工程菌种子制备方法对rEPA表达量影响的研究

由重组E.colirPE553D所表达的基因缺失突变脱毒的重组铜绿假单胞菌外毒素A(rEPA),目前大量以载体蛋白用于多种细菌多糖蛋白结合疫苗研究。rEPA的表达量受众多因素的影响,种子的制备方式就是重要因素之一。本文用长期冷冻保存的和新鲜提取的质粒分别转化宿主菌E.coliBL21(λDE3)后制备种子,并将它们和冻干保存的E.colirPE553D在相同条件下培养和诱导,经SDS-PAGE分析表明长期保存的质粒转化宿主后的重组工程菌生长速度较慢,但rEPA的表达量和新鲜质粒转化制备的工程菌基本相同,而冻干保存的工程菌E.colirPE553D几乎丧失了表达rEPA的能力。     

火山岩滤料固定化恶臭假单胞菌条件的优化及其吸附铜离子的研究

以多孔介质火山岩滤料为载体,探讨了温度、转速、反应器的底面积等因素对滤料固定化恶臭假单胞菌的影响,比较了固定化恶臭假单胞菌野生菌和重组菌吸附Cu2+的效果.结果表明,火山岩滤料固定化恶臭假单胞菌的最优条件为选择底面积较大的反应器、30℃、静置条件下吸附3.5h.固定化野生菌、重组菌滤料及空白滤料对Cu2+的吸附率依次为:74.76％、89.36％和55.09％.为多孔载体固定化微生物在废水处理中的应用提供了实验依据.     

The Measurement of Swimming Velocity of Vibrio cholerae and Pseudomonas aeruginosa Using the Video Tracking Method

The swimming velocities of two monotrichous flagellated bacteria were measured by a computer-assisted video tracking method. Tracing the moving path of the individual bacterium revealed that the bacterial cell did not swim continuously in a straight direction, but frequently changed swimming direction and velocity. The average swimming velocities calculated from the 3-sec path were 75.4 ±9.4 μm/sec in four strains of Vibrio cholerae and 513 ±8.4 μm/sec in five strains of Pseudomonas aeruginosa. These results suggest that V. cholerae swim faster than P. aeruginosa at 30 C in nutrient broth. This method is useful for a detailed analysis of bacterial movement and moving patterns in different environmental conditions.     

EFFECT OF METAL TOLERANT PLANT GROWTH PROMOTING BACTERIA ON GROWTH AND METAL ACCUMULATION IN ZEA MAYS PLANTS GROWN IN FLY ASH AMENDED SOIL

The present study was undertaken to examine the effect of the application of fly ash (FA) into Garden soil (GS), with and without inoculation of plant growth promoting bacteria (PGPB), on the growth and metal uptake by Zea mays plants. Three FA tolerant PGPB strains, Pseudomonas sp. PS5, PS14, and Bacillus sp. BC29 were isolated from FA contaminated soils and assessed for their plant growth promoting features on the Z. mays plants. All three strains were also examined for their ability to solubilize phosphate and to produce Indole Acetic Acid (IAA), siderophores, and hydrogencynide acid (HCN) production. Although inoculation of all strains significantly enhanced the growth of plants at both the concentration of FA but maximum growth was observed in plants inoculated with BC29 and PS14 at low level (25%) of FA concentration. The experimental results explored the plant growth promoting features of selected strains which not only enhanced growth and biomass of plants but also protected them from toxicity of FA.     

一株海藻糖合成酶产生菌的鉴定及产酶条件优化

目的对一株海洋来源的产海藻糖合成酶菌株进行鉴定及产酶条件的初步优化。方法通过16SrDNA基因序列的同源性分析,对一株来源于东海海水的海藻糖合成酶产生菌进行鉴定,并通过单因素分析初步研究其培养特性和最佳的发酵条件。结果该菌16SrDNA序列与GenBank中已知序列相比,最高相似度为100％,鉴定为假单胞菌属（Pseudomonas）,命名为Pseudomonassp．A50。其最佳碳源和氮源分别为2％麦芽糖和0．5％酵母膏,最佳NaCl浓度为2．5％,在初始pH7．8,接种量1％,装液量125mL／250mL,28℃,130r／min发酵48h,海藻糖合成酶活力达到最高。结论此产海藻糖合成酶菌株为假单胞菌属,优化后,海藻糖合成酶活力达到14．16U／mL。     

Evolution of novel metabolic pathways for the degradation of chloroaromatic compounds

Chlorobenzenes are substrates not easily metabolized by existing bacteria in the environment. Specific strains, however, have been isolated from polluted environments or in laboratory selection procedures that use chlorobenzenes as their sole carbon and energy source. Genetic analysis indicated that these bacteria have acquired a novel combination of previously existing genes. One of these gene clusters contains the genes for an aromatic ring dioxy-genase and a dihydrodiol dehydrogenase. The other contains the genes for a chlorocatechol oxidative pathway. Comparison of such gene clusters with those from other aromatics degrading bacteria reveals that this process of recombining or assembly of existing genetic material must have occurred in many of them. Similarities of gene functions between pathways suggest that incorporation of existing genetic material has been the most important mechanism of expanding a metabolic pathway. Only in a few cases a horizontal expansion, that is acqui sition of gene functions to accomodate a wider range of substrates which are then all transformed in one central pathway, is observed on the genetic level. Evidence is presented indicating that the assembly process may trigger a faster divergence of nearby gene sequences. Further fine-tuning, for example by developing a proper regulation, is then the next step in the adaptation.     

