Nutritional requirements and strain heterogeneity in Ashbya gossypii

Colony radial growth rates and specific growth rates of three related Ashbya gossypii strains ATCC10895, IMI31268, MUCL29450 and an unrelated strain, CBS109.26, were measured on various carbon and nitrogen sources at pH 4.5 and pH 6.5 to elucidate physiological growth requirements and strain differences. All strains grew on yeast extract or ammonium as nitrogen sources, but not on nitrate. Substantial growth at pH 4.5 was observed only on complex medium. D‐Glucose, glycerol and starch were utilised as carbon sources. Ethanol was produced during growth on glycerol. Conversion of xylose into xylitol demonstrates that the xylose reductase is active. Phenotypic differences between related strains were greater than expected. We demonstrate that A. gossypii utilizes ammonium as sole nitrogen source at pH 6.5, facilitating further physiological studies using chemically defined media in the future. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of acetic acid on xylose fermentation to xylitol by Candida guilliermondii

The effect of acetic acid concentration on xylose fermentation to xylitol by Candida guilliermondii FTI 20037 was evaluated in semisynthetic medium containing different concentrations of the acid. Increasing acetic acid concentration up to 1.0 g/1 favored xylitol yield and productivity, with maximum values of 0.82 g/g and 0.57 g/.h1, respectively. The presence of acetic acid reduced cell production at all concentration. Furthermore, acetic acid was assimilated by the yeast together with the sugars and was depleted from the medium at concentrations of less than 3.0 g/1. The ability of this yeast to assimilate acetic acid suggests that these cells act as agents of medium detoxification. This behavior may lead to a viable microbiological process of xylitol production by C. guilliermondii FTI 20037 using xylose-rich lignocel-lulosic hydrolysates in which acetic acid is commonly present, causing inhibition of fermentative activity.     

Isolation and characterization of xyl mutants in a xylose-utilizing yeast,Pichia stipitis

Summary Mutants of the xylose-utilizing yeast, Pichia stipitis, unable to grow on xylose as the sole carbon source were isolated and characterized. The mutants were deficient in either xylose reductase or xylitol dehydrogenase. By immunological means and upon analysis of revertants, both mutant types could be attributed to the structural genes XYL1 and XYL2, which code for xylose reductase and xylitol dehydrogenase, respectively. These data support previous assumptions that both NADH- and NADPH-dependent xylose reductase activity are due to one protein or gene, respectively. Revertant analysis of xyl1 mutants has revealed the existence of a second xylose reductase gene in P. stipitis. This gene is very likely cryptic. Its corresponding xylose reductase activity is NADPH-dependent.     

Characterization of a respiratory-deficient mutant of Pachysolen tannophilus

Summary A pleiotropic, respiration-deficient mutant was isolated from the petite negative yeast Pachysolen tannophilus after UV mutagenesis. The mutant is unable to utilize xylose, arabinose, galactose or glycerol, and shows no detectable respiration when grown on glucose. Cytochrome c oxidase, xylose reductase and xylitol dehydrogenase activities are lacking. Mitochondrial ultrastructre is altered. The results support the hypothesis that functioning mitochondria are necessary for xylose utilization in this organism.     

Biorhythms of activities of liver and blood dehydrogenases and changes in body weight of the rats feeding normal diet or excess of sugar substitutes

Biorhythms with higher levels of activity of sorbitol dehydrogenase and lactate dehydrogenase in blood plasma, specific activity of sorbitol dehydrogenase, lactate dehydrogenase, and malate dehydrogenase in the liver, and body weight of rats were more pronounced in the spring-summer period than in the autumn-winter period. These specific features were revealed in animals feeding a normal diet or food with 54 and 27% sugar substitute sorbitol. However, specific activity of glucose-6-phosphate dehydrogenase in the liver was higher in the autumn-winter period. Activity of sorbitol dehydrogenase in blood plasma increased by tens of times due to induction of sorbitol synthesis (substrate) in the liver. Sugar substitute xylitol is structurally similar to sorbitol, but is not the substrate for sorbitol dehydrogenase. However, the effect of xylitol on activities of lactate dehydrogenase, malate dehydrogenase, and glucose-6-phosphate dehydrogenase in the spring-summer period was similar to that of sorbitol. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 12, pp. 686–689, December, 2007     

Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene,XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant

Summary A P. stipitis cDNA library in gt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.     

