Development of a Defined Minimal Medium for the Growth of Edwardsiella ictaluri

In this report, a complete defined medium and a minimally defined medium are described for Edwardsiella ictaluri. The complete defined medium consists of 46 individual components, including a basal salt solution, glucose, magnesium sulfate, iron sulfate, six trace metals, four nucleotides, 10 vitamins, and 19 amino acids. This medium supports growth in broth and on solid media. Optimal growth at 30(deg)C was obtained at pH 7.0, and at an osmolality of 390 mosmol/kg of H(inf2)O, with a glucose concentration of 4 g/liter. The defined minimal medium reduces the 46 components of the complete medium to eight essential components, including the basal salt solution, glucose, magnesium sulfate, pantothenic acid, and niacinamide. In addition, specific amino acids that depend on the specific requirements of the individual strains of E. ictaluri are added.     

Studies on lipid production by Rhodotorula glutinis fermentation using monosodium glutamate wastewater as culture medium

Microbial lipid, as a raw material for biodiesel, can be produced by Rhodotorula glutinis with the monosodium glutamate (MSG) wastewater. The effect of adding glucose to MSG wastewater on lipid production was studied in this paper. Three different strategies, including initial addition, fed-batch addition and glucose feedback addition were attempted. The results show that addition of glucose was found favorable not only for cell growth but also for lipid synthesis. Of the three adding methods glucose feedback addition was the most effective one: about 25 g/L of biomass, 20% of lipid content and 45% of COD degradation were obtained respectively. And the components of the resulted lipid using different addition strategies were further studied.     

Theoretical and computational studies of the glucose signaling pathways in yeast using global gene expression data

We have combined DNA microarray experiments with novel computational methods as a means of defining the topology of a biological signal transduction pathway. By DNA microarray techniques, we previously acquired data on expression over time of all genes in the yeast Saccharomyces following addition of glucose to wild-type cells and to cells mutated in one or more components of the Ras signaling network. In addition, we examined the time course of expression following activation of components of the Ras signaling network in the absence of glucose addition. In this current study, we have applied a novel theoretical and computational framework to these data to identify the network topology of the glucose signaling pathway in yeast and the role of Ras components in that network. The computational approach involves clustering genes by expression pattern, postulating a signaling network topology superstructure that includes all possible component interconnections and then evaluating the feasibility of the superstructure interconnections by optimization methods using Mixed Integer Linear Programming techniques. This approach is the first rigorous mathematical framework for addressing the biological network topology issue, and the novel formulation features the introduction of discrete variables for the connectivity and logical expressions that connect the experimental observations to the network structure. This analysis yields a topology for the glucose signaling pathway that is consistent with, and an extension of, known biological interactions in glucose signaling.     

Soil‐specific response functions of organic matter mineralization to the availability of labile carbon

Soil organic matter (SOM) mineralization processes are central to the functioning of soils in relation to feedbacks with atmospheric CO₂ concentration, to sustainable nutrient supply, to structural stability and in supporting biodiversity. Recognition that labile C‐inputs to soil (e.g. plant‐derived) can significantly affect mineralization of SOM (‘priming effects’) complicates prediction of environmental and land‐use change effects on SOM dynamics and soil C‐balance. The aim of this study is to construct response functions for SOM priming to labile C (glucose) addition rates, for four contrasting soils. Six rates of glucose (3 atm% ¹³C) addition (in the range 0–1 mg glucose g^?1 soil day^?1) were applied for 8 days. Soil CO₂ efflux was partitioned into SOM‐ and glucose‐derived components by isotopic mass balance, allowing quantification of SOM priming over time for each soil type. Priming effects resulting from pool substitution effects in the microbial biomass (‘apparent priming’) were accounted for by determining treatment effects on microbial biomass size and isotopic composition. In general, SOM priming increased with glucose addition rate, approaching maximum rates specific for each soil (up to 200%). Where glucose additions saturated microbial utilization capacity (>0.5 mg glucose g^?1 soil), priming was a soil‐specific function of glucose mineralization rate. At low to intermediate glucose addition rates, the magnitude (and direction) of priming effects was more variable. These results are consistent with the view that SOM priming is supported by the availability of labile C, that priming is not a ubiquitous function of all components of microbial communities and that soils differ in the extent to which labile C stimulates priming. That priming effects can be represented as response functions to labile C addition rates may be a means of their explicit representation in soil C‐models. However, these response functions are soil‐specific and may be affected by several interacting factors at lower addition rates.     

