Dynamics of production of extracellular polysaccharides by nodule bacteria

The dynamics of the accumulation of the extracellular polysaccharides synthesized by nodule bacteria and the possibility of their assimilation by these bacteria as a source of carbon was studied. When nodule bacteria were cultured for 20 days in a medium containing glucose, an increase in the titer of the bacteria and the accumulation of extracellular polysaccharides was observed in the first three days. After this the titer of the nodule bacteria decreased with a decrease in the glucose in the medium, but the amount of extracellular polysaccharide synthesized did not increase. These data suggest that extracellular polysaccharides are not assimilated by nodule bacteria as a source of carbon and evidently are protective substances for the cells.     

Growth of cultured mammalian cells on secondary glucose sources

Mammalian cells can grow in culture at very low glucose concentrations. They can also grow using starch or maltose as secondary sources of glucose if hydrolytic enzymes (amylase and/or maltase) are available to release the glucose. The serum supplement in the culture medium provides these enzymes in amount adequate to permit growth at as rapid a rate as when free glucose is added. Owing to the relatively slow liberation of glucose from the secondary sources, the cells produce less lactic acid, and the culture medium does not become acidic.If the amount of hydrolytic enzyme in the serum supplement is reduced by heat inactivation, the rate of glucose liberation is further reduced. As a result, glucose continues to be released into the medium even at high cell densities, when all glucose added directly to control cultures has been consumed at a time. For this reason, the cells survive longer at high density on secondary glucose sources than on free glucose. Use of such a culture system should have important practical advantages in maintaining dense cultures of any mammalian cell type.Medium containing secondary glucose sources and serum whose hydrolytic enzymes have been completely inactivated should be a selective medium for the corresponding cellular enzymes. Attempts to select for cell lines able to grow using their own amylase or maltase were not successful, but calculations based on embryonic pancreatic cells, known to synthesize amylase, showed that the amount of enzyme required should be quite low in comparison with that present in the differentiated state. The possibilities of selection for a differentiated function in cell culture have been very little explored, and such an approach may be fruitful if applied to the right cell types.     

Determination of the Chemical Form of Fluorine in Tea Infusions by 19F-NMR

A novel marine ice-nucleating bacterium, KUIN-5, was isolated from a marine algae, Monostroma latissum. Strain KUIN-5 was identified as a Pseudomonas sp. from its characteristics and taxonomies; the optimum temperature and pH for its growth were 25°C and 6.0, respectively. When strain KUIN-5 was aerobically cultured in Carlucci-Pramer medium (pH 6.0) for 50 h at 25°C, the highest ice-nucleating activity of the cells among the media for marine bacteria was obtained, and the ice-nucleating temperature, T₅₀, was indicated to be ? 3.2°C. Also, the optimum concentration of NaCl for the growth in this medium, which was prepared with distilled water instead of seawater, was 2.0% (w/v) and then the ice-nucleating activity was inversely proportional to the NaCl concentration. Moreover, when strain KUIN-5 was cultured in Davis medium under optimum conditions, it produced insoluble polysaccharide (IPS) in the culture. The maximum amount of IPS production by strain KUIN-5 was 84.5 mg/ml of medium under optimum conditions. Therefore, this IPS was isolated and could be identified as cellulose, based on TLC or HPLC of the acid hydrolysate, and GC-MS of the acetylated polyalcohol prepared by periodate oxidation and Smith degradation of this polysaccharide. This is the first report of cellulose production by a marine ice-nucleating bacterium.     

