Hydroxamic Acid from Histoplasma capsulatum That Displays Growth Factor Activity

Growth factor(s) present in a spent liquid medium after culture of the yeast form of Histoplasma capsulatum enhanced both yeast and mycelial growth of nine isolates tested. Hydroxamic acid extracted from the culture fluid displayed growth factor activity.     

Factors affecting membrane fouling in filtration of Saccharomyces cerevisiae in an internal ceramic filter bioreactor

Filtration of ethanol fermentation medium and broth by using symmetric and asymmetric ceramic membranes has been studied in an internal filter bioreactor. Factors studied included membrane structure and pore size, medium sterilization, and concentrations of glucose, yeast extract in the medium, yeast cell and protein in broth. The aim was to determine the main factors responsible for the decline in filtration performance during ethanol fermentation by Saccharomyces cerevisiae. Flux index (Fi) of a new concept has been developed to evaluate the degree of flux decline during the membrane fouling process. Fi was defined as the ratio of the membrane flux at certain filtration time (t?=?t) to the initial (t?=??0) flux of pure water, not the initial (t?=?+0) flux of the test fluid. Flux with sterilized medium was approximately two-fold higher than that with unsterilized medium although the reason could not be explained clearly. Glucose, interaction between glucose and yeast extract, yeast cells, and proteins in fermentation broth were found to play an important part in membrane fouling. Fi of the symmetric membrane decreased to a less extent than that of the asymmetric membrane with increasing glucose concentration. But, the result with various yeast cell concentrations turned out to be contrary. Fouling was more serious for asymmetric membrane during the filtration of fermentation supernatant. This was thought to be due to different fouling mechanisms for the two types of membrane.     

Secreted pitfall-trap fluid of carnivorous Nepenthes plants is unsuitable for microbial growth

Background and Aims

Carnivorous plants of the genus Nepenthes possess modified leaves that form pitfall traps in order to capture prey, mainly arthropods, to make additional nutrients available for the plant. These pitchers contain a digestive fluid due to the presence of hydrolytic enzymes. In this study, the composition of the digestive fluid was further analysed with regard to mineral nutrients and low molecular-weight compounds. A potential contribution of microbes to the composition of pitcher fluid was investigated.

Methods

Fluids from closed pitchers were harvested and analysed for mineral nutrients using analytical techniques based on ion-chromatography and inductively coupled plasma–optical emission spectroscopy. Secondary metabolites were identified by a combination of LC-MS and NMR. The presence of bacteria in the pitcher fluid was investigated by PCR of 16S-rRNA genes. Growth analyses of bacteria and yeast were performed in vitro with harvested pitcher fluid and in vivo within pitchers with injected microbes.

Key Results

The pitcher fluid from closed pitchers was found to be primarily an approx. 25-mm KCl solution, which is free of bacteria and unsuitable for microbial growth probably due to the lack of essential mineral nutrients such as phosphate and inorganic nitrogen. The fluid also contained antimicrobial naphthoquinones, plumbagin and 7-methyl-juglone, and defensive proteins such as the thaumatin-like protein. Challenging with bacteria or yeast caused bactericide as well as fungistatic properties in the fluid. Our results reveal that Nepenthes pitcher fluids represent a dynamic system that is able to react to the presence of microbes.

Conclusions

The secreted liquid of closed and freshly opened Nepenthes pitchers is exclusively plant-derived. It is unsuitable to serve as an environment for microbial growth. Thus, Nepenthes plants can avoid and control, at least to some extent, the microbial colonization of their pitfall traps and, thereby, reduce the need to vie with microbes for the prey-derived nutrients.     

