两个黄芪根瘤菌新类群代表菌株的16SrNDA序列分析

对黄芪根瘤菌两个新类群中心菌株CA8561和JL84进行了16SrNDA全序列测定,并进行了系统发育学分析。结果表明,CA8561位于Rhizobium分支中,JL84位于Sinorhizobium分支中。     

黄芪根瘤菌的分类研究

采用数值分类方法研究了分离自不同地区的黄氏属根瘤菌36株,发现在80％的相似性水平上,8株菌形成了亚群8,7株菌形成了亚群9。DNA同源性测定结果表明,这两个亚群是不同于已知根瘤菌种的新的DNA同源群。其中心菌株CA8561和JL84的部分16S rRNA基因序列分析发现,CA8561菌株与所有已知根瘤菌远缘,形成了一个独立的系统发育分支。JL84菌株在快生型根瘤菌属(Rhizobium)和土壤杆菌属(Agrobacterium)形成的系统发育分支中占据了一个独立的系统发育地位。     

滇重楼寄生菌的研究

从滇重楼(Paris polyphylla var.yunnanensis)地下茎中分离和鉴定出两种细菌——蜡状芽孢杆菌(Bacillus cereus)和产碱假单胞菌(Pseudomonas alcaligenes),以及三种真菌——黑团孢霉(Periconia sp.)、白色厚顶孢霉(Pachnocybe albida)和重楼索霉(Hormomyces paridiphilus)。对蜡状芽孢杆菌、产碱假单胞菌和重楼索霉进行了液体培养并测定了胞外多糖含量,结果表明重楼索霉可分泌大量胞外多糖,这可能是导致滇重楼地下茎胶质化和多糖含量增加的原因。     

生孢噬纤维细菌的滤纸纤维素的降解过程研究

生孢噬纤维细菌(Sporocytophaga)是能够降解纤维素的滑动细菌,它可将滤纸和棉花纤维素完全降解;但其可测得的纤维素酶活性极低。为研究其纤维素降解机制,本文采用扫描电子显微镜观察了一株生孢噬纤维细菌(Sporocytophaga sp.) JL01对滤纸纤维素的降解过程,分析了在此过程中滤纸纤维素的降解与菌体形态变化之间的关系。结果表明：生孢噬纤维细菌在纤维素降解过程中,形成长度为2.5μm～4.0μm可弯曲的细长杆状细胞,该细长杆状细胞通过与纤维素分子的吸附或嵌入纤维素中完成对纤维素的降解;在降解后期,杆状细胞转化为直径约为0.6μm的小孢囊休眠细胞。     

钝齿棒杆菌天冬氨酸激酶基因的克隆和序列分析

运用PCR方法,从野生型钝齿棒杆菌株(Corynebacterium crenatum)AS1542及具有AEC抗性的突变株CD945染色体上分别扩增出天冬氨酸激酶(AK)基因(ask),构建了重组质粒。核苷酸序列分析表明,C.crenatum AS1542AK基因与C.crenatum CD945相比,第1199位的碱基由T变为C,引起酶蛋白β亚基第80位氨基酸从亮氨酸变成脯氨酸。该氨基酸的突变在蛋白结构上位于ACT结构域内,该区受赖氨酸调控。C.crenatum AS1542的AK基因的编码区核苷酸序列与C.glutamicum\,C.flavum及B.lactofermentum相比,同源性分别为97.23%、97.55%和97.62%,酶蛋白氨基酸序列的同源性分别为99.76%、99.52%和99.76%。但在AK基因的启动子上游序列部分与其它棒杆菌相比有较大差异。     

