The promoter of the beta-glucosidase gene from Kluyveromyces fragilis contains sequences that act as upstream repressing sequences in Saccharomyces cerevisiae

Summary The relationship between the promoter length of the Kluyveromyces fragilis -glucosidase gene and the level of its expression in Saccharomyces cerevisiae was studied by gene fusion between deleted promoter fragments of various lengths and the promoterless -galactosidase gene of Escherichia coli. The removal of a region from position-425 to-232 led to a tenfold increase in the expression of the gene. The same results were obtained for the reconstructed -glucosidase gene with the same promoter length. It is likely that the deletion of this part of the promoter removes negative regulatory elements which are functional in Saccharomyces cerevisiae. This increase in activity is the main event which may explain the high increase in gene expression (60-fold) previously observed for an upstream deletion obtained during subcloning experiments of the -glucosidase gene. It is also shown that the expression of the gene greatly depends upon the nature of the recipient strain, the growth phase of the cell and that of the vector carrying it.     

Transmembrane diffusion of hydrophobic antimicrobial agents and cell surface hydrophobicity in Bacteroides fragilis

The transmembrane diffusion of hydrophobic antimicrobial agents, e.g. lincomycin and clindamycin, was examined in Bacteroides fragilis which is sensitive to these agents. The results showed that these agents penetrate efficiently through the outer membrane. Cell surface hydrophobicity measured by the partition assay between water and p-xylene revealed that the cell surface of B. fragilis is more hydrophobic than that of Salmonella typhimurium or Pseudomonas aeruginosa. Furthermore, treatment with low concentrations of surfactant caused cell lysis. These results suggest that the cell surface hydrophobicity in B. fragilis plays an important role in the efficient transmembrane penetration of hydrophobic compounds. This efficiency explains the susceptibility of B. fragilis to hydrophobic antimicrobial agents.     

Behaviour of the yeast Kluyveromyces marxianus var. marxianus during its autolysis

The lactic yeast Kluyveromyces marxianus var.marxianus (formerly K. fragilis) autolyzates at faster rate than Saccharomyces cerevisiae. During K. marxianus autolysis, quite similar release kinetics were observed for intracellular space markers (potassium ions, nucleotides), cell-wall components (polysaccharides, N-acetyl-D-Glucosamine) and non specific products (amino nitrogen). By Scanning Electronic Microscopy examination, no cell burst was observed, but a variation in cell shape (from ellipsoidal to cylindrical), as well as a 43% decrease in the internal volume were observed. The mechanism proposed for S. cerevisiae autolysis appeared also likely for K. marxianus.Abbreviations NacGlc N-acetyl-D-glucosamine - x total biomass (dry cellular weight) concentration     

马克斯克鲁维酵母GX-UN120糖转运蛋白基因的克隆及表征

【背景】马克斯克鲁维酵母(Kluyveromyces marxianus)具有完整的木糖代谢途径,可以高效利用木质纤维素中的木糖,因此对其糖转运蛋白基因的研究或可有效解决酵母木糖转运的相关问题。【目的】根据马克斯克鲁维酵母DMKU3-1042中KLMA＿70145和KLMA＿80101基因位点的功能预测,获得马克斯克鲁维酵母GX-UN120相应的糖转运蛋白基因序列并探究其功能。【方法】将转运蛋白基因分别克隆表达至酿酒酵母EBY.VW4000中考察重组菌株生长特性,以此间接评价对应转运蛋白的转运能力。【结果】Km＿SUT2基因编码的糖转运蛋白可有效提高宿主细胞转运木糖、阿拉伯糖、山梨糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖、果糖、蔗糖和半乳糖。类似地,Km＿SUT3基因编码的糖转运蛋白可提高细胞转运木糖、阿拉伯糖、山梨糖、半乳糖、核糖、乳糖和葡萄糖的能力,但却不能转运甘露糖和果糖。然而在葡萄糖存在的条件下,重组菌株对各种碳源的利用均受抑制,但Km＿SUT3转运木糖和核糖过程中受葡萄糖的抑制作用较小。【结论】马克斯克鲁维酵母GX-UN120中转运蛋白Km＿SUT2和Km＿SUT3可...     

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30°C (generation time 1.1 h) or 20°C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.     

