Trehalose accumulation in a high-trehalose-accumulating mutant of Saccharomycopsis fibuligera sdu does not respond to stress treatments

The isolation of high-trehalose-accumulating mutant A11 from Saccharomycopsis fibuligera sdu has been previously described. In this paper, accumulation of trehalose under various stress conditions in S. fibuligera A11 was investigated. Neither activation of trehalose-6-phosphate synthase (SfTps1) nor change in trehalose content was observed under stress exposure of S. fibuligera A11 cells. A fragment of the Sftps1 gene in this strain was also cloned by degenerate PCR using the CoDeHOP strategy and multiply-aligned Tps1 sequences. This sequence allowed us to investigate the expression of the Sftps1 gene, which was also kept constant under the various stress conditions. Altogether, these results indicate that trehalose metabolism in S. fibuligera A11 in response to stress conditions clearly differs from that of Saccharomyces cerevisiae and most other fungi. The expression of the Sftps1 gene was not responsive to different stress treatments.     

Increased Staphylococcus-killing activity of an antimicrobial peptide,lactoferricin B,with minocycline and monoacylglycerol

This study aimed to find antibiotics or other compounds that could increase the antimicrobial activity of an antimicrobial peptide, lactoferricin B (LFcin B), against Staphylococcus aureus, including antibiotic-resistant strains. Among conventional antibiotics, minocycline increased the bactericidal activity of LFcin B against S. aureus, but methicillin, ceftizoxime, and sulfamethoxazole-trimethoprim did not have such an effect. The combination of minocycline and LFcin B had synergistic effects against three antibiotic-resistant strains of S. aureus, according to result of checkerboard analysis. Screening of 33 compounds, including acids and salts, alcohols, amino acids, proteins and peptides, sugar, and lipids, showed that medium-chain monoacylglycerols increased the bactericidal activity of LFcin B against three S. aureus strains. The short-term killing test in water and the killing curve test in growing cultures showed that a combination of LFcin B and monolaurin (a monoacylglycerol with a 12-carbon acyl chain) killed S. aureus more rapidly than either agent alone. These findings may be helpful in the application of antimicrobial peptides in medical or other situations.     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

Preliminary evaluation of antimicrobial activity of diastereomeric cis/trans-3-aryl(heteroaryl)-3,4-dihydroisocoumarin-4-carboxylic acids

Preliminary differentiating screening of the antibacterial and antifungal activity of a series of diastereomeric cis/trans-3-aryl(heteroaryl)-3,4-dihydroisocoumarin-4-carboxylic acids (3a-i) was performed by the agar diffusion method against twelve microorganism strains of different taxonomic groups. S. aureus and A. niger were the most sensitive strains to the antibiotic effect of the tested compounds, both inhibited by 10 of 12 compounds. The most potent antibacterial agent was cis-3-phenyl-3,4-dihydroisocoumarin-4-carboxylic acid (cis-3a), exhibiting activity against all seven bacterial test strains.     

Two-stage continuous fermentation of Saccharomycopsis fibuligeria and Candida utilis

Biomass production and carbohydrate reduction were determined for a two-stage continuous fermentation process with a simulated potato processing waste feed. The amylolytic yeast Saccharomycopsis fibuligera was grown in the first stage and a mixed culture of S. fibuligera and Candida utilis was maintained in the second stage. All conditions for the first and second stages were fixed except the flow of medium to the second stage was varied. Maximum biomass production occurred at a second stage dilution rate, D(2), of 0.27 h (-1). Carbohydrate reduction was inversely proportional to D(2), between 0.10 and 0.35 h (-1).     

Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera

Lipomyces kononenkoae and Saccharomycopsis fibuligera possess highly efficient alpha-amylase and/or glucoamylase activities that enable both of these yeasts to utilize raw starch as a carbon source. Eight constructs containing the L. kononenkoae alpha-amylase genes (LKA1 and LKA2), and the S. fibuligera alpha-amylase (SFA1) and glucoamylase (SFG1) genes were prepared. The first set of constructs comprised four single gene cassettes each containing one of the individual amylase coding sequences (LKA1, LKA2, SFA1 or SFG1) under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator, while the second set comprised two single cassettes containing SFA1 and SFG1 linked to their respective native promoters and terminators. The third set of constructs consisted of two double-gene cassettes, one containing LKA1 plus LKA2 under the control of the PGK1 promoter and terminator, and the other SFA1 plus SFG1 controlled by their respective native promoters and terminators. These constructs were transformed into a laboratory strain Saccharomyces cerevisiae (Sigma1278b). Southern-blot analysis confirmed the stable integration of the different gene constructs into the S. cerevisiae genome and plate assays revealed amylolytic activity. The strain expressing LKA1 and LKA2 resulted in the highest levels of alpha-amylase activity in liquid media. This strain was also the most efficient at starch utilization in batch fermentations, utilizing 80% of the available starch and producing 0.61g/100 mL of ethanol after 6 days of fermentation. The strain expressing SFG1 under the control of the PGK1 expression cassette gave the highest levels of glucoamylase activity. It was shown that the co-expression of these heterologous alpha-amylase and glucoamylase genes enhance starch degradation additively in S. cerevisiae. This study has resulted in progress towards laying the foundation for the possible development of efficient starch-degrading S. cerevisiae strains that could eventually be used in consolidated bioprocessing, and in the brewing, whisky, and biofuel industries.     