土壤来源的五个苏云金芽孢杆菌新亚种的鉴定

从中国土壤中分离的大量苏云金芽孢杆菌菌株中鉴定出H42、H43、H56、H60及H62等5种新H血清型,并进行了形态、培养特征、生化反应、晶体蛋白质成分及毒力特性等项检测鉴定,鉴定了5个苏云金芽孢杆菌新亚种： Bacillus thuringiensis subsp. jinghongiensis (H42), B.thuringiensis subsp. guiyangiensis (H43),B.thuringiensis subsp. rongseni(H56),B.thuringiensis subsp. pingluonsis(H60)及B.thuringiensis subsp. zhaodongensis(H62) 。毒力生物测定证明5个新亚种的代表菌株对棉铃虫(Heliothis armigera)\,小菜蛾(Plutella xylostella)\,柳蓝叶甲(Plagiodera versicolora)幼虫均无毒力。H42、H43、H56、H60对埃及伊蚊(Aedes aegypti)\,斑须按蚊(Anopheles stephensi)及尖音库蚊(Culex pipiens)亦均无毒;H62对埃及伊蚊无毒,但对尖音库蚊与斑须按蚊有低毒。     

西藏扎布耶茶卡盐碱湖古菌多样性的非培养技术分析

采用非培养技术,直接从西藏扎布耶茶卡盐碱湖样品中提取微生物总DNA。以样品总DNA为模板,PCR扩增湖中古菌的16S rDNA序列。扩增产物经过克隆并随机挑选60个克隆进行测序得到它们的16S rDNA部分序列,大部分序列与嗜盐碱古菌的16S rDNA相近。在系统发育树上,部分克隆与已知古菌属归于同一分支,主要分布在嗜盐菌科的Natronococcus、Natronorubrum、Natronobacterium、Natronomonas、Natrinema、Halorubrum、Haloterrigena和Halorhabdus等8个嗜盐古菌属中, 也有一些克隆形成了独立的分支。它们共同显示出扎布耶茶卡湖中的古菌具有丰富的多样性。     

透明颤菌血红蛋白在肉桂地链霉菌中的表达对其细胞生长及抗生素合成的影响

肉桂地链霉菌(S.cinnamonensis)是莫能菌素(Monensin)的产生菌。大肠杆菌链霉菌穿梭表达载体pHZ1252中的透明颤菌血红蛋白基因(vhb)位于硫链丝菌素诱导启动子P_tipA之下,它在肉桂地链霉菌中的结构不稳定,发生了重组缺失,缺失的片段包括大肠杆菌质粒部分和vhb基因。但来自阿维链霉菌(S.avermitilis)中缺失了大肠杆菌质粒部分却保留了完整的vhb基因及tipA启动子的pHZ1252,可在肉桂地链霉菌中稳定复制,不再发生缺失,经硫链丝菌素诱导表达出了有生物活性的VHb蛋白。摇瓶发酵实验证明,VHb蛋白在氧限条件下可明显促进肉桂地链霉菌的菌体生长和抗生素合成。     

脱乙酰氧基头孢菌素C合成酶/羟化酶基因cefEF的克隆及序列分析

利用氯化苄分别从真菌顶头孢(Cephalosporium acremonium)和产黄头孢(Acremonium chrysogenum)中提取总DNA,通过PCR方法扩增脱乙酰氧基头孢菌素C合成酶/羟化酶基因cefEF,结果只能从产黄头孢DNA中扩增出cefEF基因。测序结果表明,其与已报道的基因序列只有3个碱基的差异,推断的氨基酸序列只有2个氨基酸有差异,并未涉及活性中心。同时表明,国外所指的与该酶有关的顶头孢(Cephalosporium acremonium或Acremonium chrysogenum)对应的是国内的产黄头孢(Acremonium chrysogenum)。     

耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌（Deinococcus radiodurans）的recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS(DE3)中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E. coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D. radiodurans的recA基因在E. coli细胞中的表达能够完全补偿recA缺损E. coli辐射抗性能力。     

Seohaeicola westpacificensis sp. nov., A Novel Member of Genera Seohaeicola Isolated from Deep West Pacific Sea Water