Differential effect of hexoses on hamster embryo development in culture

The effects of glucose, fructose, and galactose on hamster embryo development in the absence of phosphate were studied in culture. One- and two-cell embryos were cultured to the blastocyst stage in HECM-9 medium without hexose or in medium with increasing concentrations of hexoses. Embryo development, cell number, and cell allocation were assessed in blastocysts. Blastocyst viability was determined by transfer to pseudopregnant recipients. Although 0.25 mM fructose increased mean cell number, low glucose concentrations had no stimulatory effect on development to blastocyst. Both galactose and 5.0 mM glucose were detrimental to embryos. Addition of 0.5 mM glucose increased implantation and fetal viability as compared with controls. Compared with 0.5 mM glucose, treatment with 0.25 mM fructose gave similar implantation and fetal viability, whereas 5.0 mM glucose tended to decrease implantation and significantly decreased fetal development. These data demonstrate that morphology is a poor indicator of embryo viability and that exposure of preimplantation embryos to glucose or fructose is important for embryo viability post-transfer. Although no difference in blastocyst viability was detected between embryos cultured with 0.25 mM fructose and those cultured with 0.5 mM glucose, increased cell numbers obtained with fructose suggest that fructose may be more appropriate than glucose for inclusion in culture medium.     

微量元素对大肠杆菌生长和乙酸生成的影响研究

大肠杆菌DA19的代谢特性与培养基中添加微量元素有较大的关系。在基本培养基中,当氮源限制时,添加微量元素可以在一定程度上改善DA19菌体的生长,提高菌体得率YX/G,大大减少乙酸的生成;当氮源充分时,与不添加微量元素相比,DA19在添加微量元素后,菌体浓度大大增加,虽然葡萄糖消耗速率加快,但产乙酸仍然很少,只有不添加时的13％,YX/G提高至少60％。基本培养基中添加0.1～1mL/L的微量元素混合溶液对DA19菌体生长、乙酸生成及葡萄糖消耗没有显著影响。在单独添加不同种类的微量元素时, BO33-、Zn2+、MoO42+、Cu2+没有特别明显的影响,Al3+会抑制菌体生长和葡萄糖利用,而Co2+、Mn2+、Fe2+可以改善细胞生长,特别是添加Fe2+时,细胞生长及乙酸生成等培养结果与添加微量元素混合溶液几乎相同。     

The hydrogen peroxide microvolumetric method of the OF test: the rapid determination of glucose oxidation and fermentation by gram-negative bacteria

For the first time the method of the rapid (1 hour) screening of groups of gram-negative bacteria by the OF test with glucose, carried out with the use of microvolumetric techniques, has been developed. The method is based on the use of hydrogen peroxide at non-bactericidal concentration as a component of a liquid buffer medium containing indicator and glucose and intended for the oxidation of glucose. Catalase of bacteria introduced into the medium for study ensures the rapid saturation of the medium with oxygen and the completion of the oxidation of glucose in 10-60 minutes. An equal period is necessary to achieve the complete fermentation of glucose in the same medium without hydrogen peroxide. The method has been proved to yield significant results in the joint study of the OF test on 502 strains, belonging to 21 genera of fermenting and nonfermenting gram-negative bacteria, by the hydrogen peroxide method and in Hugh-Leifson medium.     

ColRS two-component system prevents lysis of subpopulation of glucose-grown Pseudomonas putida

ColRS two-component system is well conserved in pseudomonads, but its exact role has remained obscure. Here, we report that Pseudomonas putida deficient in ColR experiences serious carbon source-specific stress that leads to the lysis of a subpopulation of bacteria growing on solid glucose medium. We observed that on glucose medium colR-deficient bacteria aggregated, produced a Congo Red-binding substance and had enhanced membrane permeability. Detection of a large amount of cytoplasmic beta-galactosidase and other proteins as well as chromosomal DNA in the growth medium of a colR mutant indicated that cell lysis took place if ColR was absent. Investigation of colony morphology revealed concavities in the centre of the colonies of colR mutant suggesting that cell lysis occurred mainly in the areas of the highest cell density. Analysis of bacteria at a single cell level by flow cytometry showed that population of glucose-grown colR-deficient cells was heterogeneous. In addition to the wild type-like population, we detected a subpopulation of cells with damaged membrane permeable to propidium iodide. Interestingly, inactivation of oprB1 encoding a glucose porin eliminated the cell lysis as well as autoaggregation and membrane leakiness of a colR mutant indicating that glucose influx could be responsible for membrane stress in the absence of ColRS system.     

