Influence of sugars and light on rhizosecretion of α-glucosidase and acid phosphatase during micropropagation of potato

The effects of carbohydrate supply and light on rhizosecretion during micropropagation of potato plantlets (Solanum tuberosum L., cv. ‘Iwa’) in liquid medium were investigated. Soluble protein content was higher in the spent medium for plantlets grown under light conditions than in the dark. For those plantlets grown under light conditions and on different sugar-supplemented media, they rhizosecreted the highest amount of soluble protein when grown in the presence of maltose, while they rhizosecreted the lowest amount of soluble protein when grown on medium containing glucose. Moreover, plantlets grown under light and on a medium containing sucrose were the most vigorous, and exhibited the highest levels of rhizosecreted acid phosphatase activity. However, there was no direct relationship between plantlet growth and rhizosecretion. When plantlets were grown in the dark and on medium containing maltose, a higher α-glucosidase activity was detected than those grown on medium containing sucrose. These results suggested that rhizosecretion of certain proteins from plantlets grown in vitro might not require exposure to light conditions.     

Regulation of glucose and maltose transport in strains ofSaccharomyces

Summary Growth of yeast cells on glucose resulted in complete inactivation of maltose transport and repression of the high affinity glucose transport system. When the cells were grown on maltose or subjected to substrate starvation, an increase in glucose and maltose transport was observed in both brewing and non-brewing yeast strains. The concentration of glucose employed in the growth medium was also observed to affect sugar transport activity. The higher the glucose concentration, the more pronounced the repressive effect. In addition, the time of growth of yeast on glucose or maltose also intermining the rate of sugar transport. These results are consistent with the repressive effect of glucose on the high affinity glucose and maltose transport systems.     

α-glucosidase in Mycoplasma mycoides subspecies capri

Abstract Mycoplasma mycoides subsp. capri utilisede maltose in medium lacking serum and hence serum saccharolytic enzymes. The presence of α-glucosidase activity was demonstrated by p-nitrophenyl-α- d -glucoside hydrolysis in toluene-treated cells. Specific activities were approx. 4-fold higher in cells grown in the presence of maltose than in cells grown with other sugars or with glucose plus maltose. Extracellular activity was < 2% of cellular activity in growing cultures. α-Glucosidase activity was also demonstrated in cells grown in medium containing serum. It is suggested that the presence of α-glucosidase might be of value in mycoplasma chatacterisation; in a previous study, α-glucosidase activity was not detected in Mycoplasma mycoides subsp. mycoides .     

Physiological role of beta-phosphoglucomutase in Lactococcus lactis

A beta-phosphoglucomutase (beta-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of beta-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h(-1), while the deletion of beta-PGM resulted in a maximum specific growth rate of 0.05 h(-1) on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as beta-glucose 1-phosphate in the medium. Furthermore, the beta-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of alpha-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the beta-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded beta-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Kinetics of alpha-amylase synthesis from Bacillus amyloliquefaciens

The microbial production of alpha-amylase from Bacillus amyloliquefaciens was investigated. The microorganism was grown using media containing glucose or maltose at 37 degrees C and under aerobic conditions in a 16-L fermentor. The alpha-amylase synthesis from maltose was not found to be inducible but was found to be subject to catabolite repression. The maltose uptake rate was observed to be the rate-limiting step compared to the conversion rate of maltose to glucose by intracellular alpha-glucosidase. The alpha-amylase activity achieved with maltose as a substrate was higher than that achieved with glucose. A slower growth rate and a higher cell density were obtained with maltose. The enzyme production pattern depended upon the nutrient composition of the medium.     

Purification and characterization of two phosphoglucomutases from Lactococcus lactis subsp. lactis and their regulation in maltose- and glucose-utilizing cells.