A model experiment to study the influence of living plants on the accumulation of soil organic matter in pastures

Summary An attempt has been made to imitate the grassland system by a perfusion apparatus containing a soil-column to which labeled glucose is continuously supplied. Experiments have also been performed with substrate supplied at the start of the experiment to imitate processes occurring in arable land. Deficiency of available nitrogen caused that more of the glucose carbon added to the soil was incorporated into soil organic matter than in the presence of a supplied nitrogen source. Even more glucose carbon was incorporated into soil organic matter when nitrogen deficiency was accompanied by a continous addition of the glucose. The results obtained indicate that the continuous addition of substrate together with nitrogen deficiency as it occurs in permanent pastures are responsible for the accumulation of soil organic matter in these soils.     

Tetanus toxin production in soy-based medium: nutritional studies and scale-up into small fermentors

AIMS: To further improve the soy-based medium, devoid of animal and dairy products, for a production of tetanus toxin by nutritional studies and to scale-up the Clostridium tetani process into small fermentors. METHODS AND RESULTS: Optimum production of tetanus toxin did not require addition of pantothenic acid, thiamine, riboflavin, pyridoxine, biotin and uracil, growth factors used by previous investigators. Furthermore, l-tyrosine and l-cysteine could be eliminated from our soy-based medium without effect. Seven carbon sources were compared with glucose in the soy-based medium, but none was found to be superior to glucose. The process was successfully scaled-up into 250-ml bottles, 1-l bottles and 1-l fermentors. CONCLUSIONS: Quite remarkably, when comparing the tetanus production process in our soy-based medium with the traditional animal/dairy-containing media, our medium does not require addition of expensive vitamins, uracil or carbon sources other than glucose. Furthermore, the l-tyrosine and l-cysteine components could be eliminated, making the medium (Hy-Soy, glucose, powdered iron and inorganic salts) much more simple and economical. The successful scale-up from test tubes into 1-l fermentors allows us to predict that further scale-up into large fermentors will be successful. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (BSE; mad cow's disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. Our vegetable-based process avoids such unfortunate possibilities. The medium, having been made simpler and less expensive, and shown to be scaleable from test tubes into small fermentors, should be excellent for large scale production of tetanus toxin.     

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.     

Production of zoogloea gum by Zoogloea ramigerawith glucose analogs

Zooglans with altered sugar composition were synthesized by Zoogloea ramigera by varying the glucose concentration and initial medium pH. The relative mol % of the sugar components, glucose and galactose, in the exopolymer made with 2% (w/v) glucose as the carbon source was 66 and 34%, respectively. By varying the glucose concentration and initial medium pH, the mol % ratios of glucose to galactose in zooglan ranged from 70 : 30 to 58 : 42. Also, glucose analogs, 3- O-methyl-D-glucose, 2-amino-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-glucose, were used as a co-substrate with glucose to produce modified zooglans. The mol % ratios of glucose to galactose in exopolymers produced by co-feeding glucose analogs ranged from 70 : 30 to 9 : 91.     

Contributions of Amino-carbonyl Reaction,Caramelization and Some Other Reaction to Browning of Saké

In the previous report, it has been concluded that the browning of Saké involves three kinds of browning reactions which are the amino-carbonyl reaction, the caramelization and some other browning reaction by components other than glucose. In the present work, the extents of contributions of the three kinds of reactions to the browning of Saké were actually estimated, respectively, by analysing its dependence on the glucose concentration. It was concluded that the browning by components other than glucose was the major factor while the amino-carbonyl reaction and the caramelization were the minor ones in the browning of Saké. In addition, the change in pH value, as well as in temperature, was observed to influence exponentially on the browning rate of Saké. An empirical equation was proposed to elucidate the way in which the three kinds of reactions contributed to the overall browning in Saké.     