易分解有机碳输入量对武夷山常绿阔叶林不同土层深度土壤激发效应的影响

土壤有机碳(SOC)的矿化在碳、氮循环过程中起着极为重要的作用。易分解有机碳的输入可以通过正(负)激发效应加快(减缓)原有SOC的矿化。然而,先前的研究更多关注易分解有机碳输入量对表层(0～20 cm)土壤激发效应的影响,而较少关注其对深层(>20 cm)土壤激发效应的影响。本研究利用¹³C标记葡萄糖(99 atom%)添加试验,研究葡萄糖添加量对武夷山常绿阔叶林表层(0～20 cm)和深层(30～40 cm)土壤激发效应的影响,并通过分析微生物群落组成的变化以及土壤可利用氮含量的变化探讨土壤激发效应产生的机理。结果表明:葡萄糖的添加抑制了表层和深层SOC的矿化(P<0.05),使SOC的矿化量分别减少了26%～61%与62%～68%,呈现负的激发效应,但激发强度因葡萄糖添加量和土层深度而异。对于表层土壤,激发强度随着葡萄糖添加量的增加而增加;而对于深层土壤,激发强度对葡萄糖添加量的响应并不敏感。而且,葡萄糖的添加并未显著影响表层和深层土壤的微生物量碳氮含量和微生物群落组成(总磷脂脂肪酸含量;细菌、真菌、放线菌磷脂脂肪酸含量以及细菌真菌比)(P>...     

Dynamic analysis of a microbial process: A systems engineering approach

A general mathematical model of the chemostat system is developed in order to define an experimental program of dynamic testing. A glucose-limited culture ofSaccharomyces cerevisiae was grown in a chemostat using chemically defined medium. The chemostat was perturbed from an initial steady state by changes in input glucose concentration, dilution rate, pH, and temperature. Dynamic responses of cell mass, glucose, cell number, RNA, and protein concentrations were measured. A number of simulation techniques were used in developing a dynamic mathematical model and in comparing the developed model with experimental data as well as the Monod model. The resulting model was found to be quantitatively accurate and superior to the Monod model. The developed model was interpreted in the light of cell physiology. Adjustment of intracellular RNA fraction was found to be rate limiting in acceleration of cell specific growth rate.     

Expression of capsule-associated genes of Cryptococcus neoformans

Cryptococcus neoformans produces an extracellular polysaccharide capsule that is related to its virulence. The production of capsular components was reported to be accelerated when cultured on media with lower amount of glucose. In this study, relationship between capsule synthesis and expression of capsule-associated genes (CAP genes) was investigated by quantitative real-time PCR analysis. Normally encapsulated strains and a stable acapsular strain were cultured in 1% polypepton medium with 0.1% or 15% glucose. The results of assessment of the capsule size showed that the capsule of yeast cells cultured in the medium with low amount of glucose was thicker than that with high amount of glucose. The CAP gene expressions of normally encapsulated strains were higher in the medium with 0.1% glucose than in the medium with 15% glucose. Furthermore, CAP10, CAP59 and CAP60 genes were expressed very low in a stable acapsular strain, and CAP64 gene was not expressed. Results of assessment of capsule size and CAP gene expressions by quantitative real-time PCR analysis indicated that CAP gene expressions might be related to the production of capsule, and that glucose concentration in culture media might be related to the expression of CAP genes.     

Microquantitative determination of glucose in solutions using microorganisms as the test system

A technique for determining small quantities of glucose in solutions and biological liquids is proposed. The assay can be performed in the presence of some other carbohydrates, except those which are transported into E. coli cells by means of the phosphoenol-transferase system. The method is based on pH-metric regictration of H+-ions which are released from bacteria as a result of glucose consumption. The suspension of cells of E coli st. M-17 was used as a test system. We can also recommend a commercial preparation of colibacterin, which contains lyophilized bacteria E. coli st. M-17 in the sucrose-gelatin medium. The sensitivity of the method is about 1 microgram of glucose in 5-10 microliter.     

