Development of novel carrier using natural zeolite and continuous ethanol fermentation with immobilized Saccharomyces cerevisiae in a bioreactor

A natural zeolite, easily vitrified and blown at 1300 °C with a high porosity and diam. of 5–100 m, was used to immobilize Saccharomyces cerevisiae at 3.6 × 10⁸ cells ml^–1 carrier. When the abilities of natural zeolite carrier were compared with glass beads, the capacity for immobilization and alcohol fermentation activity were, respectively, 2-fold higher and 1.2-fold higher than that of glass beads. Continuous alcohol fermentation was stable for over 21 d without breakage of the carrier.     

Mitogen activated protein kinases SakAHOG1 and MpkC collaborate for Aspergillus fumigatus virulence

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen‐activated protein kinases of the high‐osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild‐type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.     

Cloning and characterization of seven cDNAs for hyperosmolarity-responsive (HOR) genes of Saccharomyces cerevisiae

Yeast cells can respond and adapt to osmotic stress. In our attempt to clarify the molecular mechanisms of cellular responses to osmotic stress, we cloned seven cDNAs for hyperosmolarity-responsive (HOR) genes from Saccharomyces cerevisiae by a differential screening method. Structural analysis of the clones revealed that those designated HOR1, HORS, HOR4, HOR5 and HOR6 encoded glycerol-3-phosphate dehydrogenase (Gpd1p), glucokinase (Glklp), hexose transporter (Hxtlp), heat-shock protein 12 (Hsp12p) and Na⁺, K⁺, Li⁺-ATPase (Enalp), respectively. HOR2 and HOR7 corresponded to novel genes. Gpdlp is a key enzyme in the synthesis of glycerol, which is a major osmoprotectant in S. cerevisiae. Cloning of HOR1/GPD1 as a HOR gene indicates that the accumulation of glycerol in yeast cells under hyperosmotic stress is, at least in part, caused by an increase in the level of GPDH protein. We performed a series of Northern blot analyses using HOR cDNAs as probes and RNAs prepared from cells grown under various conditions and from various mutant cells. The results suggested that all the HOR genes are regulated by common signal transduction pathways. However, the fact that they exhibited certain distinct responses indicated that they might also be regulated by specific pathways in addition to the common pathways. Ca²⁺ seemed to be involved in the signaling systems. In addition, Hog1p, one of the MAP kinases in yeast, appeared to be involved in the regulation of expression of HOR genes, although its function seemed to be insufficient for the overall regulation of expression of these genes.     

中国脊髓灰质炎Ⅱ型疫苗相关分离株病毒性状的观察

对１９９４年中国分离的１３株脊髓灰质炎Ⅱ型疫苗相关株进行了ＰＣＲ－ＲＦＬＰ分析,发现７株为重组病毒,毒力较疫苗株有回复,在Ⅱ型脊髓灰质炎病毒基因序列上,对于神经毒力有重要影响的第４８１位核苷酸发生突变,另一个被视为重要位点的２９０８位核苷酸无一发生变化,反而在２９０９位核苷发生了高频率的点突变,意味着２９０９位点在中国Ⅱ型疫苗相关株的自然变异中可能起着重要作用。     

Improvement in thermostability and psychrophilicity of psychrophilic alanine racemase by site-directed mutagenesis

A psychrophilic alanine racemase from Bacillus psychrosaccharolyticus has a higher catalytic activity than a thermophilic alanine racemase from Bacillus stearothermophilus even at 60 °C in the presence of pyridoxal 5′-phosphate (PLP), although the thermostability of the former enzyme is lower than that of the latter one [FEMS Microbial. Lett. 192 (2000) 169]. In order to improve the thermostability of the psychrophilic enzyme, two hydrophilic amino acid residues (Glu150 and Arg151) at a surface loop surrounding the active site of the enzyme were substituted with the corresponding residues (Val and Ala) in the B. stearothermophilus alanine racemase. The mutant enzyme (ER150,151VA) showed a higher thermostability, and a markedly lower K_m value for PLP, than the wild type one. In addition, the catalytic activities at low temperatures and kinetic parameters of the two enzymes indicated that the mutant enzyme was more psychrophilic than the wild type one. Thus, the psychrophilic alanine racemase was improved in both psychrophilicity and thermostability by the site-directed mutagenesis. The mutant enzyme may be useful for the production of stereospecifically deuterated NADH and various -amino acids.     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.