conversion by recombinant, XYL1-expressing Saccharomyces cerevisiae: a comparison of different sugars and ethanol as cosubstrates.

Conversion of xylose to xylitol by recombinant Saccharomyces cerevisiae expressing the XYL1 gene, encoding xylose reductase, was investigated by using different cosubstrates as generators of reduced cofactors. The effect of a pulse addition of the cosubstrate on xylose conversion in cosubstrate-limited fed-batch cultivation was studied. Glucose, mannose, and fructose, which are transported with high affinity by the same transport system as is xylose, inhibited xylose conversion by 99, 77, and 78%, respectively, reflecting competitive inhibition of xylose transport. Pulse addition of maltose, which is transported by a specific transport system, did not inhibit xylose conversion. Pulse addition of galactose, which is also transported by a specific transporter, inhibited xylose conversion by 51%, in accordance with noncompetitive inhibition between the galactose and glucose/ xylose transport systems. Pulse addition of ethanol inhibited xylose conversion by 15%, explained by inhibition of xylose transport through interference with the hydrophobic regions of the cell membrane. The xylitol yields on the different cosubstrates varied widely. Galactose gave the highest xylitol yield, 5.6 times higher than that for glucose. The difference in redox metabolism of glucose and galactose was suggested to enhance the availability of reduced cofactors for xylose reduction with galactose. The differences in xylitol yield observed between some of the other sugars may also reflect differences in redox metabolism. With all cosubstrates, the xylitol yield was higher under cosubstrate limitation than with cosubstrate excess.     

Carbohydrate utilization patterns and substrate preferences in Bacteroides thetaiotaomicron

Specific growth rates of Bacteroides thetaiotaomicron NCTC 10582 with either glucose, arabinose, mannose, galactose or xylose as sole carbon sources were 0.42/h, 0.10/h, 0.38/h, 0.38/h and 0.16/h respectively, suggesting that hexose metabolism was energetically more efficient than pentose fermentation in this bacterium. Batch culture experiments to determine whether carbohydrate utilization was controlled by substrate-induced regulatory mechanisms demonstrated that mannose inhibited uptake of glucose, galactose and arabinose, but had less effect on xylose. Arabinose and xylose were preferentially utilized at high dilution rates (D > 0.26/h) in carbon-limited continuous cultures grown on mixtures of arabinose, xylose, galactose and glucose. When mannose was also present, xylose was co-assimilated at all dilution rates. Under nitrogen-limited conditions, however, mannose repressed uptake of all sugars, showing that its effect on xylose utilization was strongly concentration dependent. Studies with individual D-ZU-14C]-labelled substrates showed that transport systems for glucose, galactose, xylose and mannose were inducible. Measurements to determine incorporation of these sugars into trichloroacetic acid-precipitable material indicated that glucose and mannose were the principal precursor monosaccharides. Xylose was only incorporated into intracellular macromolecules when it served as growth substrate. Phosphoenolpyruvate:phosphotransferase systems were not detected in preliminary experiments to elucidate the mechanisms of sugar uptake, and studies with inhibitors of carbohydrate transport showed no consistent pattern of inhibition with glucose, galactose, xylose and mannose. These results indicate the existence of a variety of different systems involved in sugar transport in B. thetaiotaomicron.     

Utilization of some monosaccharides by two species of Colletotrichum

Summary Utilization of 8 monosaccharides, viz., glucose, fructose, galactose, mannose, sorbose, arabinose, xylose and rhamnose, by some plant pathogenic isolates ofColletotrichum gloeosphorioides andC. dematium has been studied with the help of paper chromatography. Among hexoses, the rate of utilization of glucose, fructose and mannose was fast, whereas, that of galactose was comparatively slow. The rate of assimilation of sorbose was very slow at early stages of incubation, although at later stages this rate showed marked enhancement. The pentoses were utilized readily. The dry weight of mycelial mats showed an increase up to the end of final incubation period (15 days), on sugars which were slowly assimilated. In cases where the sugars were consumed up rapidly, the dry weight at later stages of incubation either became nearly stationary or recorded slight fall.     

Characterization and regulation of galactose transport in Neurospora crassa

Two galactose uptake systems were found in the mycelia of Neurospora crassa. In glucose-grown mycelia, galactose was transported by a low-affinity (Km = 400 mM) constitutive system which was distinct from the previously described glucose transport system I (R. P. Schneider and W. R. Wiley, J. Bacteriol. 106:479--486, 1971). In carbon-starved mycelia or mycelia incubated with galactose, a second galactose transport activity appeared which required energy, had a high affinity for galactose (Km = 0.7 mM), and was shown to be the same as glucose transport system II. System II also transported mannose, 2-deoxyglucose, xylose, and talose and is therefore a general monosaccharide transport system. System II was derepressed by carbon starvation, completely repressed by glucose, mannose, and 2-deoxyglucose, and partially repressed by fructose and xylose. Incubation with galactose yielded twice as much activity as starvation. This extra induction by galactose required protein synthesis, and represented an increase in activity of system II rather than the induction of another transport system. Glucose, mannose, and 2-deoxyglucose caused rapid degradation of preexisting system II; fructose and xylose caused a slower degradation of activity.     