Two distinct forms of phosphoglucomutase were found in Lactococcus lactis subsp. lactis, strains 19435 and 65.1, growing on maltose: beta-phosphoglucomutase (beta-PGM), which catalyzes the reversible conversion of beta-glucose 1-phosphate to glucose 6-phosphate in the maltose catabolism, and alpha-phosphoglucomutase (alpha-PGM). beta-PGM was purified to more than 90% homogeneity in crude cell extract from maltose-grown lactococci, and polyclonal antisera to the enzyme were prepared. The molecular mass of beta-PGM was estimated by gel filtration to be 28 kDa; its isoelectric point was 4.8. The corresponding values for alpha-PGM were 65 kDa and 4.4, respectively. The expression of both PGM enzymes was investigated under different growth conditions. The specific activity and amount of beta-PGM per milliliter of cell extract increased with time in lactococci grown on maltose, but the enzyme was absent in lactococci grown on glucose, indicating enzyme synthesis to be induced by maltose in the growth medium. When glucose was added to maltose-grown lactococci, both the specific activity and amount of beta-PGM per milliliter of cell extract decreased rapidly. This suggests that synthesis of beta-PGM is repressed by glucose in the medium. Although the specific activity of alpha-PGM did not change during growth on maltose or glucose, lactococcal strain 19435 showed a much higher specific activity of both alpha- and beta-PGM than strain 65.1 when grown on maltose.     

The effect of anaerobiosis on the induced synthesis of β-galactosidase in non-growingEscherichia coli

Depending on conditions of aeration maltose and glucose were found to exhibit different effects on the inducible synthesis of β-galactosidase in aerobically grown cells ofEscherichia coli starving for an exogenous source of nitrogen; both saccharides repressed the synthesis of the enzyme under aerobic conditions, while the above-mentioned saccharides were essential for the enzyme synthesis under anaerobic conditions. The presence of maltose in the medium resulted in the repression of the enzyme synthesis in anaerobically grown cells starving for an exogenous nitrogen source under anaerobic conditions. The synthesis of β-galactosidase-specific messenger RNA was completely blocked and the synthesis of the enzyme proper considerably inhibited in aerobically grown cells incubated anaerobically in a medium without nitrogen and carbon sources.     

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     

Growth defects of Escherichia coli cells which contain the gene of an alpha-amylase from Bacillus coagulans on a multicopy plasmid

An alpha-amylase gene from Bacillus coagulans has previously been cloned in Escherichia coli and shown to direct the synthesis of an enzymically active protein of 60,000 Dal (Cornelis et al., 1982). In one particular E. coli host, strain HB101, amylase was found to accumulate in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into E. coli 188 which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for beta-galactosidase, a cytoplasmic marker. Abnormally large amounts of both alpha-amylase and beta-galactosidase were found in the culture fluid of cells grown in rich medium. Furthermore a severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by two types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. These results suggest that the synthesis and export of B. coagulans alpha-amylase is deleterious to E. coli, especially in media containing maltose or glycerol as sole carbon source.     

Carbohydrate utilization by Trichomonas gallinae: effects on growth and metabolic activity under nongrowth conditions

Trichomonas gallinae used 13 of 29 carbohydrates for growth. Quantitative relationships between final populations, acid production, and cellular glycogen contents varied depending on the substrate. The effect of growth on different carbohydrates on the subsequent utilization of carbohydrates by cells under nongrowth conditions was studied by measuring carbohydrate uptake, changes in cellular glycogen content, and gas production. Two major utilization patterns were found. Cells grown on maltose or starch used these substrates well, but cells grown on other sugars did not. All cells used glucose, fructose, galactose, and mannose, but cells grown on maltose or starch did not use them as well as cells grown on other sugars. All cells used ribose slightly but not xylose or arabinose. Turanose, a disaccharide yielding high populations in growth medium, was not used under nongrowth conditions.     

Relative importance of maltose and sucrose supplied during a 2-step potato microtuberization process

A 2-stage in vitro tuberization process comprising first micropropagation via nodal explants and then tuber induction in the resultant in vitro plantlets was studied using 2 cultivars of potato, Iwa and Daeji. In particular, the effects on both plantlet growth and subsequent in vitro tuberization of Murashige and Skoog (1962) basal medium containing either sucrose or maltose, each at 3 % (w/v), used for micropropagation were investigated. Sucrose and maltose were found to be equally effective in supporting development of vigorous plantlets from the nodal explants of both potato cultivars. Upon transfer to a medium with an optimised level of sucrose (i.e. 8 %, w/v) for in vitro tuberization, only the plantlets previously grown in the sucrose-containing medium were capable of forming more microtubers of the larger size category (greater than 0.5 g). The relative importance of sucrose supply at the mircropropagation stage was further confirmed when the resultant plantlets grown in the 3 % sucrose-containing medium were transferred to an in vitro tuberization medium containing either sucrose or maltose, each at 8 % (w/v). In this experiment, maltose and sucrose had indistingushable effects on in vitro tuberization.     