Adaptation and reutilization of Candida guilliermondii cells for xylitol production in bagasse hydrolysate

The xylitol productivity increased by about 15% with the use of cells of Candida guilliermondii FTI 20037 previously recycled through four consecutive batch cultures and adapted to the sugar cane bagasse hemicellulosic hydrolysate. Furthermore, the more concentrated the hydrolysate, the more necessary was the adaptation of the cells, owing to the presence of toxic substances at high concentration which inhibited the xylose-xylitol conversion by the yeast.     

Effect of xylitol and other carbon sources on Streptococcus pneumoniae biofilm formation and gene expression in vitro

Xylitol inhibits the growth of Streptococcus pneumoniae. In clinical trials, xylitol decreased the occurrence of acute otitis media in day-care children, but did not decrease nasopharyngeal carriage of pneumococci. We hypothesized that xylitol inhibits biofilm formation of pneumococci, and measured biofilm formation and gene expression levels of the capsule gene cpsB and two other genes: autolysin encoding gene lytA and competence gene comA in different growth media in vitro. Twenty pneumococcal isolates were grown on polystyrene plates for 18 h in test media containing 0.5% xylitol, 0.5% glucose, 0.5% xylitol and 0.5% glucose, 0.5% fructose, 0.5% xylitol and 0.5% fructose or brain heart infusion (BHI) medium supplemented with 10% horse serum. Gene expression levels were measured after 5 h of growth using a relative quantification method with calibrator normalization. Exposure to xylitol lowered OD values, which were used as an indication of biofilm, compared with BHI medium, but when the medium was supplemented with glucose or fructose, biofilm formation was enhanced and the inhibitory effect of xylitol on biofilm formation was not observed. Xylitol also lowered lytA expression levels. Changes in biofilm formation in response to different sugar compounds may partly explain the efficacy of xylitol to prevent acute otitis media in previous clinical trials.     

Xylitol Concentrations in the Saliva of Children After Chewing Xylitol Gum or Consuming a Xylitol Mixture

Xylitol prevents otitis media when given to children regularly five times per day. To find a more convenient dosing schedule, an enzymatic assay was used to measure xylitol concentrations in the saliva of 65 children after giving them xylitol chewing gum or syrup in doses equal to those used in clinical trials. Although concentrations high enough to have an antimicrobial effect were attained, the xylitol disappeared from the saliva within 15 min. This finding indicates that high peak concentrations are more important for efficacy than the amount of time the xylitol concentration exceeds that needed to produce an antimicrobial effect. A schedule with the same single doses given less frequently could be clinically effective in preventing otitis media. Electronic Publication     

Improving Oral Health and Modulating the Oral Microbiome to Reduce Bloodstream Infections from Oral Organisms in Pediatric and Young Adult Hematopoietic Stem Cell Transplantation Recipients: A Randomized Controlled Trial

Bloodstream infections (BSIs) from oral organisms are a significant cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT) recipients. There are no proven strategies to decrease BSIs from oral organisms. The aim of this study was to evaluate the impact of daily xylitol wipes in improving oral health, decreasing BSI from oral organisms, and modulating the oral microbiome in pediatric HSCT recipients. This was a single-center 1:1 randomized controlled trial in pediatric HSCT recipients age >2 years. Age-matched healthy children were enrolled to compare the oral microbiome. The oral hygiene standard of care (SOC) group continued to receive the standard oral hygiene regimen. The xylitol group received daily oral xylitol wipes (with .7 g xylitol) in addition to the SOC. The intervention started from the beginning of the transplantation chemotherapy regimen and extended to 28 days following transplantation. The primary outcome was oral health at interval time points, and secondary outcomes included BSIs from oral organisms in the first 30 days following transplantation, oral microbiome abundance, and diversity and oral pathogenic organism abundance. The study was closed early due to efficacy after an interim analysis of the first 30 HSCT recipients was performed (SOC group, n = 16; xylitol group, n = 14). The xylitol group had a significantly lower rate of gingivitis at days 7, 14, and 28 following transplantation (P = .031, .0039, and .0005, respectively); oral plaque at days 7 and 14 (P = .045 and .0023, respectively); and oral ulcers >10 mm at day 14 (P = .049) compared with the SOC group. The xylitol group had no BSI from oral organisms compared with the SOC group, which had 4 (P = .04). The xylitol group had significantly lower abundance of potential BSI pathogens, such as Staphylococcus aureus (P = .036), Klebsiella pneumoniae (P = .033), and Streptococcus spp (P = .011) at the day after transplantation compared with the SOC group. Healthy children and young adults had significantly increased oral microbiome diversity compared with all HSCT recipients (P < .001). The addition of xylitol to standard oral care significantly improves oral health, decreases BSI from oral organisms, and decreases the abundance of pathogenic oral organisms in pediatric and young adult HSCT recipients.     