不同碳源对大肠杆菌lpdA突变菌累积丙酮酸的影响

为了利用大肠杆菌构建模式"细胞工厂",必须了解在构建过程中各种因素的影响。本研究选用敲除了lpdA基因的大肠杆菌作为模型细胞,考察了该突变菌在合成培养基中利用葡萄糖、果糖、木糖和甘露糖累积丙酮酸的能力。结果显示,在初始糖浓度为10g/L的情况下,lpdA突变菌可以很好地利用葡萄糖、果糖、木糖和甘露糖转化丙酮酸,其得率分别达到了0.884g/g、0.802g/g、0.817g/g和0.808g/g,且在以葡萄糖、果糖和木糖发酵时,丙酮酸的积累过程与细胞生长偶联。甘露糖发酵的情况则不同:菌浓度很快达到平台期,随后丙酮酸积累和甘露糖消耗都表现为线性变化。当在考察了不同的接种量对lpdA突变菌发酵葡萄糖的影响时发现,大接种量能加快葡萄糖消耗速率、丙酮酸的积累速率和细胞生长速率,但丙酮酸得率却明显下降。这些结果对构建以大肠杆菌为母体的模式"细胞工厂"有参考价值。     

An improved method for analysis of biomass sugars and galacturonic acid by anion exchange chromatography

The most accurate analysis method for sugars in biomass, based on gas chromatography, requires a time consuming and laborious sample derivatation to trimethylsilanes or alditol acetates. In comparison, sample preparations for sugar analysis by liquid chromatography are simple water dilutions. However, HPLC methods either require long analysis times, use of expensive solvents, or do not give good resolution of sugars. A gradient method developed previously using a Dionex PA-1 column and pulsed-amperometric detection was modified to reduce analysis time from 75 to less than 40?min and provide good resolution of arabinose, rhamnose, galactose, xylose, glucose, fructose, sucrose, cellobiose, and galacturonic acid in both standards and hydrolyzed citrus waste biomass.     

The influence of aeration and hemicellulosic sugars on xylitol production by Candida tropicalis

The influence of other hemicellulosic sugars (arabinose, galactose, mannose and glucose), oxygen limitation, and initial xylose concentration on the fermentation of xylose to xylitol was investigated using experimental design methodology. Oxygen limitation and initial xylose concentration had considerable influences on xylitol production by Canadida tropicalis ATCC 96745. Under semiaerobic conditions, the maximum xylitol yield was 0.62 g/g substrate, while under aerobic conditions, the maximum volumetric productivity was 0.90 g/l h. In the presence of glucose, xylose utilization was strongly repressed and sequential sugar utilization was observed. Ethanol produced from the glucose caused 50% reduction in xylitol yield when its concentration exceeded 30 g/l. When complex synthetic hemicellulosic sugars were fermented, glucose was initially consumed followed by a simultaneous uptake of the other sugars. The maximum xylitol yield (0.84 g/g) and volumetric productivity (0.49 g/l h) were obtained for substrates containing high arabinose and low glucose and mannose contents.     

Production of a New Acidic Polysaccharide,Succinoglucan by Alcaligenes faecalis var. myxogenes

A slimy non-spore-forming bacterium strain 10C3 isolated from soil was motile with peritrichous flagella and named Alcaligenes faecalis var. myxogenes. Studies were made on the conditions necessary for maximal production of a new acidic succinoglucan polysaccharide by this strain in shaken cultures. Much production was observed with sucrose, glucose, xylose, galactose, cellobiose, maltose, fructose, mannose and rhamnose. The yield was greatest with sucrose and decreased in order with the above sugars from about 36 to 23 per cent. The most suitable medium contained 4 per cent sugar, 0.5 per cent yeast extract and one per cent calcium carbonate in tap water. The optimum temperature was 28°C.     

A sensitive GLC-method for component sugars andO-glycosidic linkage monosaccharides of cartilage proteoglycans

A method is described for the measurement ofN-acetylgalactosamine,N-acetylglucosamine, galactose, mannose and xylose present in the different carbohydrate chains of cartilage proteoglycans (PG). Bovine articular cartilage PG samples corresponding to the minimum of 1 nmol of each monosaccharide were reproducibly quantified following hydrolysis with 2 M HCl and derivatization into alditol acetates. An on-column injection mode and an OV-1701 fused silica capillary column were used for chromatography.Alkaline borohydride treatment of the PG was exploited to reduce the acid labile xylose in the base of the chondroitin sulphate chain into more stable xylitol, allowing the assay of chondroitin sulfate chain length as anN-acetylgalactosamine/xylose ratio. A novel procedure is described for the measurement of the galactosaminitol evolving from the protein linkage of oligosaccharides and of keratan sulphate.     