Diauxic growth in Azotobacter vinelandii.

Azotobacter vinelandii exhibited diauxie when grown in a medium containing both acetate and glucose as carbon sources. Acetate was used as the primary carbon source during the acetate-glucose diauxie. Uptake of acetate was constitutively expressed during both diauxic phases of growth. Induction of the glucose uptake system was inhibited in the presence of acetate. Acetate was also the preferred growth substrate for A. vinelandii grown in a medium containing either fructose, maltose, xylitol, or mannitol. The tricarboxylic acid cycle intermediates citrate, isocitrate, and 2-oxoglutarate inhibited glucose utilization in cells grown in glucose medium containing these substrates, and diauxic growth was observed under these growth conditions. Temporal expression of isocitrate-lyase, ATPase, and nitrogenase was exhibited during acetate-glucose diauxie.     

Viability,cell division and microcallus formation of barley microspores in culture

Barley microspores were viable when cultured in a sugarless medium. Adding 2g of glucose to 1l of this medium resulted in a significant reduction in the frequency of viable microspores. The frequency of viable microspores was further reduced when 50g of cellobiose, glucose, maltose, melezitose, raffinose or sucrose were added to 1l of the culture medium containing 2g/l glucose. Adding 50g of melibiose, Ficoll, polyethylene glycol (PEG) or a combination 50g each of Ficoll and PEG to 1I of the medium containing 2g/l glucose had very little effect on the viability of the microspores.Up to 66% of the viable microspores were able to divide and many of these developed into microcalli in the basal medium complemented with melibiose, maltose, melezitose, raffinose, Ficoll, PEG or a combination of Ficoll with PEG. Sucrose, cellobiose and glucose added in large quantities inhibited cell division in microspores or destabilized the microspores and only very few of them developed into microcalli.The microcalli in the PEG, Ficoll, Ficoll-PEG and melibiose media were smaller in size than those grown in the melezitose, maltose and raffinose media. Sustained cell division and microcallus formation were observed in a medium with melibiose or maltose as sole source of sugars.Abbreviations 2.4-D 2,4-dichlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - PEG Polyethylene glycol     

Physiological Role of β-Phosphoglucomutase in Lactococcus lactis

A β-phosphoglucomutase (β-PGM) mutant of Lactococcus lactis subsp. lactis ATCC 19435 was constructed using a minimal integration vector and double-crossover recombination. The mutant and the wild-type strain were grown under controlled conditions with different sugars to elucidate the role of β-PGM in carbohydrate catabolism and anabolism. The mutation did not significantly affect growth, product formation, or cell composition when glucose or lactose was used as the carbon source. With maltose or trehalose as the carbon source the wild-type strain had a maximum specific growth rate of 0.5 h⁻¹, while the deletion of β-PGM resulted in a maximum specific growth rate of 0.05 h⁻¹ on maltose and no growth at all on trehalose. Growth of the mutant strain on maltose resulted in smaller amounts of lactate but more formate, acetate, and ethanol, and approximately 1/10 of the maltose was found as β-glucose 1-phosphate in the medium. Furthermore, the β-PGM mutant cells grown on maltose were considerably larger and accumulated polysaccharides which consisted of α-1,4-bound glucose units. When the cells were grown at a low dilution rate in a glucose and maltose mixture, the wild-type strain exhibited a higher carbohydrate content than when grown at higher growth rates, but still this content was lower than that in the β-PGM mutant. In addition, significant differences in the initial metabolism of maltose and trehalose were found, and cell extracts did not digest free trehalose but only trehalose 6-phosphate, which yielded β-glucose 1-phosphate and glucose 6-phosphate. This demonstrates the presence of a novel enzymatic pathway for trehalose different from that of maltose metabolism in L. lactis.     