Expression of glucose transporter isoform GLUT-2 and glucokinase genes in human brain

The glucose transporter isoform-2 (GLUT-2) and glucokinase are considered to be components of a glucose sensor system controlling several key processes, and hence may modulate feeding behaviour. We have found GLUT-2 and glucokinase mRNAs in several brain regions, including the ventromedial and arcuate nuclei of the hypothalamus. GLUT-2, glucokinase and glucokinase regulatory protein mRNAs and proteins were present in these areas as determined by biochemical approaches. In addition, glucose-phosphorylating activity with a high apparent Km for glucose that displayed no product inhibition by glucose-6-phosphate was observed. Increased glycaemia after meals may be recognized by specific hypothalamic neurones due to the high Km of GLUT-2 and glucokinase. This enzyme is considered to be the true glucose sensor because it catalyses the rate-limiting step of glucose catabolism its activity being regulated by interaction with glucokinase regulatory protein, that functions as a metabolic sensor.     

Detoxification of a lignocellulosic biomass slurry by soluble polyelectrolyte adsorption for improved fermentation efficiency

This study investigated the detoxification of a dilute acid pretreated Ponderosa pine slurry using the polyelectrolyte polyethyleneimine (PEI). The addition of polyelectrolyte to remove enzymatic and/or fermentation inhibitory compounds, that is, acetic acid, furfural, and 5-hydroxymethylfurfural (HMF), was performed either before or after enzymatic hydrolysis to determine the optimal process sequence. Negligible acetic acid, glucose, and xylose were removed regardless of where in the process the polymer addition was made. Maximum furfural and HMF separation was achieved with the addition of PEI to a clarified pre-enzymatic hydrolysis liquor, which showed that 88.3% of furfural and 66.4% of HMF could be removed. On the other hand, only 23.1% and 13.4% of furfural and HMF, respectively, were removed from a post-enzymatic hydrolysis sample; thus, the effects of enzymes, glucose, and wood solids on inhibitor removal were also investigated. The presence of solid particles >0.2 μm and unknown soluble components <10 kDa reduced inhibitory compound removal, but the presence of elevated glucose levels and enzymes (cellulases) did not affect the separation. The fermentability of detoxified versus undetoxified hydrolysate was also investigated. An ethanol yield of 92.6% of theoretical was achieved with Saccharomyces cerevisiae fermenting the detoxified hydrolyzate, while no significant ethanol was produced in the undetoxified hydrolyzate. These results indicate that PEI may provide a practical alternative for furan removal and detoxification of lignocellolosic hydrolysates, and that application before enzymatic hydrolysis minimizes separation interferences.     

Concentration-dependent repression of the soluble and membrane components of the Streptococcus mutans phosphoenolpyruvate: sugar phosphotransferase system by glucose.

Growth of Streptococcus mutans Ingbritt in continuous culture (pH 7.0, dilution rate of 0.1 h-1) at medium glucose concentrations above 2.6 mM resulted in repression of the sugar-specific membrane components, enzyme IIGlc (EIIGlc) and EIIMan, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In one experiment, significant repression (27-fold) was observed with 73 mM glucose when the glycolytic capacity of the cells was reduced by only 2-fold and when the culture was still glucose limited. In a more comprehensive experiment in which cells were grown in continuous culture at eight glucose concentrations from 2.6 to 304 mM, in addition to repression of specific EII activities for glucose, mannose, 2-deoxyglucose, and fructose, synthesis of the general protein, EI, was repressed at all glucose levels above 2.6 mM to a maximum of 4-fold at 304 mM glucose when the culture was growing with excess glucose (i.e., nitrogen limited). The other PTS general protein, HPr, was less sensitive to the exogenous glucose level but was nevertheless repressed fourfold under glucose-excess conditions. The Km for glucose for EIIGlc increased from 0.22 mM during growth at 3.6 mM glucose (glucose limited) to 0.48 mM at 271 mM glucose (glucose excess). The shift from heterofermentation to homofermentation during growth with increasing glucose levels suggests the involvement of glycolytic intermediates, ATP, or another high-energy phosphate metabolite in regulation of the synthesis of the PTS components in S. mutans.     