具有a-淀粉酶分泌活性以及低双乙酰产量的啤酒酵母工程菌的构建

扣囊复膜酵母(Saccharomycopsis fibuligera)因具有较强的a-淀粉酶以及葡聚糖酶活性, 使其在以淀粉为唯一碳源的培养基上能够良好的生长。从其基因组中克隆了a-淀粉酶的编码区, 构建了由酵母磷酸甘油酸激酶基因(PGK1)启动子、酿酒酵母a-因子信号序列以及扣囊复膜酵母a-淀粉酶基因编码序列组成的基因表达盒。将该表达盒插入到质粒pPLZ-2的ILV2基因序列内部, 使其两翼具有ILV2基因的同源区。将该表达盒通过同源重组的方式整合到啤酒酵母工业菌株YSF-5的a-乙酰乳酸合成酶(AHAS)基因ILV2内部。在以淀粉为唯一碳源的培养基上进行转化子的筛选。通过多对引物PCR、a-淀粉酶活性以及AHAS活性分析对转化子进行鉴定, 得到一株具有a-淀粉酶分泌表达活性、较低AHAS活性, 并且发酵液中双乙酰产量也相对较低的啤酒酵母工程菌。该菌株在非选择压力条件下连续培养50代后仍然保持其遗传稳定性。还对pH、温度以及金属离子对该转化菌株的a-淀粉酶活性的影响进行了研究。由于所构建的菌株不含有非酵母来源的DNA, 所以生物安全性相对较高, 对酵母育种以及啤酒生产工业都具有较为重要的意义。     

Synthesis and beta-lactamase inhibitory activity of new 6beta-cysteinesulfonamidopenicillanic acids

New 6beta-cysteinesulfonamidopenicillanic acids and their sulfoxides were synthesized by sulfonylation of 6beta-aminopenicillanic acid or its (S)-sulfoxide with (R)-N-benzyloxycarbonylcysteinesulfonyl chloride ethyl ester (2a, 1b) and (R)-N-benzyloxycarbonylcysteinesulfonyl chloride benzyl ester (2a, 2b). The corresponding 6beta-cysteinesulfonamidopenicillanic acids sulfones 1c and 2c were prepared by oxidation of the sulfoxides 1b and 2b with potassium permanganate in aqueous medium. When combined with ampicillin some of the compounds reduced the minimal inhibitory concentrations of ampicillin against beta-lactamase producing strains.     

Salmonella vaccines secreting measles virus epitopes induce protective immune responses against measles virus encephalitis

In the present study we describe a live vaccine against measles virus (MV) infection on the basis of attenuated Salmonella typhimurium aroA secreting MV antigens via the Escherichia coli alpha-hemolysin secretion system. Two well-characterized MV epitopes, a B-cell epitope of the MV fusion protein (amino acids 404-414) and a T-cell epitope of the MV nucleocapsid protein (amino acids 79-99) were fused as single or repeating units to the C-terminal secretion signal of the E. coli hemolysin and expressed in secreted form by the attenuated S. typhimurium aroA SL7207. Immunization of MV-susceptible C3H mice revealed that S. typhimurium SL7207 secreting these antigens provoked a humoral and a cellular MV-specific immune response, respectively. Mice vaccinated orally with a combination of both recombinant S. typhimurium strains showed partial protection against a lethal MV encephalitis after intracerebral challenge with a rodent-adapted, neurotropic MV strain.     

Development of a screening method for the indentification of a novelSaccharomyces cerevisiae mutant over-expressingTrichoderma reesei cellobiohydrolase II

In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) fromTrichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme fromSaccharomycopsis fibuligera significantly hindered its expression and secretion inSaccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes inS. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) inS. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenisedS. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain ofS. cerevisie, WM91-CBGL1, which exhibited up to 200 U L^?1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with theT. resei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.     

Chemical and serological studies of liposaccharides of bacteria of the genus Azospirillum

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPS(Cd), LPS(Sp59b), LPS(Sp7), LPS(S17), and LPS(KBC1) preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, Ab(Sp59b) and Ab(Cd) were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.     

犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCV DXMV)纤突蛋白基因进行了克隆和序列测定.该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白.与GenBank中已发表的11个CCV毒株S基因相比,S基因核苷酸序列同源性在40.2%～99.5%之间;推导的氨基酸序列同源性在15.9%～99.0%之间.DXMV株S基因变异区主要集中在该基因前1/2处,其中350-370、439-478、1718-1818三个区域碱基变异较大,而1060-1700区却十分保守.基于S全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和US patent株亲源关系最近.推导的DXMV株S蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc-1弱毒多一个;其中第566-568位糖基化位点为多数强毒拥有而弱毒没有的.另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究.     