氦氧饱和高气压暴露对铜绿假单胞菌PAO1基因表达的诱导调节

研究氦氧饱和高气压暴露对铜绿假单胞菌基因表达谱的系统影响,对急性毒力基因表达的调节。用全基因组DNA芯片分析技术比较菌株暴露前后基因表达谱差异;RT-PCR方法验证部分差异表达基因;用分光光度法在细胞水平验证弹性蛋白酶含量;小鼠染毒法观察暴露组细菌毒力在整体动物水平的变化。基因表达谱分析结果表明,铜绿假单胞菌暴露12 h差异表达基因达243个、72 h差异表达基因为1 168个。72 h差异表达基因中与细菌应激响应、蛋白折叠、转录调节、菌毛和鞭毛合成、毒力因子调节与合成、细菌外膜蛋白和抗原合成的基因大量上调;部分基因的RT-PCR验证结果与芯片结果一致;细胞水平验证结果显示暴露72 h细菌毒力表型增强。因此,氦氧饱和高气压暴露对铜绿假单胞菌基因表达谱有明显影响,对急性侵袭性感染毒力因子基因表达水平有正向诱导调节作用。     

孔雀石绿脱色菌恶臭假单胞菌菌株M6的分离、鉴定及其生长特性研究

从养殖池污泥中分离了6株对孔雀石绿具有脱色能力的菌株, 经过进一步在孔雀石绿营养肉汤中富集培养及其脱色率的比较, 筛选出对孔雀石绿具有较强脱色能力的优良菌株M6。菌株M6在30°C、150 r/min条件下对孔雀石绿的脱色率为97.14％, 通过革兰氏染色、电镜对其形态进行了观察, 用ATB细菌鉴定仪对其进行了生理生化鉴定; 通过对其16S rDNA序列进行PCR扩增和测序, 与NCBI中收录的与其同源性较高的菌株进行了聚类分析并构建了系统发育树。此外, 对其生长特性也进行了研究。实验结果表明, 菌株M6革兰氏染色阴性, 杆形, 端生一根鞭毛, 大小约为0.45 mm×0.84 mm, 在孔雀石绿营养琼脂平板上形成的菌落特征为圆形、浅蓝色、粘稠、不易挑取; 菌株M6的16S rDNA序列与GenBank基因库中假单胞菌属的细菌菌株的16S rDNA序列有98％~99％的高度同源性, 菌株M6与恶臭假单胞菌OW-16(登录号：DQ112328.1)的亲缘关系最近。结合传统的形态与生理生化特性鉴定以及16S rDNA序列分析鉴定的结果, 判定菌株M6为恶臭假单胞菌(Pseudomonas putida)(登录号：EU348741.1)。此外, 菌株M6在30°C、150 r/min条件下摇床振荡培养的生长曲线为：0 h~4 h为生长延迟期, 4 h~64 h为对数生长期, 64 h~80 h为稳定期, 80 h以后为衰亡期; 其最适生长pH值为7, 最适生长温度为30°C, 在转速为50 r/min~250 r/min条件下, 其浓度随转速的增加而增大。     

一株苯酚降解菌的鉴定及其降解特性

【目的】鉴定从某化工厂附近土样中分离到的一株耐高浓度苯酚的菌株T10,通过优化菌株的培养条件提高菌株对苯酚的降解率。【方法】根据菌株的形态、生理生化鉴定及16S rDNA测序分析确定其种属,以液体摇瓶培养菌株T10对苯酚的降解率为指标,对菌株的生长条件进行优化。【结果】菌株T10属恶臭假单胞菌(Pseudomonas putida)。添加葡萄糖、蛋白胨能有效缩短T10菌的生长周期,并使苯酚的降解率提高1.7倍。在菌体初始接种浓度为10%、温度为30°C、转速为180 r/min条件下,对初始苯酚浓度、pH和装液量的响应面优化结果如下:初始苯酚浓度3 000 mg/L、pH 7.5和装液量80 mL/250 mL,苯酚去除率最高可达到87.56%。【结论】T10菌能够耐受较高浓度的含酚废水,并且对苯酚有较强的降解能力,为下一步利用生物法处理含酚废水提供科学依据。     

Pseudomonas aeruginosa infection stimulates mitogen‐activated protein kinases signaling pathway in human megakaryocytes

Pseudomonas aeruginosa is a major cause of nosocomial infections and contributes to higher mortality in hospitalized individuals. Infection by P. aeruginosa triggers host immune response through activation of pathogen recognition receptors, which are present in innate cells. Several studies have reported the mechanism of P. aeruginosa induced innate immunity in multiple cell types. But so far there is no reports on response of megakaryocytes to P. aeruginosa infection. Hence, our aim was to investigate the precise role and signaling mechanism of megakaryocytes during P. aeruginosa infection. In this study, we used Mo7e cells as representatives of human megakaryocyte and found that P. aeruginosa infection induces cytotoxicity in these cells. We further demonstrated that P. aeruginosa infection modulates p38 and extracellular signal regulated kinase pathways in Mo7e cells. Protein expression profiling in P. aeruginosa lipopolysaccharide‐treated Mo7e cells revealed upregulation of importin subunit β and downregulation of metabolic enzymes. Our results suggest that P. aeruginosa infection regulates mitogen‐activated protein kinases signaling pathway and importin in Mo7e cells and that this is a potential mechanism for nuclear translocation of nuclear factor binding near the κ light‐chain gene in B cells and c‐Jun N‐terminal kinases to induce cell cytotoxicity.