Membrane-mediated killing of Saccharomyces cerevisiae by glycoproteins from Torulopsis glabrata.

Cell-free supernatants from cultures of Torulopsis glabrata contained glycoprotein toxins that killed sensitive and killer strains of Saccharomyces cerevisiae with single-hit kinetics. Growing S. cerevisiae treated with the toxins showed a leakage of cellular potassium, partial dissipation of the adenosine triphosphate pool, and a coordinate shutdown of macromolecular synthesis. These pool efflux-stimulating toxins have been partially purified and at least three toxic glycoproteins have been separated. Pool efflux-stimulating toxin activity was stable from pH 3 through 7, though killing was maximal close to pH 4.     

Biosynthesis of the Naturally Occurring Nucleoside Antibiotics from Adenosine

Abstract

9-β-D-Arabinofuranosyladenine (ara-A, l), first isolated from the culture filtrates of StreDtomyces antibioticus, has a broad spectrum of activity against DNA viruses in cell culture and is successfully used in therapy of herpes simplex encephalitis, neonatal herpes, herpes zoster and chronic myelogenous leukemia¹. 2′-Chlorodeoxycoformycin (2′CldCF, 2), 2′-amino-Zt-deoxyadenosine (3) and nucleocidin (4) have been isolated from the culture medium of Fctinomadura and S. clavus, respectively.     

Diagnostic and clinical characteristics of mycotic vaginitis caused by Candida glabrata (Torulopsis glabrata)

In this study an evaluation of frequency of occurrence of C. glabrata, its diagnosis, sensitivity to antifungal drugs and its significance in pathogenesis of mycotic vaginosis was performed. Strains belonging to C. glabrata genus constituted 12.1% of total of 852 isolated strains and 39.2% of strains other than C. albicans. During fungal vaginosis caused by C. glabrata Lactobacillus sp. was present and normal pH values of vaginal secretion were seen. In direct preparations single or few leukocytes were observed and usually numerous blastospores were present. During evaluation of the sensitivity of C. glabrata strains to antimycotic agents a decreased sensitivity of these strains to clotrimazole and ketoconazole was found what speaks for their low usefulness in the treatment of mycotic vaginosis. Significance of C. glabrata in pathogenesis of mycotic vaginosis is not questioned since release of complaints and clinical symptoms in patients with positive therapeutic effect is seen and their persistence in a group of patients with treatment failure.     

Increasing glycolytic flux in Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation

AIMS: This study aimed at further increasing the pyruvate productivity of a multi-vitamin auxotrophic yeast Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation. METHODS AND RESULTS: We examined two strategies to decrease the activity of F0F1-ATPase. The strategies were to inhibit F0F1-ATPase activity by addition of oligomycin, or to disrupt F0F1-ATPase by screening neomycin-resistant mutant. The addition of 0.05 mmol l(-1) oligomycin to the culture broth of T. glabrata CCTCC M202019 resulted in a significantly decreased intracellular ATP level (35.7%) and a significantly increased glucose consumption rate (49.7%). A neomycin-resistant mutant N07 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the F0F1-ATPase activity of the mutant N07 decreased about 65%. As a consequence, intracellular ATP level of the mutant N07 decreased by 24%, which resulted in a decreased growth rate and growth yield. As expected, glucose consumption rate and pyruvate productivity of the mutant N07 increased by 34% and 42.9%, respectively. Consistently, the activities of key glycolytic enzymes of the mutant N07, including phosphofructokinase, pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase, increased by 63.7%, 28.8% and 14.4%, respectively. In addition, activities of the key enzymes involved in electron transfer chain of the mutant N07 also increased. CONCLUSIONS: Impaired oxidative phosphorylation in T. glabrata leads to a decreased intracellular ATP production, thereby increasing the glycolytic flux. SIGNIFICANCE AND IMPACT OF THE STUDY: The strategy of redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation provides an alternative approach to enhance the glycolytic flux in eukaryotic micro-organisms.     