Cytosolic Ca2+-Concentrations and Distributions in Rhizoids of Chara fragilis Desv. Determined by Ratio Analysis of the Fluorescent Probe Indo-1

Rhizoids of Charafragilis Desv. were iontophoretically loaded with the Ca²⁺-sensitive ratio dye indo-1. After loading, the rhizoids regained their preinjection-membrane potential within 2 to 5 min and survived the procedure for more than 24 h, but their growth in length was permanently inhibited. Microfluorimetric measurements of the indo-1 fluorescence-ratio showed spontaneous fluctuations of the cytoplasmic Ca²⁺-concentration, usually declining from high values after loading to 425 ± 80 nM (± SD, n = 7) as determined by in-vitro calibration. Increasing the extracellular K⁺-concentration (0.1 mM to 10 mM) or Ca²⁺-concentration (1 mM to 10 mM) led to increases of 100 to 200 nM in cytoplasmic Ca²⁺-concentration. The spatial distribution of cytosolic Ca²⁺ in the rhizoid tips was visualised in ratio images computed from low-light video-pictures. These images showed a fairly homogeneous distribution of Ca²⁺ throughout the tip cytoplasm with concentrations being in the same range as determined by microfluorimetry. A tip-to-base gradient in cytoplasmic Ca²⁺, thought to be a prerequisite for cell polarity and tip growth, was found in only 1 out of 16 successfully microinjected cells. Additionally, a progressive compartmentalization of the fluorochrome indo-1, probably in the proplastids and the very abundant endoplasmic reticulum of the rhizoids, was observed.     

A unified stoichiometric model for oxidative and oxidoreductive growth of yeasts

Yeasts degrade glucose through different metabolic pathways, where the choice of the pathway is dependent on the nature of the limitation in the various substrates. When oxygen is limiting in addition to glucose, yeasts often grow according to a mixture of oxidative and reductive metabolism. Oxygen may be limiting either by supply or by inherent biological restrictions such as the respiratory bottleneck in Saccharomyces cerevisiae or by both. A unified model incorporating both supply and biological limitations is proposed for the quantitative prediction of growth rates, consumption and production rates, as well as key metabolite concentrations during mixed oxidoreductive metabolism occuring as a result of such oxygen limitations. This simple unstructured model can be applied to different yeast strains while at the same time requiring a minimum number of measured parameters. "Estimators" are utilized in order to predict the presence of supply-side or biological limitations. The values of these estimators also characterize the relative importance of oxidative to total metabolism. Results from the aerobic and oxygen-limited chemostat cultures were used to corroborate the model predictions. During these experiments, the heat released by the yeast cultures was also monitored on-line. The model correctly predicted the overall stoichiometry, steady-state concentrations, and rates including heat dissipation rates measured in the various situations of oxygen limitations. Direct continuous measurements such as heat can be used in conjunction with the unified model for on-line proces control. (c) 1992 John Wiley & Sons, Inc.     

脆弱拟杆菌BF839治疗寻常型银屑病：一项单臂、开放初步临床试验

银屑病被认为是一种T细胞主导的炎症性疾病,其发病与肠道菌群失调密切相关。脆弱拟杆菌 (Bacteroides fragilis,BF) 可通过调节T细胞的细胞因子表达起抗炎作用。目前尚无脆弱拟杆菌用于治疗银屑病的相关报道,文中率先探究脆弱拟杆菌BF839对银屑病的治疗效果。选择2019年4月至2019年10月广州医科大学附属第二医院就诊的27例银屑病患者,维持原治疗不变,口服脆弱拟杆菌BF839 12周,对比治疗前后银屑病皮损面积与严重程度指数 (Psoriasis area and severity index,PASI) 评分,统计治疗12周后药物减停率。结果表明,12周试验完成率为96.3% (26/27),12周PASI30 (PASIN定义为治疗后PASI评分下降≥N%的患者比例) 为65.4%,PASI50为42.3%,PASI75为19.2%;治疗前PASI评分为9.1±5.9,治疗12周后PASI评分为5.8±4.9,具有显著统计学差异 (P<0.01);治疗12周后皮肤瘙痒程度用视觉模拟量表 (Visual analog scale,VAS) 评分有效率为42.3%,治疗前VAS评分为2.9±2.2,治疗12周后VAS评分为2.3±2.1,无显著统计学差异 (P>0.05)。患者治疗12周内不良反应率为3.8% (1/26),其中便秘1例,药物减停率为60.0%。以上结果提示脆弱拟杆菌BF839可能对银屑病治疗有一定疗效,可降低PASI评分及药物使用率,不良反应少,值得进一步研究。