A convenient procedure for producing gram-quantities of brucine l-guluronate and brucine d-mannuronate

A comparative investigation of the individual protein-bound carbohydrate components and the distribution of carbohydrates in the electrophoretic glycoprotein fractions was carried out in sera from 20 normal subjects and 30 schizophrenic patients matched for age and sex. The mean concentration of each of the protein-bound carbohydrate components was significantly elevated in schizophrenics. The electrophoretic patterns for serum glycoprotein showed increases in alpha-2 and beta globulins in schizophrenics. The serum glycoproteins contained glucose and l-arabinose, in addition to mannose, galactose, fucose, sialic acid, and a trace of xylose. The identity of glucose and arabinose was confirmed by g.l.c.-electron-impact mass spectrometry and by specific enzymic reactions. The contents of glucose and arabinose were higher in serum glycoproteins from schizophrenic patients. This elevation of serum glycoprotein paralleled serum glycosaminoglycan elevation previously reported by us, but was opposite to decrease of urinary glycoprotein in schizophrenics.     

Effect of growth media on cell envelope composition and nitrile hydratase stability in Rhodococcus rhodochrous strain DAP 96253

Rhodococcus is an important industrial microorganism that possesses diverse metabolic capabilities; it also has a cell envelope, composed of an outer layer of mycolic acids and glycolipids. Selected Rhodococcus species when induced are capable of transforming nitriles to the corresponding amide by the enzyme nitrile hydratase (NHase), and subsequently to the corresponding acid via an amidase. This nitrile biochemistry has generated interest in using the rhodococci as biocatalysts. It was hypothesized that altering sugars in the growth medium might impact cell envelope components and have effects on NHase. When the primary carbon source in growth media was changed from glucose to fructose, maltose, or maltodextrin, the NHase activity increased. Cells grown in the presence of maltose and maltodextrin showed the highest activities against propionitrile, 197 and 202?units/mg cdw, respectively. Stability of NHase was also affected as cells grown in the presence of maltose and maltodextrin retained more NHase activity at 55?°C (45 and 23?%, respectively) than cells grown in the presence of glucose or fructose (19 and 10?%, respectively). Supplementation of trehalose in the growth media resulted in increased NHase stability at 55?°C, as cells grown in the presence of glucose retained 40?% NHase activity as opposed to 19?% without the presence of trehalose. Changes in cell envelope components, such mycolic acids and glycolipids, were evaluated by high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), respectively. Changing sugars and the addition of inducing components for NHase, such as cobalt and urea in growth media, resulted in changes in mycolic acid profiles. Mycolic acid content increased 5 times when cobalt and urea were added to media with glucose. Glycolipids levels were also affected by the changes in sugars and addition of inducing components. This research demonstrates that carbohydrate selection impacts NHase activity and stability. Cell envelope components such as mycolic acids are also influenced by sugars and inducers such as cobalt and urea. This is information that can be useful when implementing rhodococcal catalysts in industrial applications.     