Strain JL2247^T, an aerobic, Gram-negative, gliding motile bacterium, was isolated from the western Pacific at the depth of 2,000 m. The cell was spindle-shaped with two narrow poles, and flagella were not observed. The colony was circular, translucent, and milky. This strain showed catalase-positive and oxidase-negative reactions. Its optimal growth conditions were at 32 °C, pH 7.3, and 3 % NaCl. The predominant polar lipids were phosphatidylcholine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylmonomethylethanolamine. The major fatty acids were summed feature 8 (18:1 w7c and/or 18:1 w6c) and Cyclo C_19:0 ω8c and the major respiratory quinone was Q-10. The DNA G+C content of strain JL2247^T was 72.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain JL2247^T fell into the genus Seohaeicola, family Rhodobacteraceae, order Rhodobacterales, class Alphaproteobacteria, sharing the highest similarity with the only species Seohaeicola saemankumensis SD-15^T (96.4 % similarity). From the phenotypic, genotypic, and chemotaxonomic data, strain JL2247^T represents a novel species of the genus Seohaeicola and the name is proposed as Seohaeicola westpacificensis sp. nov. The type strain is JL2247^T (=CGMCC 1.12198^T = JCM18883).     

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. IV. Degradation by heat and proteolysis of normal and variant carbonic anhydrase isozymes of Macaca nemestrina

Studies were undertaken on the heat denaturation and proteolytic degradation by alpha-chymotrypsin of the normal red cell carbonic anhydrase isozyme, CA II, and two electrophoretic variants of carbonic anhydrase I, CA Ia and CA Ib, of the pigtail macaque. The heat degradation results showed a difference of about 40-fold in the rate constants between CA Ia and CA Ib, which is due to the marked thermostability of CA Ib compared to CA Ia. The enthalpies and entropies of activation were calculated from the heat denaturation constants. These values were compared, on enthalpy-entropy compensation plots, with those values previously determined for the human CA I and CA II isozymes. They were highly correlated and clearly fell into two distinct clusters, separated by about 200 kJ mol-1; one group comprising the macaque and human CA I isozymes and the other the CA II isozymes. The proteolytic degradation results showed that CA Ia is degraded about 2.5 times more rapidly than CA Ib by alpha-chymotrypsin. Thus, the characteristic 3/1 ratio of CA Ib/CA Ia in mature red cells could be accounted for by the greater susceptibility of CA Ia to degradation at some stage in red cell development.     

Oxidation sensitivity may be a useful tool for the detection of the hematotoxic potential of newly developed molecules: application to antipsychotic drugs.

Some antipsychotic agents have been found to produce agranulocytosis and aplastic anemia. The oxidation phenomena and/or the formation of free radicals has been suggested to be causally related to various hematological disorders, e.g., agranulocytosis. Using five experimental conditions, we tested the oxidative potential of compounds with and without a history of hematological side effects, e.g., agranulocytosis and aplastic anemia. A statistical analysis was undertaken for each experimental condition and a multivariate analysis combining all results was performed. Two peroxidase-induced free radical models did not successfully discriminate between drugs with and without a history of causing hematologic problems (<70%). The lipid peroxidation system provided even less satisfactory discrimination, with only 56.25% correct classification. However, an 87.5% correct classification was obtained when using the oxidation potentials of these drugs determined at pH 4.7 and at pH 7.4. A multivariate analysis taking into account the five variables provided 87.5% success in classification. The two clusters were better discriminated in terms of a "distance coefficient." In a second analysis, the putative antipsychotic pyridobenzodiazepine analogues (JL5, JL8, JL18, and JL25) were classified in the cluster of toxic compounds, while the oxa- and thiazepine analogues (JL2, JL3, and JL13) were classified as nontoxic compounds. On the other hand, a few metabolites of clozapine and fluperlapine were classified in the toxic compound group. The procedure described herein is, to our knowledge, the first which classifies molecules of different structures as well as different pharmacological profiles according to their hematotoxic potential. Such a procedure could be used to predict drug-induced hematological side effects.     