Problemas clínicos en micología médica: problema número 49

The case of a 59-year-old female born in Buenos Aires (Argentina) is presented. She had been diagnosed with HIV in 2007 and received highly active antiretroviral therapy until 2011; she also suffered from diabetes type 2. She had received empirical treatment (pyrimethamine-clindamycin) for cerebral toxoplasmosis. Fifteen days later she suffered a drug-induced skin disorder and was treated in the Dermatology Service of the Hospital Muñiz with corticosteroids. After five weeks she was readmitted to the Infectious Disease Unit due to asthenia, weight loss, left hip pain and weakness in all four limbs. Septic arthritis and aseptic hip necrosis were ruled out. Blood cultures were positive for Staphylococcus aureus and Escherichia coli. The patient received intravenous antibiotics, but before being discharged Acinetobacter baumannii was isolated from blood, catheter and urine cultures, and a new series of antibiotics were prescribed. On the 3rd day she presented encephalic facies, changes of behaviour and disorientation, without nuchal rigidity, Kernig and Brudzinski signs or focal signs. An X-ray computed tomography did not show parenchymal lesions. A yeast identified as Candida albicans was isolated in a cerebrospinal fluid culture. The same yeast was recovered in a new cerebrospinal fluid sample. The isolate was susceptible to amphotericin B and susceptible dose dependent to fluconazole. The patient was treated with amphotericin B (0.7 mg/kg plus 800 mg fluconazole daily). Three weeks later, new cerebrospinal fluid cultures were negative. Unfortunately, the patient died soon afterwards.     

Observations on cellular immunity and parasitism in the tussock moth

Cellular responses to the introduction of foreign objects into the haemocoele of both control and parasitized tussock moth larvae were examined. In normal larvae, the response to large foreign objects such as Hyposoter fugitivus eggs and Sephadex beads was encapsulation, accompanied by a rapid and sustained increase in the total haemocyte count. Smaller objects such as yeast cells were cleared into nodules within a matter of minutes; nodulation too was accompanied by an increased total haemocyte count.In larvae parasitized by the braconid wasp Cotesia melanoscela, both encapsulation and nodulation were permanently suppressed. Inhibition of these normal cellular defence reactions was accompanied by a reduction in the total haemocyte count, the appearance of debris in the haemocoele, and by nuclear pycnosis in an unidentified population of cells; however, since extensive nuclear pycnosis also occurred in haemopoietic tissue, it is assumed that prohaemocytes may represent a target cell population. All of the observed effects required the presence of both C. melanoscela calyx fluid and venom in the host animal. Phagocytosis as an immune response remained essentially intact, and was capable of completely clearing both yeast and Escherichia coli cells injected into the haemocoele.     

Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127-6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1? deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1? mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.     

Grinding Microorganisms with a Peristaltic Pump

The Randolph Co. model 610 peristaltic, ⅛-hp pump was effective for preparative purposes in disrupting baker's yeast and spores of Bacillus globigii when suspended with glass beads. Best results were obtained with use of a slurry just fluid enough to flow through tubing while stirred. Beads of 0.2 and 0.1 mm were used to best advantage for the yeast cells and spores, respectively. Yeast cells were disrupted completely within 15 min, and the spores in 10 to 30 min. Temperature and surface denaturation are readily controlled, and the system is easily modified for use with large quantities of microorganisms.     

A Comparison of the Beneficial Effects of Live and Heat-Inactivated Baker’s Yeast on Nile Tilapia: Suggestions on the Role and Function of the Secretory Metabolites Released from the Yeast

Yeast is frequently used as a probiotic in aquaculture with the potential to substitute for antibiotics. In this study, the involvement and extent to which the viability of yeast cells and thus the secretory metabolites released from the yeast contribute to effects of baker’s yeast was investigated in Nile tilapia. No yeast, live yeast or heat-inactivated baker’s yeast were added to basal diets high in fishmeal and low in soybean (diet A) or low in fishmeal and high in soybean (diet B), which were fed to fish for 8 weeks. Growth, feed utilization, gut microvilli morphology, and expressions of hsp70 and inflammation-related cytokines in the intestine and head kidney were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Gut alkaline phosphatase (AKP) activity was measured after challenging the fish with Aeromonas hydrophila. Results showed that live yeast significantly improved FBW and WG (P < 0.05), and tended to improve FCR (P = 0.06) of fish compared to the control (no yeast). No significant differences were observed between inactivated yeast and control. Live yeast improved gut microvilli length (P < 0.001) and density (P < 0.05) while inactivated yeast did not. The hsp70 expression level in both the intestine and head kidney of fish was significantly reduced by live yeast (P < 0.05) but not inactivated yeast. Live yeast but not inactivated yeast reduced intestinal expression of tnfα (P < 0.05), tgfβ (P < 0.05 under diet A) and il1β (P = 0.08). Intestinal Lactococcus spp. numbers were enriched by both live and inactivated yeast. Lastly, both live and inactivated yeast reduced the gut AKP activity compared to the control (P < 0.001), indicating protection of the host against infection by A. hydrophila. In conclusion, secretory metabolites did not play major roles in the growth promotion and disease protection effects of yeast. Nevertheless, secretory metabolites were the major contributing factor towards improved gut microvilli morphology, relieved stress status, and reduced intestinal inflammation of Nile tilapia fed diets supplemented with baker’s yeast.     

