Screening and breeding of high taxol producing fungi by genome shuffling

To apply the fundamental principles of genome shuffling in breeding of taxol-producing fungi, Nodulisporium sylviform was used as starting strain in this work. The procedures of protoplast fusion and genome shuffling were studied. Three hereditarily stable strains with high taxol production were obtained by four cycles of genome shuffling. The qualitative and quantitative analysis of taxol produced was confirmed using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and LC-MS. A high taxol producing fungus, Nodulisporium sylviform F4-26, was obtained, which produced 516.37 μg/L taxol. This value is 64.41% higher than that of the starting strain NCEU-1 and 31.52%―44.72% higher than that of the parent strains.     

Production, Purification and Characterization of Alkaline β-Glucosidase Isolated from Agrobacterium sp. PJD-1-1

In order to screen novel β-glucosidase producing strains from environment, one targeted novel strain PJD-1-1 producing β-glucosidase were isolated from putrefied sugarcane leaves with screening and spreading plate. 16S rDNA analysis revealed it was a novel Agrobacterium sp. When the strain was incubated at initial pH 7.0, 20 ℃ with lactose as carbon and NaNO₃ as nitrogen sources, the maximum enzyme activity was 3.92 U/mg. β-glucosidase from this strain was purified using (NH₄)₂SO₄ precipitation followed by dextran gel filtration chromatography and ion exchange chromatography. A purifying fold of 4.85 with gaining rate of 8.0% was obtained. SDA-PAGE analysis of the purified enzyme showed that it was a clear and pure band with molecular mass of ca. 40 kDa. The most optimum activity of the enzyme was at 50 ℃ and pH at 8.0. The enzyme could maintain stability under the conditions below 50 ℃. Hg²⁺ and Ag⁺ heavily inhibited the enzyme activity suggesting that the active catalytic sites of the enzymes might possess thiol radical. Ba²⁺, Ca²⁺, Pb²⁺, Co²⁺, Zn²⁺, Mn²⁺, Na⁺, K⁺, EDTA, and urea had no obvious effects on the enzyme activity. It is concluded that the novel strain Agrobacterium sp. PJD-1-1 producing β-glucosidase was successfully screened from putrefied sugar cane leaves. The produced enzyme had thermal stability, alkaline feature and metal ions tolerance made it useful in the food and broad potential applications in other fields.     

嗜杀酿酒酵母毒素蛋白及其杀伤质粒的研究

Killer toxin from \%Saccharomyces cerevisiae\% SK was isolated by ultrafiltration of culture supernatants and purified by poly(ethylene glycol). The toxin migrates as one single protein band on SDS-PAGE and its molecular weight is 15kD. The SK toxin has the greatest lethal effect on the sensitive yeast strain in the lat lag phase. Extraction and purification of killer heretity factor(dsRNA) from SK found that M dsRNA plasmid and L dsRNA plasmid have different molecular lengths being 1.7kb and 4.0kb.     

Construction of a trivalent candidate vaccine against Shigella species with DNA recombination

In this work asd gene of Shigella flexneri 2a strain T32 was replaced by Vibrio cholerae toxin B subunit (ctxB) gene with DNA recombination in vivo and in vitro. The resulting derivative of T32, designed as FWL01, could stably express CtxB, but its growth in LB medium depended on the presence of diaminopimelic acid (DAP). Then form I plasmid of Shigella sonnei strain S7 was labeled with strain T32 asd gene and mobilized into FWL01. Thus a trivalent candidate oral vaccine strain, designed as FSW01, was constructed. In this candidate strain, a balanced-lethal system was constituted between the host strain and the form I plasmid expressing S, sonnei O antigen. Therefore the candidate strain can express stably not only its own O antigen but also CtxB and O antigen of S. sonnei in the absence of any antibiotic. Experiments showed that FSW01 did not invade HeLa cells or cause keratoconjunctivitis in guinea pigs. However, rabbits immunized FSW01 can elicit significant immune responses. In mice and rhesus monkey     

Antibacterial, antifungal and cytotoxic activity of terrestrial cyanobacterial strains from Serbia

Cyanobacteria are known to be a rich source of biologically active compounds some of which can have pharmaceutical importance. In this work we present the screening results of cyanobacterial strains for their antibacterial, antifungal, and cytotoxic activity. Cyanobacterial strains were isolated from various soil types in province of Vojvodina and Central Serbia, Republic of Serbia. The screening included 9 strains of Anabaena and 9 strains of Nostoc. Both, extracellular products (from the culture liquid) and cellular crude lipophilic extracts were tested against 13 bacterial strains and 8 fungal strains. Cytotoxic activity was tested against three human cell lines. Methanol extracts were prepared according to ?stensvik. Antibacterial and antifungal activities were determined measuring inhibition zone, 48 h after inoculation. The cytotoxic activity was determined by sulforhodamine B (SRB) colorimetric assay. Of all cyanobacterial strains tested, 52% showed some antifungal and 41% antibacterial activity. Two out of six tested strains possessed cytotoxic activity. The cytotoxic activity of Anabaena strain S12 was found both in culture liquid and crude cell extract. It occurred specifically between the 21st and 42nd day of cultivation against HeLa and MCF7 cells, but had no activity against cell line derived from a healthy tissue. A high percentage of the active strains among the tested strains justify the effort of screening cyanobacteria that are isolated from terrestrial environments. The most promising strains for the fur- ther study are Anabaena strain S12 which showed strong cytotoxic and antibacterial activity and Ana- baena strain S20 which produces a potent antifungal compound. The future work, besides further screening and chemical identification of the active compounds, should also include the development of culture techniques that would lead to more efficient production of biologically active compounds.     