Zur Frage einer Wirkung von Xylit auf die Insulinsekretion des Menschen

Zusammenfassung Bei zehn freiwilligen Verauchspersonen wurde das Verhalten von Seruminsulin, Glucose, freien Fettsäuren und Serumphosphat nach Xylitinfusion mit 0,8 g/kg Körpergewicht untersucht. Das leichte Ansteigen des Seruminsulins direkt nach Beendigung der Infusion ist statistisch nicht abzusichern; der tierexperimentell von anderen Autoren erhobene Befund, daß Xylit die Insulinsekretion steigert, konnte demnach nicht bestätigt werden. Die Blutglucosewerte werden anfänglich kaum beeinflußt und liegen erst am Ende des Beobachtungszeitraumes (120 min nach Beendigung der Xylitinfusion) signifikant unterhalb der Werte vor Infusionsbeginn. Serumphosphat und freie Fettsäuren fallen nach Xylitinjektion rasch ab.

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Summary The behaviour of serum insulin, glucose, free fatty acids and phosphate was studied in ten volunteers after infusion of 0,8 g/kg body weight of xylitol. A slight increase in serum insulin immediately after termination of the infusion was not statistically significant. Therefore, the findings of other authors in animals, namely that xylitol elicits an increase in insulin secretion, were not confirmed. The blood glucose level was at first hardly influenced and only at the end of the observation period (120 min after termination of the xylitol infusion) were the levels significantly lower than the original values. Serum phosphate and free fatty acids were considerably reduced after the xylitol infusion.

     

Lactatbildung aus Zuckern und Zuckeralkoholen in Erythrocyten

Zusammenfassung Erythrocyten können Lactat bilden aus Glucose, Fructose, Sorbit, Xylit und Ribit. Mannit wird nicht umgesetzt. Die Kombinationen Xylit + Glucose, Xylit + Fructose und Sorbit + Glucose ergeben additive Lactatbildung. Bei den Kombinationen Xylit + Sorbit, Xylit + Ribit und Glucose + Fructose addiert sich die Lactatbildung nicht.Über Konsequenzen dieser Befunde für die parenterale Therapie wird diskutiert.

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Summary There is a production of lactic acid in erythrocytes from glucose, fructose, sorbitol, xylitol, and ribitol. Mannitol has no effect. Combinations of xylitol + glucose, xylitol + fructose, and sorbitol + glucose lead to an additive formation of lactic acid, whereas combinations of xylitol + sorbitol, xylitol + ribitol, and glucose + fructose have no additive effects. On the bearing of these data for the problem of parenteral energy supply is discussed.

     

Adrenal function of the rat in relation to peroral administration of xylitol: depression of aldosterone

The effect of peroral administration of xylitol (5% or 20% in food) on adrenal function was investigated in thirty-five Long-Evans male rats. The control rats were fed either a non-substituted stock diet or a 20% glucose diet. Glucose elevated and 20% xylitol reduced the growth of the rats (P less than 0.001), but 5% xylitol had no effect on the body weight. The concentrations of serum glucose and lactic acid decreased in rats fed 20% and 5% xylitol, respectively, but those of insulin, glucagon, corticosterone and aldosterone were not affected. In the adrenal glands, 20% xylitol loading was associated with increased epinephrine (P less than 0.05) and norepinephrine (P less than 0.001), but with decreased aldosterone (P less than 0.001) concentrations. The weights and histological picture of adrenal glands were normal. The urinary pH of xylitol-fed rats decreased significantly (P less than 0.01). Although peroral xylitol affected the levels of aldosterone and catecholamines, a normal glucocorticoid metabolism was permitted. The reduced aldosterone levels were regarded as secondary reactions, possibly resulting from alterations in electrolyte and/or acid-base balance. The increased catecholamine synthesis may be associated with the promoting effect of xylitol on intestinal calcium absorption.     