Studies on choanephoraceae. VI. The utilization of mono- and oligosaccharides

Summary The utilization of some mono- and oligosaccharides by the members of Choanephoraceae has been studied in detail. The filtrate was analysed by using circular paper chromatography. Amongst the seven monosaccharides tested, viz., glucose, galactose, fructose, mannose, xylose, sorbose and rhamnose, the first five were completely utilized within the specified period, while sorbose and rhamnose remained in the medium throughout the incubation period. A mixture of glucose, galactose and fructose was found to support better growth of all the present species, than that when these sugars were supplied singly. Out of the four oligosaccharides tested, only maltose could be hydrolysed, and it was completely consumed within the specified period. The other three oligosaccharides, viz., sucrose, lactose and raffinose were not hydrolysed and they remained in the medium throughout the incubation period.     

Comparison of carbohydrate substrate preferences in eight species of bifidobacteria

Eight species of bifidobacteria were tested for their abilities to grow on a range of monosaccharides (glucose, arabinose, xylose, galactose and mannose). In contrast to the other sugars, glucose and galactose were utilized by all species and, in general, specific growth rates were highest on these sugars. Different substrate preferences were observed between species when the bacteria were grown in the presence of all five monosaccharides. For example, glucose and xylose were coutilized by Bifidobacterium longum, whereas glucose repressed uptake of all other sugars in B. bifidum and B. catenulatum. Galactose was the preferred substrate with B. pseudolongum. In B. angulatum, glucose and galactose were utilized simultaneously. B. breve did not grow on arabinose when this sugar provided the sole source of energy. However, glucose and arabinose were preferentially taken up during growth on sugar mixtures.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Golgi Apparatus Mediated Polysaccharide Secretion by Outer Root Cap Cells of Zea mays. III. Control by Exogenous Sugars

Sugars supplied to germinating seedlings of maize (Zea mays L.) regulate the secretion of polysaccharides by the outer cells of the root cap. The polysaccharide secreted by these cells adheres to the root tip as a droplet and the size of the droplet was used to quantitate polysaccharide secretion. The polysaccharide contains glucose, galacrose, and galacturonic acid residues with smaller quantities of mannose, arabinose, xylose, fucose and rhamnose. These sugars supplied to maize seedlings had marked effects on the rate of polysaccharide secretion by root tips. The effects on secretion were independent of the growth rates of the roots. Glucose, fucose and xylose increased droplet size 1.5–2 fold (as did sucrose, maltose, lacrose, fructose and ribose) whereas galactose, arabinose and galacturonic acid were inhibitory. Mannose increased dropler size 5–7 fold. The marked effect of mannose on polysaccharide secretion was due to an increased rate of secretion combined with a longer phase of extrusion of polysaccharide into the forming droplet. The effect of mannose was partially reversed by inorganic phosphate and other sugars (except for fucose which had no effect or promoted secretion in the presence of mannose). In contrast to sucrose, mannose stimulated secretion in a maize variety having a high sugar endosperm (high endogenous sugar). The results suggest that regulation of secretion by mannose is due to an alteration of normal sugar metabolism; whereas stimulation of secretion by sucrose and other sugars may be due to an increased availability of sugars for metabolism.     

Effect of some disaccharides, hexoses and pentoses on nitrate reductase, glutamine synthetase and glutamate dehydrogenase in excised pea roots

The effects of exogenous sucrose, lactose, d -glucose, d (-)fructose, d -galactose, d -mannose, l -sorbose, l -arabinose and d -xylose on nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) levels, on anaerobic nitrite production and on respiratory O₂ consumption were studied in excised roots of pea (Pisum sativum L. cv. Raman). Sucrose, glucose and fructose increase NR and GS levels and decrease GDH level (when compared with roots cultures without any sugar) at all concentrations used, but the extent of this effect varies. NR induction is enhanced by all sugars within the concentration range studied. Precultivation of roots with mannose and galactose results in an increase in anaerobic nitrite production in a medium consisting of phosphate buffer and KNO₃. GS reaches its maximum at lower sugar concentrations, this fact being especially clear-cut with galactose. The decrease in GS level observed in roots cultured without sucrose is enhanced by higher sorbose concentrations. The increase in GDH level occurring in roots cultured without sucrose is depressed by low galactose and mannose concentrations but enhanced by high galactose, mannose, xylose and a wide range of sorbose concentrations. Lactose exerts only slight influence on the enzymes. The effects of sugars are in no case consistent with their effect on respiratory O₂ consumption which is most pronounced with NR. The above results show that the effects of sugars on NR, GS and GDH are not mediated by one universal mechanism.     

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.