Bt Research(BT)

<正>Bt Research(ISSN 1925-1939)is an open access,peer reviewed journal published online by BioP ublisher.The Journal is publishing high quality original research on all aspects of Bacillus thuringiensis and their toxins affecting the living organisms,as well as environmental risk and public policy relevant to Bt modified organisms.Topics include(but are not limited to)Bt strain identification,novel Bt toxin discovery and bioassay,transgenic Bt plants,insecticidal mechanism of Bt toxin as well as     

Bt Research(BT)

<正>Bt Research(ISSN 1925-1939)is an open access,peer reviewed journal published online by Bio Publisher.The Journal is publishing high quality original research on all aspects of Bacillus thuringiensis and their toxins affecting the living organisms,as well as environmental risk and public policy relevant to Bt modified organisms.Topics include(but are not limited to)Bt strain identification,novel Bt toxin discovery and bioassay,transgenic Bt plants,insecticidal mechanism of Bt toxin as well as     

Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

Construction of a trivalent candidate Shigella vaccine strain with host-vector balanced-lethal system

A trivalent live Shigella vaccine candidate FSD01 against S. flexneri 2a, S. sonnei and S. dysen-teriae I was constructed. This candidate strain was based on the S. flexneri 2a vaccine T32. By homologous recombi-nation exchange, the chromosomal asd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while another asd gene of S. mutans was employed to construct an Asd complementary vector. This combination of asd 'host/ Asd vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S. sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the     

Identification and characterization of Acidithiobacillus ferrooxidans with high activity and resistance isolated from ancient mine area

A highly active ferrous iron oxidation Acidithiobacillus ferrooxidans strain SY, was isolated from an ancient copper mining area in Daye, Hubei Province. Analysis of 16S rDNA sequence showed that the strain has high similarity to the sequence of A. ferrooxidans (DQ 062116.1). Physiological and biochemical determinations showed that the strain was a chemical energy autotrophically with the optimal growth pH at 2.0 and optimal growth temperature at 30 ℃. The MTC of the strain to resist Cu²⁺, Cd²⁺, Ni²⁺, and Zn²⁺ were respectively at 300, 350, 700, and 800 (mmol/L), demonstrated it has high resistance against multiple heavy metal ions. Bioleaching data of SY showed its bioleaching rate on native ore was as high as 84.28%, higher than bioleaching rate on the ore from other mining areas, showing it has very high superiority on bioleaching native ore. The strain SY has great potential in the application of native minerals bioleaching.     

Penicillic Acid Production by Blue-Eye Fungi on Various Agricultural Commodities

Of 10 Penicillium species reported to cause blue-eye disease of corn, four (P. martensii, P. palitans, P. cyclopium, P. puberulum) were found capable of producing the mycotoxin penicillic acid on various agricultural commodities. Commodities with high protein contents did not support toxin synthesis. The extent of toxin production varied with the strain of mold, the commodity, and the temperature; low temperatures (1 to 10 C) favored toxin accumulation.     

低能氮离子注入诱变选育高产结冷胶菌株的研究

结冷胶是由伊乐假单胞杆菌生成的具有重要商业价值的微生物胞外多糖。本研究为了提高伊乐假单孢菌的产结冷胶水平,采用低能氮离子注入的方法选育高产菌株。经过初筛和复筛得到一株高产结冷胶菌株Pseudomonas elodea A3-5。该菌株传代稳定,生长迅速,在7 L发酵罐内生长状态优于出发菌株,分子量和品质都较高,分批发酵结冷胶产量达到16.5g/L,较出发菌株明显提高了25%。     

海水中DHA产生菌的筛选及一株高产菌的鉴定

从海水中筛选产DHA的微生物,共采集海水样品280余份,用苏丹黑染色法得到160株产油脂菌株,在对60株脂肪粒较大的微生物用索氏抽提法提取油脂后,初筛得到油脂含量高于8%的菌株7株。对10株油脂产量较高的菌株进行复筛,编号7-3的菌株油脂含量达到15.9%,DHA在油脂中的含量达到45.2%,选用7-3作为目的菌株。对7-3进行形态特征、培养特征及生理生化特征鉴定,初步判定菌株7-3为酒香酵母属(Brettanomyces sp.)。     

