Green fluorescent protein as a novel indicator of antimicrobial susceptibility in Aureobasidium pullulans.

Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfish Aequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686-690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r(2) > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1 gfp blastospores (pH 5 buffer). These biocides each caused a rapid (< 25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 microg of available chlorine ml(-1) and 500 microg ml(-1), respectively. Further, loss of GFP fluorescence from A. pullulans cells was highly correlated with a decrease in the number of viable cells (r(2) > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulans was attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with > 95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.     

Biosynthesis of Novel Exopolymers by Aureobasidium pullulans

Aureobasidium pullulans ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 × 10⁶ to 2.12 × 10⁶ to 0.85 × 10⁶ to 0.77 × 10⁶ with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 ± 3 to 29 ± 2 mol%, and the molecular weight increased from 2.73 × 10⁶ to 4.86 × 10⁶. There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 ± 3:13 ± 3 to 28 ± 2:72 ± 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.     

Construction of a New Bacterial Cloning Vector Using a Mutant Green Fluorescent Protein as an Indicator

A new bacterial cloning vector, pGreenLD, derived from the triple substitution mutated Aequorea v/ctor/a green fluorescent protein(GFP-S65A, V68L, S72A), when expressed in E. coli produced colonies which showed yellow-green colour under daylight and strong green fluorescence under long-wave ultraviolet light. It can be a useful vector for selecting foreign DNA fragment which was inserted into multiple cloning site based on the loss of the yellow-green color/green fluorescence of E. coli cells attributable to the insertional inactivation of GFP production.     

Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Background

Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.

Methodology/Principal Findings

Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.

Conclusions and Significance

The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.     

绿色荧光蛋白

来源于水母Aequorea victoria的绿色荧光蛋白(green fluorescent protein, GFP)现已成为在生物化学和细胞生物学中研究和开发应用得最广泛的蛋白质之一. 其内源荧光基团在受到紫外光或蓝光激发时（λ_max=395 nm, 小峰在479 nm）可高效发射清晰可见的绿光. GFP的高分辨率晶体结构为了解和研究蛋白质结构和光谱学功能关系提供了一个极好的机会. GFP已成为一个监测在完整细胞和组织内基因表达和蛋白质定位的理想标记. 通过突变和蛋白质工程构建的GFP嵌合蛋白在生理指示剂、生物传感器、光化学领域以及生产发光纤维等方面展示了广阔前景.     

Aureobasidium pullulans as a biocontrol agent of postharvest pathogens of apples in Uruguay

Aureobasidium pullulans was the microorganism most frequently recovered from the surface of apple fruit (cv. Red Delicious) stored in commercial cold chambers for 6 months. In the present work, 10 isolates of Aureobasidium pullulans were assayed to determine if they could control blue and grey mold disease of apple during cold storage. Although nine of 10 isolates, significantly reduced the percentage of decayed wounds when compared to the control, one of them, designated isolate ApB, showed the highest levels of protection. ApB was able to grow in a wide range of temperatures lower than 35°C, which is an important human health safety factor. ApB was resistant to thiabendazole, iprodione and imazalil, the most commonly commercially applied fungicides in postharvest treatment of apples in Uruguay. Regarding the mechanisms of action of the selected biocontrol agent, lytic enzymes did not seem to play a central role. ApB depleted iron from nutrient media, which may be an important aspect of its ability to inhibit Botrytis cinerea. Further experiments are needed, however, to determine if the depletion of iron is caused by the production of siderophores, by the immobilization of iron in an insoluble pigment, or a combination of both.     

Production of extracellular polyols in Aureobasidium pullulans

Aureobasidium pullulans produced extracellularly considerable amounts of polyols in the media with sucrose, glucose, fructose and mannose as sole carbon source during the late exponential and stationary phase of growth. The maximum yield of polyol was about 23% in the 20%(w/v) sucrose medium, of which mannitol was the main polyol associated with minute quantities of glycerol. Stress solutes such as NaCl and KCl did not promote polyol production.     