Effects of elevated glucose levels on interactions of cardiac fibroblasts with the extracellular matrix

Exposure of fibroblasts to high glucose levels promotes a fibrotic response characterized by increased expression of extracellular matrix components including interstitial collagens. Little is known about the effects of glucose levels on other aspects of fibroblast function. Fibroblasts in the myocardium are surrounded by an extensive extracellular matrix composed predominantly of type I collagen. Interactions between fibroblasts and the myocardial extracellular matrix are thought to affect heart function by altering ventricular diastolic properties. The purpose of the present study was to determine the effects of elevated glucose levels on the interactions between heart fibroblasts and the collagenous extracellular matrix. Studies were performed to determine the effects of relative glucose levels on the ability of fibroblasts to migrate on and contract a three-dimensional collagenous substratum. These experiments illustrated that exposure of cardiac fibroblasts to high glucose levels (25 mM) resulted in decreased migratory activity of fibroblasts on a collagen matrix and decreased fibroblast proliferation. In addition, high glucose stimulated collagen and collagen-binding integrin expression and contraction of three-dimensional collagen gels by cardiac fibroblasts. These studies illustrate that altered glucose levels induce important changes in the interactions of cardiac fibroblasts with the collagenous extracellular matrix. Xiaoyi Zhang and James A. Stewart, Jr. are co-first authors.     

Vacuole import and degradation pathway: Insights into a specialized autophagy pathway

Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.     

Identification of catalytic residues in the beta-glucoside permease of Escherichia coli by site-specific mutagenesis and demonstration of interdomain cross-reactivity between the beta-glucoside and glucose systems

beta-Glucoside Enzyme II (IIBgl) of the Escherichia coli phosphotransferase system transports and phosphorylates beta-glucosides, whereas the glucose Enzyme II-III pair (IIGlc-IIIGlc) transports and phosphorylates glucose as well as certain aliphatic alpha- and beta-glucosides. Comparisons of their respective amino acid sequences previously revealed that both systems are homologous and must be evolutionarily related. To gain more insight into the details of the transport mechanism, we made use of the observed homologies among phosphotransferase system permeases to design a suitable set of site-specific mutants within the gene encoding IIBgl. This set was used to study in vivo fermentation and to analyze in vitro P-enolpyruvate-dependent sugar phosphorylation as well as sugar phosphate-dependent sugar transphosphorylation. The following results were obtained. (i) IIBgl transports and phosphorylates glucose as well as aryl- and alkyl-beta-glucosides; (ii) histidyl 547 is essential for the phosphorylation of IIBgl by the histidine-containing phosphoryl carrier protein of the phosphotransferase system (HPr) (first phosphorylation site); (iii) both cysteyl 24 and histidyl 306 are essential for the transfer of the phosphoryl group to the sugar; (iv) replacement of Cys-24 by serine leads to uncoupling of sugar transport from phosphorylation; and (v) histidyl 183 is important for substrate specificity. Our studies also revealed heterologous phosphoryl transfer between the beta-glucoside and glucose permease components which probably occurs as follows: 1) HPr-P----IIBgl (His-547)----IIGlc----alkyl-alpha- or -beta-glucosides or glucose (but not aryl-beta-glucosides) and 2) HPr-P----IIIGlc----IIBgl (Cys-24 or His-306)----alkyl- or aryl-beta-glucosides or glucose (but not methyl-alpha-glucoside). In addition to the essential residues noted above, several residues in IIBgl were identified which when mutated reduced the in vitro catalytic efficiency of the enzyme more than 10-fold. Thus, aspartyl 551 and arginyl 625 appeared to function together with histidyl 547 in phosphoryl transfer involving the first phosphorylation site in the permease, whereas histidyl 183 appeared to function together with cysteyl 24 and histidyl 306 in phosphoryl transfer involving the second phosphorylation site in the permease.     

Glycoproteins from ulva lactuca

Ulva lactuca yielded glycoprotein materials which differed significantly in their carbohydrate and protein moieties. The electrophoretic patterns of the isolated materials showed the presence in each of glycoprotein and polysaccharide components. All the glycoprotein components moved towards the cathode.The glycoprotein material isolated by extraction with NaOH was fractionated on a DEAE-cellulose column into four glycoprotein components and one protein component. All the glycoprotein components contained glucuronic acid, xylose, and rhamnose in different proportions, and in addition two of them contained glucose.