一种产邻苯二酚菌株高通量筛选方法

目的：对产邻苯二酚菌株进行筛选。方法：采用前期筛得的产邻苯二酚菌为出发菌株,通过硫酸二乙酯诱变的方法使该菌株突变,同时建立96孔板培养和酶标仪检测方法对产邻苯二酚菌株进行高通量筛选。结果：硫酸二乙酯的终浓度为0.1%,诱变时间为8 min的条件下,突变菌致死率为84.5%,突变效果最好。筛选培养基中吸光值（495nm）和富集培养基中菌液浊度值（OD630）的加和值较大的突变菌株产邻苯二酚能力高。通过诱变和筛选得到的菌株,产邻苯二酚浓度可达0.87mg/ml,比出发菌株的提高了262.5%。经过形态学和生理生化反应,初步鉴定该菌株属于假单胞菌属（Pseudomonas sp.）。结论：硫酸二乙酯诱变和96孔板筛选的方法能以高通量方式快速筛选出产邻苯二酚菌株。     

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae)

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.     

Comparative proteomics between natural Microcystis isolates with a focus on microcystin synthesis

ABSTRACT: BACKGROUND: Microcystis aeruginosa is a species of cyanobacteria commonly found in a number of countries and frequently related to animal poisoning episodes due to its capacity to produce the cyanotoxin known as microcystin. Despite vast literature on microcystin structures and their deleterious effects, little is known about its synthesis by cyanobacteria. Therefore, this study used proteomic tools to compare two M. aeruginosa strains, contrasting them for microcystin production. RESULTS: 2-DE gels were performed and 30 differential protein spots were chosen. Among them, 11 protein spots were unique in the toxin producing strain and 8 in the non-toxin producing strain, and 14 protein spots were shown on both 2-DE gels but expressed differently in intensity. Around 57% of the tandem mass spectrometry identified proteins were related to energy metabolism, with these proteins being up-regulated in the toxin producing strain. CONCLUSIONS: These data suggest that the presence of higher quantities of metabolic enzymes could be related to microcystin metabolism in comparison to the non-toxin producing strain. Moreover, it was suggested that the production of microcystin could also be related to other proteins than those directly involved in its production, such as the enzymes involved in the Calvin cycle and glycolysis.     

Synthetic DNA probes for detection of genes for enterotoxins A, B, C, D, E and for TSST-1 in staphylococcal strains.

A dot blot hybridization technique with oligonucleotide probes was developed for the specific detection of the TSST-1 gene and the staphylococcal enterotoxin (SE) genes A, B, C, D and E. For each toxin gene a probe sequence was chosen from the previously determined sequence. A total of 145 staphylococcal strains (133 Staphylococcus aureus and 12 coagulase-negative staphylococci (CNS) were studied by this genotypic method and by two phenotypic assays (gel immunodiffusion and ELISA). An excellent correlation (96%) was observed between the genotypic and phenotypic assays. DNA from two CNS strains hybridized with a probe without detection of the corresponding toxin (SEB for one strain and SEC for the other strain). One Staph. aureus strain was shown to be an SEC producer, but was not detected by the corresponding probe. Gene probe and immunological assays seem to be complementary methods for studies of staphylococcal strains producing (or potentially producing) TSST-1 or enterotoxins.     

南方红豆杉产IAA内生芽胞杆菌的分离、鉴定及产脂肽类化合物研究

通过组织研磨法对南方红豆杉(Taxus wallichiana var. mairei)新鲜组织进行内生细菌的分离，利用16S rRNA测序技术对分离的内生细菌进行鉴定分析，使用高效液相色谱-高分辨飞行质谱测定菌液提取物IAA含量，利用HPLC-TOF-MS对提取物脂肽类化合物进行分离、鉴定，结合数据库筛选菌株产脂肽类化合物种类及含量差异。结果表明，从南方红豆杉根茎叶中共分离鉴定出内生细菌31株，筛选出产IAA菌株20株，其中编号为KLBMPTC10的芽胞杆菌属菌株产IAA含量高达210.3955 μg·L^-1。在对产IAA菌株所产脂肽类化合物的研究中发现，菌株KLBMPTC10是所有产IAA内生细菌中脂肽类化合物含量最丰富的，其产Iturin A2相对含量为26.06%，明显优于其他产IAA菌株。芽胞杆菌属菌株KLBMPTC10同时具备高产IAA和脂肽类化合物的能力，可作为研究菌株进一步开发。     

ADP-ribosylation in Clostridium difficile toxin-treated cells is not related to cytopathogenicity of toxin B

ADP-ribosylation of a protein in human fibroblasts treated with partially purified Clostridium difficile toxin B was previously reported. Here we show that the same protein was ADP-ribosylated also in human fibroblasts exposed to supernatant from a C. difficile strain producing neither toxin A nor toxin B. Furthermore, in Chinese hamster ovary and in Vero cells, showing toxin B-induced cytopathogenic effect, the protein was not significantly ADP-ribosylated. The results indicate that the ADP-ribosylation is unrelated to the cytopathogenic effect of toxin B. It appears to be caused by another unidentified factor from C. difficile, and the substrate may correspond to a protein modified endogenously in cells exposed to stressful situations. Cellular actin was not ADP-ribosylated by toxin B.     

Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.