Development of a novel recombinant strain Zygosacharomyces rouxii JL2011 for 1,3-propanediol production from glucose

1,3-propanediol (1,3-PDO) is one of the most important industrial chemicals due to its highly desired properties and its wide applications as a key component of the emerging polymer industry. Biotechnology route has been one of the most interesting methods for 1,3-PDO production, whereas, the dha genes were essential to 1,3-PDO biosynthesis. In this study, we cloned and placed the dha cassettes under the control of a glyceraldehyde 3-phosphate dehydrogenase gene promoter pGAP and homologous ZrFPS1 gene promoter pZrfps1; these two promoters were further integrated into the chromosome of Z. rouxii JL2011 to generate recombinant strain JL2011-GZ and JL2011-ZZ, respectively. The results showed that the two strains could produce 1,3-PDO from glucose with a final yield of 6.9 and 10.3 g/l, respectively. The engineered strain JL2011-ZZ showed a 2.3- and 1.5-fold increase in the specific activities and final concentration of 1,3-PDO, respectively, with respect to JL2011-GZ. Batch fermentation with aerobic/micro-aerobic combined strategy of JL2011-ZZ resulted a titer of 17.1 g/l and a yield from glucose of 8.6 %. These results demonstrated that JL2011-ZZ would be a potential strain for 1,3-PDO production from glucose.     

内生枯草芽孢杆菌JL4在葡萄叶上的定殖及其对葡萄霜霉病的防治

为了明确枯草芽孢杆菌JL4在葡萄叶表面和内部的定殖情况,研究定殖与防治效果的关系,采用电击转化的方法将含有GFP基因的质粒pGFP78导入枯草芽孢杆菌JL4中,并得到成功表达GFP 的生防菌JL4-gfp,测试了标记菌株的稳定性及其对葡萄霜霉病菌的抑制作用.采用叶片喷雾法接种,用抗生素平板稀释分离回收,检测生防菌JL4-gfp在葡萄叶片的定殖情况,并将采回的叶片在室内接种葡萄霜霉菌孢子囊悬浮液进行生防测定.结果表明: 标记菌株在经过10次传代培养后,仍具有良好的发光表型,能稳定表达GFP蛋白,并且标记菌株JL4-gfp对葡萄霜霉菌保持了原有的抑菌作用;用抗生素平板稀释分离回收,检测到JL4-gfp菌株在葡萄叶片表面的定殖量在接种后的0、3和7 d分别为3.6×10⁵、2.7×10⁵和3.1×10³ CFU·g^-1;叶片内部的定殖在接种3 d后达到最大(9.6×10⁴ CFU·g^-1),然后下降,14 d后已经检测不到接种菌株;室内生防测定结果显示,喷雾后3 d对葡萄霜霉病的防治效果达88.0%以上,但7 d后则无明显防效.JL4-gfp的定殖量与其防治葡萄霜霉病的效果呈正相关,其有效定殖量临界值为10⁵ CFU·g^-1.     

应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。     

Recombinant Trypanosoma cruzi antigens and Chagas' disease diagnosis: analysis of a workshop

Abstract A workshop organized by the Ibero-American Project of Biotechnology evaluated the diagnostic potential of several cloned Trypanosoma cruzi recombinant antigens for Chagas' disease serodiagnosis. A set of recombinants, Antigen 2, Antigen 13, SAPA, H49, A13, JL5, JL7, JL8, JL9, and RA1 provided by three different South American laboratories were probed with a panel of 236 South American serum samples. Antigens JL7, H49, Antigen 2, and A13 scored as the best diagnostic recombinant reagents. The results suggested that the main advantage of using cloned peptides for chronic Chagas' disease diagnosis resided in their highly specific immunoreactive properties.     

应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。