Expression of a thermostable cellulase gene from a thermophilic anaerobe in Saccharomyces cerevisiae

A cellulase gene from a thermophilic anaerobe was recloned in the yeast Saccharomyces cerevisiae. The maximum level of the gene expression in the recombinant yeast was 4.4 times higher than that in the Escherichia coli transformant harboring the same plasmid. Cellulase activity was observed only within the yeast cells. To compare the enzymatic properties of cellulase produced by the yeast and E. coli transformants, cellulases were purified to homogeneous state by only three purification steps of heat treatment, and cellulose affinity and ion exchange chromatographies. The molecular weights of the enzymes produced by the yeast and E. coli were 3.8 × 10⁴ and 4.0 × 10⁴, respectively by SDS-polyacrylamide gel electrophoresis. Neither of the enzymes was glycosylated. Although the molecular weights were slightly different, enzymatic properties and thermostability were almost indistinguishable between the enzymes produced by the yeast and E. coli transformants.     

Amino acid incorporation on plant ribosomes: The transfer system

Pre-formed Vicia faba phenylalanyl-tRNA was active in a TYMV-RNA-directed Transfer System, whereas a similar tRNA preparation from yeast was not. Thus, lack of charging of yeast tRNA by enzymes from Phaseolus was not the only reason why yeast tRNA would not function in this Transfer System. In the poly U-directed Transfer System; where both types of tRNA were active, the pH and ionic parameters governing the reaction with yeast tRNA were more stringent.     

In vitro assembly of minicellulosomes with two scaffoldins on the yeast cell surface for cellulose saccharification and bioethanol production

The present paper reports in vitro strategies for assembly of minicellulosomes with two miniscaffoldins on the Saccharomyces cerevisiae cell surface. It was carried out through incubation of the yeast cells displaying scaffoldins with Escherichia coli lysates containing recombinant cellulases, or using a four-population yeast consortium. The results showed that the display level of miniscaffoldin II was distinctly increased by moving the cellulases production into E. coli or other yeast cells, indicating that the metabolic burden of the yeast host was decreased. The yeast consortium did not show any cellulolytic activity, while the E. coli lysates-treated yeast, whose anchoring miniscaffoldin length was optimized, was able to produce ～1138 mg/L ethanol from microcrystalline cellulose within 4 days. We also confirmed that the yeast-associated minicellulosome moreover showed both higher thermal stability and lower protease accessibility than free minicellulosome. This research promotes the application of S. cerevisiae as a consolidated bioprocessing (CBP) microorganism in cellulosic bioethanol production.     

Genetic transformation of the yeast Rhodotorula gracilis ATCC 26217 by electroporation

We examined the genetic transformation of the biotechnologically relevant yeast Rhodotorula gracilis ATCC 26217 by electroporation. To evaluate the yeast transformation, we created a genomic integration cassette that was targeted to the yeast orotidine-5′-phosphate decarboxylase gene (URA3 gene) locus and composed of the zeocin-resistant gene (Sh ble gene) with the URA3 promoter and terminator of the yeast. The yeast was unable to grow on medium containing 2.0 μg/mL zeocin, even with the inoculation of a large number of cells (approximately 1.0 × 10⁸ cells/plate). Using the integrative cassette and zeocin-containing medium, we successfully obtained yeast transformants by electroporation, and the highest transformation efficiency of approximately 40 colony-forming units/μg DNA was obtained with a 0.6-kV electrical pulse. No homologous integration of the cassette at the URA3 gene locus was detected by the analyses of uracil auxotrophy and genomic PCR of transformants, suggesting that this method is a useful tool for randomly mutating the yeast genome.     