Population of Aerobic Heterotrophic Nitrogen-Fixing Bacteria Associated with Wetland and Dryland Rice

Nitrogen-fixing activity and populations of nitrogen-fixing bacteria associated with two varieties of rice grown in dryland and wetland conditions were measured at various growth stages during the dry season. Acetylene reduction activities were measured both in the field and for the hydroponically grown rice, which was transferred from the field to water culture 1 day before assay. The activities measured by both methods were higher in wetland than in dryland rice. The population of nitrogen-fixing heterotrophic bacteria associated with rhizosphere soil, root, and basal shoots was determined by the most probable number method with semisolid glucose-yeast extract and semisolid malate-yeast extract media. The number of nitrogen-fixing bacteria was higher in wetland conditions than in dryland conditions. The difference between two conditions was most pronounced in the population associated with the basal shoot. The glucose medium gave higher counts than did the malate medium. Colonies were picked from tryptic soy agar plates, and their nitrogen-fixing activity was tested on a semisolid glucose-yeast extract medium. The incidence of nitrogen-fixing bacteria among aerobic heterotrophic bacteria in association with rhizosphere soil, root, and basal shoots was much lower in dryland rice than in wetland rice.     

The effect of energy substrate concentration and amino acids on the in vitro development of preimplantation porcine embryos

As the pig becomes increasingly used for biomedical research, an effective and efficient in vitro culture system is essential. This study aimed to improve the commonly used porcine embryo culture medium, NCSU23, by altering the energy substrates and adding amino acids, using electrically activated diploid parthenotes from oocytes obtained from the ovaries of prepubertal and adult animals. Morphological development to day 6 and blastocyst cell number were examined. Glucose (5.56 mM) was replaced by pyruvate and lactate (0.2 mM and 5.7 mM, respectively) for either the entire culture period or for the first 48 h only. Blastocyst rates were not different between any of the treatments, and were similar for prepubertal and adult oocytes. When the embryos were cultured with pyruvate and lactate for the first 48 h and then glucose, there was a significant increase in blastocyst cell number compared to glucose only. Blastocysts produced using pyruvate and lactate for the entire time tended to have more cells than those exposed to glucose only and less than those who were cultured in pyruvate and lactate for the first 48 h and then glucose. Nonessential amino acids added for the first 48 h and nonessential and essential amino acids added for the remaining time significantly increased blastocyst cell number only when the embryos were grown in pyruvate and lactate followed by glucose. Blastocyst rates were not different between any of the treatments, and this result was the same when using sow or gilt oocytes. The modified medium was then tested using in vitro matured and fertilized embryos from sow oocytes. Blastocyst rates and cell number were significantly increased in the modified medium compared to those grown in unmodified NCSU23. This shows that altering energy substrates and adding amino acids can increase the quantity and cell number of IVP blastocysts compared with NCSU23.     

A 31P-NMR study of Propionibacterium acnes, including effects caused by near-ultraviolet irradiation

161.8 MHz 31P-NMR spectra were recorded from the light sensitive skin bacterium Propionibacterium acnes. The cells were grown anaerobically on synthetic phosphate-buffered Eagle's medium or on a complex yeast extract medium. The spectra showed a large accumulation of polyphosphates when grown on Eagles medium. A splitting of the inorganic phosphate peak indicated a difference between internal and external pH of the cells. Addition of glucose to the cell suspension gave rise to a change in the pH gradient across the cell membrane, as reported for other Gram-positive bacteria. A decrease in the polyphosphate peak was observed after addition of glucose. A lethal dose of broad-band near-ultraviolet light (corresponding to a 10% survival in a survival test), increased the amount of polyphosphates visible in the NMR-spectra. The addition of glucose to irradiated cells decreased the pH in the external solution, but no splitting of the inorganic phosphate peak could however be observed. 31P-NMR can, therefore, be used to study immediate near-ultraviolet-induced effects at the cellular level, at least in the case of P. acnes.     