Significant increase of glycolytic flux in Torulopsis glabrata by inhibition of oxidative phosphorylation

This study was aimed at increasing the glycolytic flux of the multivitamin-auxotrophic yeast Torulopsis glabrata by disturbing oxidative phosphorylation. We examined two different strategies to impede oxidative phosphorylation. The first strategy was disruption of the activity of the electron transfer chain (ETC), by either of two approaches. One was separately adding, at 10 mg L1, specific inhibitors of complex I (rotenone) or of the bc1 complex (antimycin A) to the culture broth of T. glabrata CCTCC M202019, which resulted in significantly decreased intracellular ATP levels (43% and 27.7%) and significantly increased rates of glucose consumption (qs) and pyruvate production (qp); another approach was breeding a respiratory-deficient mutant RD-16, in which cytochromes aa3 and b in the ETC were deleted after ethidium bromide mutagenesis, to reduce the ETC activity constitutively. The second strategy was inhibiting F0F1-ATP synthase with 0.05 mM oligomycin. Also, a neomycin-resistant mutant with 65% decreased F0F1-ATPase activity was studied. With the two strategies, the specific activity of phosphofructokinase (R2=0.9971), the average specific glucose consumption rate (R2=0.9967) and the average specific pyruvate production rate (R2=0.965) were closely correlated with the intracellular ATP level, all of them being increased at a lower intracellular ATP level.     

var1 Gene on the mitochondrial genome of Torulopsis glabrata

We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene (var1Sc). An open reading frame that could encode a protein of 339 amino acids was found with 72.7% amino acid and 85.3% nucleotide sequence homology to the S. cerevisiae var1 gene. The T. glabrata gene (var1Tg) is transcribed yielding two stable RNAs, a more abundant 13.5 S RNA and a less abundant 18 S species. We have also identified a candidate for a T. glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria. The var1Tg gene is even more A + T-rich (93%) than var1Sc (89.6%) and has conserved the strong codon bias of var1Sc. Major differences between the two sequences were found. Significant among these are that no GC clusters are found in var1Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1Sc have deletions at the corresponding sites in var1Tg. These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S. cerevisiae mitochondrial DNA.     

Redistribution of carbon flux in Torulopsis glabrata by altering vitamin and calcium level

Manipulation of cofactor (thiamine, biotin and Ca(2+)) levels as a potential tool to redistribute carbon flux was studied in Torulopsis glabrata. With sub-optimization of vitamin in fermentation medium, the carbon flux was blocked at the key node of pyruvate, and 69 g/L pyruvate was accumulated. Increasing the concentrations of thiamine and biotin could selectively open the valve of carbon flux from pyruvate to pyruvate dehydrogenase complex, the pyruvate carboxylase (PC) pathway and the channel into the TCA cycle, leading to the over-production of alpha-ketoglutarate. In addition, the activity of PC was enhanced with Ca(2+) present in fermentation medium. By combining high concentration's vitamins and CaCO(3) as the pH buffer, a batch culture was conducted in a 7-L fermentor, with the pyruvate concentration decreased to 21.8 g/L while alpha-ketoglutarate concentration increased to 43.7 g/L. Our study indicated that the metabolic flux could be redistributed to overproduce desired metabolites with manipulating the cofactor levels. Furthermore, the manipulation of vitamin level provided an alternative tool to realize metabolic engineering goals.     

Effect of nitrogen source and nitrogen concentration on the production of pyruvate by Torulopsis glabrata