N-glycosylation deficiency enhanced heterologous production of a Bacillus licheniformis thermostable α-amylase in Saccharomyces cerevisiae

Expression of foreign enzymes in yeast is a traditional genetic engineering approach; however, useful secretory enzymes are not produced in every case. The hyperthermostable α-amylase encoded by the AmyL gene of Bacillus licheniformis was expressed in Saccharomyces cerevisiae; however, it was only weakly produced and was degraded by the proteasome. To determine the cause of low α-amylase production, AmyL was expressed in a panel of yeast mutants harboring knockouts in non-essential genes. Elevated AmyL production was observed in 44 mutants. The knockout genes were classified into six functional categories. Remarkably, all non-essential genes required for N-linked oligosaccharide synthesis and a gene encoding an oligosaccharyl transferase subunit were identified. Immunoblotting demonstrated that differently underglycosylated forms of AmyL were secreted from oligosaccharide synthesis-deficient mutants, while a fully glycosylated form was produced by wild-type yeast, suggesting that N-linked glycosylation of AmyL inhibited its secretion in yeast. Mutational analysis of six potential N-glycosylation sites in AmyL revealed that the N33Q and N309Q mutations remarkably affected AmyL production. To achieve higher AmyL production in yeast, all six N-glycosylation sites of AmyL were mutated. In wild-type yeast, production of the resulting non-glycosylated form of AmyL was threefold higher than that of the glycosylated form.     

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel⁻. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel⁰ baker's yeast strain. The Mel⁺ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel⁺ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel⁺ strain and may prove useful for commercial production of α-galactosidase.     

Elicitation of sanguinarine production in two-phase cultures of Eschscholtzia californica

Large increases in sanguinarine production were observed in suspension cultured Eschscholtzia californica cells treated with elicitors prepared from yeast extract, Colletotrichum lindemuthianum and Verticillum dahliae. Compounded silicone fluid, an ideal accumulation phase for the two-phase culture of E. californica, accumulated a large amount of sanguinarine produced in a specific manner and increased sanguinarine production. Elicitation in combination with two-phase culture additionally increased net sanguinarine production, as well as the sanguinarine concentration in the accumulation phase.     

Ribosomal RNA from the yeast and mycelial phases ofHistoplasma capsulatum

Purified yeast and mycelial phase rRNA fromHistoplasma capsulatum was heat-formaldehyde deantured and analyzed by polyacrylamide gel electrophoresis. An average difference of 6.9% was seen in electrophoretic migration between the yeast and mycelial phase denatured 17 S rRNAs that was not apparent for the native 17 S rRNAs. This difference was statistically significant and could not be accounted for by random variation among gels electrophoresed in parallel. A significant difference was not found between the 25 S rRNAs of yeast and mycelial phase cells. Molecular weights of 1.28 × 10⁶ for both the yeast and mycelial phase 25 S rRNA, and 0.79 × 10⁶ and 0.73 × 10⁶ for the yeast and mycelial phase 17 S rRNA, were estimated by using denaturing conditions which minimize conformational contributions to electrophoretic migration in polyacrylamide gels. In addition, we find that both the yeast and mycelial phase 17 S and 25 S rRNAs ofH. capsulatum are smaller than their respective counterparts inS. cerevisiae. The results indicate that a structural difference may exist, at least between the 40 S subunits ofH. capsulatum yeast and mycelial phase ribosomes.     

Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast

Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker’s yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified as N-(E)-p-coumaroyl-3-hydroxyanthranilic acid by NMR. This compound is an amide condensation product of p-coumaric acid, which was supplied to the yeast, with 3-hydroxyanthranilic acid, which was unexpectedly recruited from the yeast metabolism by the HCT enzyme. N-(E)-p-coumaroyl-3-hydroxyanthranilic acid has not been described before, and it shows structural similarity to avenanthramides, a group of inflammation-inhibiting compounds present in oat. When applied to mouse fibroblasts, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid induced a reduction of intracellular reactive oxygen species, indicating a potential therapeutic value for this novel compound.