Microplate assay for detection of bacterial contamination in tissue culture media

In the present study, a rapid and simple colorimetric technique has been described to determine the presence of bacteria in tissue culture medium used in animal cell culture. The microplate assay is based on utilization of MTT [3(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] by bacteria resulting in formation of formazan crystals which can be measured colorimetrically. Contaminated medium, a standard gram-negative and gram-positive bacteria produce formazan from MTT which is related to the bacterial load. The assay has utility in screening tissue culture reagents to detect the presence of bacteria.     

Active transport and accumulation of iodide by newly isolated marine bacteria

Iodide (I(-))-accumulating bacteria were isolated from marine sediment by an autoradiographic method with radioactive (125)I(-). When they were grown in a liquid medium containing 0.1 microM iodide, 79 to 89% of the iodide was removed from the medium, and a corresponding amount of iodide was detected in the cells. Phylogenetic analysis based on 16S rRNA gene sequences indicated that iodide-accumulating bacteria were closely related to Flexibacter aggregans NBRC15975 and Arenibacter troitsensis, members of the family Flavobacteriaceae. When one of the strains, strain C-21, was cultured with 0.1 microM iodide, the maximum iodide content and the maximum concentration factor for iodide were 220 +/- 3.6 (mean +/- standard deviation) pmol of iodide per mg of dry cells and 5.5 x 10(3), respectively. In the presence of much higher concentrations of iodide (1 microM to 1 mM), increased iodide content but decreased concentration factor for iodide were observed. An iodide transport assay was carried out to monitor the uptake and accumulation of iodide in washed cell suspensions of iodide-accumulating bacteria. The uptake of iodide was observed only in the presence of glucose and showed substrate saturation kinetics, with an apparent affinity constant for transport and a maximum velocity of 0.073 muM and 0.55 pmol min(-1) mg of dry cells(-1), respectively. The other dominant species of iodine in terrestrial and marine environments, iodate (IO(3)(-)), was not transported.     

Degradation and biosynthesis of the glucose transporter protein in chicken embryo fibroblasts transformed by the src oncogene.

The rate of glucose transport in cultured fibroblasts is regulated to a number of physiological variables, including malignant transformation by src, glucose starvation, and stimulation with mitogens. Much of this transport regulation can be accounted for by variations in the amount of transporter protein in the cells. To determine the mechanisms by which levels of the transporter are regulated, we measured the rates of synthesis and degradation of the transporter by pulse-chase experiments and immunoprecipitation of the transporter. We found that transformation by the src oncogene results in a large decrease in the rate at which the transporter protein is degraded but that it does not appreciably increase the rate of transporter biosynthesis. On the other hand, glucose starvation and mitogen stimulation increase the rate of transporter biosynthesis, although a role for control of degradation is possible in these circumstances also. Variations in the rate of glucose transport or the amount of the transporter are not associated with phosphorylation of the transporter protein.     

Metabolite level from energy metabolism in Escherichia coli cells during growth on various substrates

In the present work, an attempt has been made to analyse some parameters of the intracellular energy metabolism and to determine whether they are related to the cell growth rate. The authors measured intracellular concentrations of adenine nucleotides, the content of glycogen, an energy "depot" of glucose, the rate of its synthesis, and the content of glutathione which specifies the redox state in the cytoplasm of E. coli cells upon continuous cultivation on a minimal nutrient medium containing various carbon sources (glucose, glycerol, lactate, and acetate). It was found that the specific contents of adenine nucleotides and reduced glutathione were independent of the cell growth rate. Based on the results obtained, it is assumed that a possible reason for changes in the duration of the sell cycle of the bacteria cultured on different substrates is an alteration in the content of reserve carbohydrates.