Xylose reductase and xylitol dehydrogenase activities of D-xylose-xylitol-fermenting Candida guilliermondii

The effect of the inoculum age and initial xylose concentration on the activities of xylose reductase (XR) and xylitol dehydrogenase (XD) of Candida guilliermondii FTI 20037 were investigated. Under the experimental conditions used, the XR and XD activities were, respectively. NADPH and NAD or NADP dependent. The NADP/NADPH ratio for XR was not substantially influenced by the age of inoculum or the initial xylose concentration. The K_m of XR (0.18 M) was about 4-folds smaller than the K_m of XD NAD-dependent. A renewable xylose-xylitol conversion was obtained with 40 h aged inoculum of Candida guilliermondii FTI 20037 grown in a medium containing an initial xylose concentration of 120g/l.     

Molecular investigations on the dilemma of nitrogen-fixing characteristics of Azotomonas

The ability of Azotomonas to reduce atmospheric nitrogen has been an issue of controversy since this genus was included in the family Azotobacteraceae. Here we report the investigations on the nitrogen fixation characteristics of Azotomonas fluorescens strain ATCC13544 and show that it can grow in both liquid medium and on solid agar plates made with modified BURK nitrogen-free medium supplemented with ammonium acetate as a nitrogen source. In contrast, when ammonium acetate was omitted from this medium, the cells did not show any noticeable growth. Southern blotting analysis of the chromosome of Azotomonas strain ATCC13544 did not show any hybridization with a probe made of DNA sequences corresponding to the nifH gene of Azotobacter vinelandii. Based on these results, we conclude that the Azotomonas fluorescens ATCC13544 does not contain the conventional genetic information on its chromosome that is needed to utilize atmospheric nitrogen as the sole source nitrogen to support its growth.     

Change in gene abundance in the nitrogen biogeochemical cycle with temperature and nitrogen addition in Antarctic soils

The microbial community (bacterial, archaeal, and fungi) and eight genes involved in the nitrogen biogeochemical cycle (nifH, nitrogen fixation; bacterial and archaeal amoA, ammonia oxidation; narG, nitrate reduction; nirS, nirK, nitrite reduction; norB, nitric oxide reduction; and nosZ, nitrous oxide reduction) were quantitatively assessed in this study, via real-time PCR with DNA extracted from three Antarctic soils. Interestingly, AOB amoA was found to be more abundant than AOA amoA in Antarctic soils. The results of microcosm studies revealed that the fungal and archaeal communities were diminished in response to warming temperatures (10 °C) and that the archaeal community was less sensitive to nitrogen addition, which suggests that those two communities are well-adapted to colder temperatures. AOA amoA and norB genes were reduced with warming temperatures. The abundance of only the nifH and nirK genes increased with both warming and the addition of nitrogen. NirS-type denitrifying bacteria outnumbered NirK-type denitrifiers regardless of the treatment used. Interestingly, dramatic increases in both NirS and NirK-types denitrifiers were observed with nitrogen addition. NirK types increase with warming, but NirS-type denitrifiers tend to be less sensitive to warming. Our findings indicated that the Antarctic microbial nitrogen cycle could be dramatically altered by temperature and nitrogen, and that warming may be detrimental to the ammonia-oxidizing archaeal community. To the best of our knowledge, this is the first report to investigate genes associated with each process of the nitrogen biogeochemical cycle in an Antarctic terrestrial soil environment.     

Stimulatory effects of Azotobacter chroococum exudates in culture and soil on its nitrogen fixation

The effect of the exudates of Azotobacter chroococcum in nitrogen-free liquid medium and in sterile soil, on its ability to fix nitrogen was studied. Cell-free culture filtrates and water extract of sterile soil inoculated with Azotobacter were fractionated into ether soluble and insoluble aliquots, concentrated and chromatographically separated into ten consecutive zones. These were eluted and tested for their effect on nitrogen fixation by Azotobacter. UV-fluorescence, and chemical tests for indoles, reducing substances, amino acids, and hydroxyl groups were made. Detection of gibberellins and water soluble vitamins was attempted. A biological test for activity of indolic compounds was carried out. The results showed that Azotobacter produces biologically active indolic compounds in culture and soil, acting as a significant stimulation for the process of nitrogen fixation. Not all the zones of the chromatogram which were stimulatory for nitrogen fixation were the same for coleoptile growth and vice versa. Amino acids in some cases were associated with non-significant stimulation of nitrogen fixation. An indication was obtained suggesting the possible stimulation of nitrogen fixation by gibberellins produced in culture and soil by Azotobacter. Suggestion was given for studying the effect of individual gib berellic compounds on the nitrogen fixation process, with a view to their interaction with amino acids and indolic compounds.