Study of the chemical makeup of the mycelium from an active strain of Act. rimosus and from an inactive mutant in relaiton to oxytetracycline biosynthesis]

Chemical composition of the mycelium of the active and inactive mutants of Act. rimosus grown under conditions favourable for oxytetracycline biosynthesis on the starch or maltose medium and under favourable conditions on the glucose medium was studied. It was shown that according to its chemical composition the above strains did not practically differ. When grown on the starch medium the mycelium of both strains contained great amounts of carbohydrates and comparatively small amounts of nucleic acids and nitrogen. Replacement of starch in the medium by glucose or maltose induced significant changes in the mycelium composition: the synthesis of intracellular polysaccharides was markedly suppressed and the synthesis of nucleic acids and nitrogen containing compounds increased. RNA was the main nucleic acid in both strains on starch and glucose media. The content of DNA was low and did not practically change. The mycelium of both strains contained small amounts of lipids which did not significantly change during the process of cultivation and did not correlate with the antibiotic activity.     

Characterization of Porins Isolated from the Outer Membrane of Serratia liquefaciens

A major outer membrane protein with an apparent molecular weight of 42 kDa was purified from Serratia liquefaciens grown on Brain Heart Infusion medium. The same protein was obtained when the cells were grown on a synthetic medium supplemented with 2% glucose. The amino acid composition of this protein revealed it to be hydrophilic. The pore-forming ability of the 42-kDa protein was determined by the liposome swelling assay. This assay demonstrated that the protein forms nonspecific channels with a diameter between 1.16 and 1.6 nm. An additional protein with a molecular weight of 47 kDa was obtained on synthetic medium supplemented with maltose. This protein exhibited specific pore-forming ability to maltose and maltodextrins, but was also permeable to other compounds, according to their size. When bacteria were grown on Nutrient Broth medium, two outer membrane proteins with molecular weights of 41 kDa and 42 kDa were produced by the bacteria. All three types of proteins represent monomers of respective oligomers. The monomers did not exhibit pore-forming ability when incorporated into liposomes. We, therefore, propose that the oligomer is the functional unit of a porin capable of forming permeability channels in the outer membrane of Serratia liquefaciens. These results indicate that S. liquefaciens contains several porins exhibiting specific osmoregulation or that are induced by a specific nutrient, where the 42-kDa outer membrane protein of this bacterium is certainly a major porin. Received: 6 July 1998 / Accepted: 19 August 1998     

Amylase secretion by sweet potato,Ipomoea batatas (L.) Lam., cell cultures grown on various carbon sources

Cell cultures of sweet potato grown in media containing sucrose, glucose, maltose, or starch secreted amylase into the growth medium. The growth rate of cells was not appreciably affected by the carbon source employed for growth, although cells grown on sucrose had a slightly longer lag period before exponential growth occurred. Amylase levels inside the cells were not affected by carbon source, but the amount of amylase released into the medium was drastically affected. Maltose-grown cells released the most amylase while sucrose-grown cells released the least. Cells grown in the light released about twice as much amylase as cells grown in the dark when grown on glucose, maltose, or starch.Three amylase electrophoretic forms were found in the storage root tissue from which all cultures were derived. Cells grown in culture exhibited either two or three amylase forms, depending on the carbon source. The slowest migrating root amylase was found only in cells grown on starch. The root amylase having intermediate mobility was present in all cultures, as was a form having higher mobility than the most mobile root form. The fastest migrating electrophoretic form from the root was not present in any of the cells.Paper No. 8466 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned.     

Conditions with high intracellular glucose inhibit sensing through glucose sensor Snf3 in Saccharomyces cerevisiae

Gene expression in micro‐organisms is regulated according to extracellular conditions and nutrient concentrations. In Saccharomyces cerevisiae, non‐transporting sensors with high sequence similarity to transporters, that is, transporter‐like sensors, have been identified for sugars as well as for amino acids. An alternating‐access model of the function of transporter‐like sensors has been previously suggested based on amino acid sensing, where intracellular ligand inhibits binding of extracellular ligand. Here we studied the effect of intracellular glucose on sensing of extracellular glucose through the transporter‐like sensor Snf3 in yeast. Sensing through Snf3 was determined by measuring degradation of Mth1 protein. High intracellular glucose concentrations were achieved by using yeast strains lacking monohexose transporters which were grown on maltose. The apparent affinity of extracellular glucose to Snf3 was measured for cells grown in non‐fermentative medium or on maltose. The apparent affinity for glucose was lowest when the intracellular glucose concentration was high. The results conform to an alternating‐access model for transporter‐like sensors. J. Cell. Biochem. 110: 920–925, 2010. © 2010 Wiley‐Liss, Inc.