Comparison of the resistance of industrial and laboratory strains of Saccharomyces and Zygosaccharomyces to lignocellulose-derived fermentation inhibitors

Low-molecular weight aliphatic acids, furaldehydes and a broad range of different aromatic compounds are known to inhibit the fermentation of lignocellulose hydrolysates by yeasts. In this work, a cocktail of different lignocellulose-derived inhibitors was used to compare the inhibitor resistance of eleven different industrial and laboratory Saccharomyces cerevisiae strains and two Zygosaccharomyces strains. The inhibitor cocktail was composed of two aliphatic acids, formic and acetic acid, two furaldehydes, furfural and 5-hydroxymethylfurfural (HMF), and two aromatic compounds, cinnamic acid and coniferyl aldehyde. Fermentations were performed under oxygen-limited conditions and with different levels (100, 75, 50, 25 and 0%) of the inhibitor cocktail present. The ethanol yield on initial glucose, the volumetric and specific ethanol productivity, the biomass yield and the glucose consumption rates were used as criteria for the performance of the strains. The results revealed major differences in inhibitor resistance between yeast strains within the same species. The ethanol yield of the S. cerevisiae strain that was least affected decreased only with 10% at an inhibitor cocktail concentration of 100%, while the decrease in ethanol yield for the most sensitive S. cerevisiae strain was more than 50% already at an inhibitor cocktail concentration of 25%. Ethanol formation was generally less affected than growth and ethanol yield less than ethanol productivity. The two most resistant strains were an S. cerevisiae strain isolated from a spent sulphite liquor plant and one of the laboratory S. cerevisiae strains. Additional fermentations with either HMF or coniferyl aldehyde revealed that the degree of resistance of different yeast strains was highly dependent on the inhibitor used. A mutant strain of S. cerevisiae displaying enhanced resistance against coniferyl aldehyde compared with the parental strains was identified.     

The evaluation of lipolytic activity of strains of Staphylococcus epidermidis and Staphylococcus haemolyticus

A total of 103 isolates of CNS (66 strains of S. epidermidis and 37 strains of S. haemolyticus) were investigated. Lipolytic activity of staphylococcal strains was determined by Tryptic Soy Agar containing Tween 20 or Tween 60. The 95.4% strains of staphylococci demonstrated the lipolytic activity on Tween 20 agar and the 89.4% of strains of staphylococci degradation ester of fatty acids on Tweens 60 agar. We detected that S. epidermidis strains (respectively 95,4%, 89,4%) produced lipases more frequently than S. haemolyticus strains (respectively 72,9%, 59,4%). Studies suggest that source of isolation from clinical materials (blood, wound and pus) does not have an influence on the ability hydrolysis esters.     

Catabolite repression of amylase synthesis in yeast

Amylase synthesis by the yeasts Saccharomycopsis fibuligera and Schwanniomyces castellii and alluvius is repressed by glucose. Steady state continuous culture data for amylase activity, E, biomass concentration, X, and reducing sugar concentration, S, were fitted to the three-parameter catabolite repression model \documentclass{article}\pagestyle{empty}\begin{document}$ \frac{E}{X} = \frac{{[1 + a(S/X)]}}{{[1 + b(S/X)]}}, $\end{document} and biomass productivity, DX, and amylase productivity, DE, were determined for S. castellii and S. alluvius.     

Immunochemical analysis and possible biological role of an Aeromonas hydrophila surface array protein in septicaemia.

The biochemical, immunological, and biological properties of an S layer purified from an Aeromonas hydrophila strain (AH-342) involved in a case of bacteraemia were investigated. The S layer selectively removed from the cell surface was composed of a single acidic (pI 4.56) protein subunit (surface array protein, SAP) with a molecular mass of approximately 52 kDa. Amino acid analysis of this 52 kDa protein indicated a molecule composed of 498 amino acids with 46% hydrophobic residues. No cysteine residues were detected. The first 35 residues of the N-terminus were sequenced by Edman degradation; only 4-24% homology was noted between this sequence and those previously published for SAPs of Aeromonas salmonicida (A450) and a strain of A. hydrophila (TF7) originally isolated from a moribund fish. Polyclonal antibodies raised against AH-342 SAP were genospecific, reacting only against S layers produced by A. hydrophila strains and not those from Aeromonas veronii. Acute serum from the bacteraemic patient from whom AH-342 was isolated reacted strongly with the SAP of AH-342 in immunoblot studies. Purified SAP, when intraperitoneally co-inoculated with SAP- strains of A. hydrophila into Swiss-Webster mice, could reduce the 50% lethal dose by approximately 30-70 fold. The results suggest that the SAP of A. hydrophila strains may play an important role in systemic dissemination after invasion through the gastrointestinal mucosa.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.