The effect of nitrogen sources including yeast extract, peptone, soybean hydrolyzate and some inorganic nitrogen sources, as well as the nitrogen concentration on the fermentative production of pyruvate by Torulopsis glabrata WSH-IP12 was investigated. The addition of yeast extract greatly inhibited pyruvate accumulation, while peptone was shown to be the most favorable nitrogen source. In flask culture, 15 g l(-1) peptone was needed to consume 80 g l(-1) glucose with 23.4 g l(-1)of pyruvate accumulated. Pyruvate production was markedly dependent on the ratio of carbon to nitrogen (C:N), its production was improved by increasing the concentration of glucose and peptone proportionally and reduced by exclusively increasing the glucose concentration. In a glucose fed-batch culture, cell growth and pyruvate production slowed after 28 h. However, cell growth and pyruvate production recovered after further nitrogen, in the form of peptone and ammonium sulfate, was added to the culture. A final concentration of pyruvate of 54.5 g l(-1) was achieved at 64 h (yield to glucose consumed of 0.471 g g(-l)). By using aqueous ammonia instead of potassium hydroxide for pH control, 57.3 g l(-1) pyruvate with a yield of 0.498 g g(-1) was produced by 55 h. This result further indicates that nitrogen level plays an important role in the production of pyruvate.     

Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy

The effect of agitation speeds on the performance of producing pyruvate by a multi-vitamin auxotrophic yeast, Torulopsis glabrata, was investigated in batch fermentation. High pyruvate yield on glucose (0.797 g g(-1)) was achieved under high agitation speed (700 rpm), but the glucose consumption rate was rather low (1.14 g l(-1) h(-1)). Glucose consumption was enhanced under low agitation speed (500 rpm), but the pyruvate yield on glucose decreased to 0.483 g g(-1). Glycerol production was observed under low agitation speed and decreased with increasing agitation speed. Based on process analysis and carbon flux distribution calculation, a two-stage oxygen supply control strategy was proposed, in which the agitation speed was controlled at 700 rpm in the first 16 h and then switched to 500 rpm. This was experimentally proven to be successful. Relatively high concentration of pyruvate (69.4 g l(-1)), high pyruvate yield on glucose (0.636 g g(-1)), and high glucose consumption rate (1.95 g l(-1)h(-1)) were achieved by applying this strategy. The productivity (1.24 g l(-1) h(-1)) was improved by 36%, 23% and 31%, respectively, compared with fermentations in which agitation speeds were kept constant at 700 rpm, 600 rpm, and 500 rpm. Experimental results indicate that the difference between the performances for producing pyruvate under a favorable state of oxygen supply (dissolved oxygen concentration >50%) was caused by the different regeneration pathways of NADH generated from glycolysis.     

Production of water-soluble surface-active exolipids by Torulopsis apicola

Summary Several Torulopsis yeasts were screened for production of extracellular surface-active compounds. One strain, Torulopsis apicola IMET 43747, was studied in greater detail. Both on nalkanes and on carbohydrates it produced a mixture of water-soluble biosurfactants with remarkable interfacial activities and surface-tension values around 30 mN m^-1 and interfacial tension below 1 mN m^-1. Most of the biosurfactants are produced in the late exponential and in the early stationary growth phase. Production was increased by using hydrophobic compounds as the carbon source. The yields on n-alkanes were influenced by the concentrations of both the carbon source and the yeast extract. The effects of one purified biosurfactant on microbial growth on nalkanes and its antibacterial and antiphagal activities reveal new physiological aspects of biosurfactant generation by T. apicola.     

New Techniques for the Estimation of Naturally Occurring Brassinosteroids

We have developed enzyme-linked immunosorbent assays (ELISAs) for measuring 24-epicastasterone and related brassinolide analogs, with detection ranges of 0.005 to 50 pmoles. Polyclonal antibodies used in these assays were raised against 24-epicastasterone carboxymethyloxime-bovine serum albumin conjugates and were found to have high specificity for 24-epibrassinosteroids. Natural brassinosteroids (BRs), such as brassinolide and 24-epibrassinolide, exhibited relatively high cross-reactivities with the generated antibodies, whereas other BR analogs with β-oriented hydroxyl groups at C-2, C-3, C-22, and C23 lacked immunoreactivity. Through the use of internal standardization, dilution assays, recovery of authentic [³H]24-epicastasterone, and immunohistograms, the ELISAs have been shown to be applicable for estimating 24-epibrassinosteroid levels in crude plant extracts. To analyze brassinosteroids in tissues from young bean (Phaseolus vulgaris L., cv. Pinto), Daucus carota ssp.sativus plants and Arabidopsis thaliana L. Heynh. seedlings, and rape (Brassica napus L.) pollen, the extracts were fractionated by high performance liquid chromatography (HPLC) and the resulting fractions were analyzed by the ELISA method. Immunohistogram ELISA analysis of HPLC fractions indicated that major peaks of immunoreactivity co-chromatographed with the labeled and unlabeled 24-epibrassinolide. A highly sensitive electrospray ionization mass spectrometry (MS) technique (LOD: 50 fmol) was also developed and the results obtained by the HPLC-ELISA and HPLC-MS approaches were compared.     