The Electromotive Protein Prestin as a Sensitive Core of the Fluorescent Voltage Indicator

Russian Journal of Bioorganic Chemistry - In this study, we first used prestin, an electromotive protein of the mammalian auditory analyzer, as a voltage-sensitive core of a genetically encoded...     

β-Mannanolytic system of Aureobasidium pullulans

A xylanolytic yeast strain Aureobasidium pullulans NRRL Y 2311-1, was found to produce all enzymes required for complete degradation of galactomannan and galactoglucomannan. The enzymes differed in function and cellular localization: endo-β-1,4-mannanase was secreted into the culture fluid, β-mannosidase was strictly intracellular, and α-galactosidase and β-glucosidase were found both extracellularly and intracellularly. Among these enzyme components, only extracellular β-mannanase and intracellular β-mannosidase were inducible. The production of β-mannanase and β-mannosidase was 10- to 100-fold higher in galactomannan medium than in medium with one of the other carbon sources. β-mannanase and β-mannosidase were coinduced in glucose-grown cells by galactomannan, galactoglucomannan, and β-1,4-manno-oligosaccharides. The natural inducer of extracellular β-mannanase and intracellular β-mannosidase appeared to be β-1,4-mannobiose. Synthesis of both enzymes was completely repressed by glucose, mannose, or galactose. The synthetic glycoside methyl β-d-mannopyranoside served as a nonmetabolizable inducer of both β-mannosidase and β-mannanase. Received: 24 June 1996 / Accepted: 26 September 1996     

Exopolysaccharide biosynthesis by a fast-producing strain of Aureobasidium pullulans

Summary The mutant strain Aureobasidium pullulans ICCF-68 was able to produce in batch fermentation on a glucose medium of 80 g/l, exopolysaccharide at high volumetric productivity and final concentration (1.05 g/l.h and 50.2 g/l, respectively). A specific pH pattern and very high oxygen requirement were shown.     

绿色荧光蛋白——现代细胞生物学与分子生物学研究领域的新标记物

从多管水母属A equoren v ictu ria 分离出的绿色荧光蛋白(GFP) , 因其特有的生物化学性质及该基因在异源细胞内的表达产物亦能产生强烈的绿色荧光, 使其在现代细胞生物学和分子生物学研究领域的应用具有广阔前景。本文就其研究进展及其应用进行简要综述。     

Paint Deterioration as a Result of the Growth of Aureobasidium pullulans on Wood

Growth of Aureobasidium was noted on both painted and unpainted wood surfaces but not on painted glass slides, indicating that the source of carbon and energy for growth was not associated with paint components but was probably supplied by the wood. Several aromatic compounds related to aromatic extractives of wood were shown to support growth of this fungus.     

绿色荧光蛋白在蛋白质研究中的应用

绿色荧光蛋白（green fluorescent protein,GFP）自发现以来,由于具有自发荧光等特性,在分子生物学和细胞生物学领域得到广泛应用。GFP作为一种报道分子,在研究蛋白质相互作用和构象变化、检测蛋白质表达、蛋白质和细胞荧光示踪中,起到了重要的作用。该文通过对绿色荧光蛋白特性的分析．介绍其作为荧光标记在蛋白质研究中的应用,并展望进一步的研究前景。     

Fifty Aureobasidium pullulans genomes reveal a recombining polyextremotolerant generalist

The black yeast Aureobasidium pullulans is a textbook example of a generalistic and ubiquitous fungus thriving in a wide variety of environments. To investigate whether A. pullulans is a true generalist, or alternatively, whether part of its versatility can be attributed to intraspecific specialization masked by cryptic diversification undetectable by traditional phylogenetic analyses, we sequenced and analysed the genomes of 50 strains of A. pullulans from different habitats and geographic locations. No population structure was observed in the sequenced strains. Decay of linkage disequilibrium over shorter physical distances (<100 bp) than in many sexually reproducing fungi indicates a high level of recombination in the species. A homothallic mating locus was found in all of the sequenced genomes. Aureobasidium pullulans appears to have a homogeneous population genetics structure, which is best explained by good dispersal and high levels of recombination. This means that A. pullulans is a true generalist that can inhabit different habitats without substantial specialization to any of these habitats at the genomic level. Furthermore, in the future, the high level of A. pullulans recombination can be exploited for the identification of genomic loci that are involved in the many biotechnologically useful traits of this black yeast.     

Aureobasidium pullulans as a source of liamocins (heavy oils) with anticancer activity

Liamocins are structurally unique, heavier-than-water “oils” produced by certain strains of Aureobasidium pullulans. The aim of the current study is to identify new sources of liamocins and evaluate their potential as anticancer agents. Nine strains of A. pullulans from phylogenetic clades 8, 9, and 11 were examined for the first time for production of liamocins. Strains in these clades have only been isolated from tropical environments, and all strains tested here were from various locations in Thailand. Strains RSU 9, RSU 21, and RSU 29, all from clade 11, produced from 7.0 to 8.6 g liamocins/l from medium containing 5 % sucrose. These are the highest yields of liamocins that we have found thus far. These strains also produced from 9.4 to 17 g pullulan/l. The structural identity of liamocins was confirmed by matrix-assisted laser desorption/ionization mass spectrometry; differential spectra were obtained in which the dominant ion was either at about m/z 805.5 or m/z 949.6, consistent with the structure of liamocins. Liamocins from A. pullulans strains RSU 9 and RSU 21 inhibited two human breast cancer cell lines and a human cervical cancer cell line (IC₅₀ values of 32.2 ± 1.4 to 63.1 ± 2.4 μg liamocins/ml) but were not toxic to a normal cell line. Liamocins weakly inhibited a strain of Enterococcus faecalis, but did not inhibit strains of Lactobacillus fermentum, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Thus, A. pullulans phylogenetic clade 11 is a promising source of liamocins, and these compounds merit further examination as potential anticancer agents.     

Biosynthesis of novel exopolymers by Aureobasidium pullulans

Aureobasidium pullulans ATCC 42023 was cultured under aerobic conditions with glucose, mannose, and glucose analogs as energy sources. The exopolymer extracts produced under these conditions were composed of glucose and mannose. The molar ratio of glucose to mannose in the exopolymer extract and the molecular weight of the exopolymer varied depending on the energy source and culture time. The glucose content of exopolymer extracts formed with glucose and mannose as the carbon sources was between 91 and 87%. The molecular weight decreased from 3.5 x 10(6) to 2.12 x 10(6) to 0.85 x 10(6) to 0.77 x 10(6) with culture time. As the culture time increased, the glucose content of the exopolymer extract formed with glucosamine decreased from 55 +/- 3 to 29 +/- 2 mol%, and the molecular weight increased from 2.73 x 10(6) to 4.86 x 10(6). There was no evidence that glucosamine was directly incorporated into exopolymers. The molar ratios of glucose to mannose in exopolymer extracts ranged from 87 +/- 3:13 +/- 3 to 28 +/- 2:72 +/- 2 and were affected by the energy source added. On the basis of the results of an enzyme hydrolysis analysis of the exopolymer extracts and the compositional changes observed, mannose (a repeating unit) was substituted for glucose, which gave rise to a new family of exopolymer analogs.     

Aureobasidium pullulans can utilize simple aromatic compounds as a sole source of carbon in liquid culture

M.W. SCHOEMAN AND D.J. DICKINSON. 1996. Ferulic acid acted as an adequate sole carbon source for Aureobasidium pullulans isolate 105 in liquid culture. A corresponding depletion of ferulate, indicated by depression of the culture filtrate's absorbance at 280 nm, was observed over 4 weeks of incubation. Growth of isolate 105 and six other isolates of A. pullulans was also observed on three other aromatic compounds, catechol, protocatechuic acid and vanillic acid. However, none of the isolates grew on dioxane lignin. These results may contribute to a better understanding of the way in which A. pullulans colonizes painted wooden surfaces.