Purification, characterization, cloning and expression of pyruvate decarboxylase from Torulopsis glabrata IFO005

In the production of pyruvate and optically active alpha-hydroxy ketones by Torulopsis glabrata, pyruvate decarboxylase (PDC, EC 4.1.1.1) plays an important role in pyruvate metabolism and in catalyzing the biotransformation of aromatic amino acid precursors to alpha-hydroxy ketones. In this paper, we have purified and characterized PDC from T. glabrata IFO005 and cloned the corresponding gene. A simple, rapid and efficient purification protocol was developed that provided PDC with high specific activity. Unlike other yeast or higher plant enzymes, known as homotetramers (alpha(4) or beta(4)) or heterotetramers (alpha(2)beta(2)), two active isoforms of PDC purified from T. glabrata IFO005 were homodimeric proteins with subunits of 58.7 kDa. We isolated the T. glabrata PDC gene encoding 563 amino acid residues and succeeded in overproducing the recombinant PDC protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract. Recombinant PDC from E. coli was purified as a homotetramer. Targeted gene disruption of PDC confirmed that T. glabrata has only one gene of PDC. This PDC gene showed about 80% homology with the genes of other yeasts, and amino acid residues involved in the allosteric site for pyruvate in other yeast PDCs were conserved in T. glabrata PDC. Both native PDC and recombinant PDC were activated by pyruvate and exhibited sigmoidal kinetics similar to those of Saccharomyces cerevisiae and higher plants. They also exhibited the similar catalytic properties: low thermostability, similar pH stability and optimal pH, and complete inhibition by glyoxylate.     

Repression of Hybrid Dysgenesis in Drosophila Melanogaster by Individual Naturally Occurring P Elements

Individual P elements that were genetically isolated from wild-type strains were tested for their abilities to repress two aspects of hybrid dysgenesis: gonadal dysgenesis and mutability of a double-P element-insertion allele of the singed locus (sn(w)). These elements were also characterized by Southern blotting, polymerase chain reaction amplification and DNA sequencing. Three of the elements were 1.1-kb KP elements, one was a 1.2-kb element called D50, and one was a 0.5-kb element called SP. These three types of elements could encode polypeptides of 207, 204, and 14 amino acids, respectively. Gonadal dysgenesis was repressed by two of the KP elements (denoted KP(1) and KP(6)) and by SP, but not by the third KP element (KP(D)), nor by D50. Repression of gonadal dysgenesis was mediated by a maternal effect, or by a combination of zygotic and maternal effects generated by the P elements themselves. The mutability of sn(w) was repressed by the KP(1) and KP(6) elements, by D50 and by SP, but not by KP(D); however, the SP element repressed sn(w) mutability only when the transposase came from complete P elements and the D50 element repressed it only when the transposase came from the modified P element known as Δ2-3. In all cases, repression of sn(w) mutability appeared to be mediated by a zygotic effect of the isolated P element. Each of the isolated elements was also tested for its ability to suppress the phenotype of a P-insertion mutation of the vestigial locus (vg(21-3)). D50 was a moderate suppressor whereas SP and the three KP elements had little or no effect. These results indicate that each isolated P element had its own profile of repression and suppression abilities. It is suggested that these abilities may be mediated by P-encoded polypeptides or by antisense P RNAs initiated from external genomic promoters.