Innovation for ascertaining genomic islands in PAO1 and PA14 of Pseudomonas aeruginosa

Based on three distinct traits of genomic islands, a novel approach was developed to search for and determine genomic islands in special strains. Two genomic islands in Pseudomonas aeruginosa PAO1 and 7 genomic islands in Pseudomonas aeruginosa PA14 were defined with this method. Among the 9 genomic islands, 4 islands had been characterized before, while the other 5 islands were initially determined. The insert sites of 6 genomic islands are tRNA sequences, direct repeats of PA14GI-3 are relative to tRNA^Leu, and direct repeats of PA14GI-2 are at the 3＇ end of bifunctional GMP synthase/ glutamine amidotransferase. Only direct repeats of PA14GI-4 are not clear. Among the 5 newly-found genomic islands, it was supposed that PA14GI-2 is a genomic island related to Hg^2＋ uptake, PA14GI-3 is a secretory activity genomic island, PA14GI-6 is a pathogenicity island, and functions of PA14GI-1 and PA14GI-5 are not clear. Finally, the tyrosine type integrases in PAOIGI-1, PA14GI-5 and PA14GI-7 were analyzed, and their binding and restriction sites were predicted.     

荧光假单胞菌产木聚糖酶的纯化和性质

有硫酸铵分级沉淀,葡聚糖凝胶分离技术,对荧光假单胞菌（Ｐｓｅｕｄｏｍｏｎａｓ ｆｌｕ．）所产的半纤维素酶进行分离纯化,得到单一组分的木聚糖酶。通过凝胶电泳法,测得其摩尔式量为３３０００。研究表明,该酶的最适ｐＨ值为６．０,最适温度６０℃。酶的动力学研究测出米氏常数Ｋｍ值为４．７６ｍｇ／ｍｌ。     

实验动物绿脓杆菌的检测方法研究

目的研究和建立实验动物及环境样品绿脓杆菌(Pseudomonas aeruginosa)快速、准确的鉴定方法。方法分别用VITEK2 Compact全自动分析系统和常规生化方法对绿脓杆菌参考菌株、绿脓杆菌分离株和恶臭假单胞菌分离株进行鉴定试验,并比较不同鉴别培养基的鉴定效果。结果可以用PIA琼脂培养基/CFC琼脂培养基和乙酰胺琼脂培养基为鉴定培养基,采用以氧化酶、明胶液化和42℃生长试验为主的常规鉴定方法,也可采用VITEK2Compact全自动微生物分析系统直接鉴定绿脓杆菌或将其用于常规鉴定结果的确认。结论 VITEK2 Compact全自动微生物分析系统适于绿脓杆菌快速、准确的鉴定或用于对常规鉴定结果的确认。     

微生物转化过程中利用OD值实时监测细菌生物量变化的研究

通过对不同稀释度菌液OD值的测定，结合平板计数结果，建立了一种假单胞菌于重(y)-0D值(x)之间的回归方程：y=301．77x-0．2546(R^2=0．9875)和活菌数(y)-OD值(x)之间的回归方程：y=2．0037x 0．0212(R^2=0．9929)．证明用OD值进行细菌生物量的实时检测具有简便、迅速、准确等优点．     

恶臭假单胞菌萘质粒ⅠNL基因的克隆及酶切图谱

恶臭假单胞菌（ＰｓｅｕｄｏｍｏｎａｓｐｕｔｉｄａＧ７）携带的质粒ＮＡＨ７，经限制性内切酶ＥｃｏＲＩ切割后，产生含有萘降解酶全部基因（２４，６ｋｂ）的片段，此片段插入到载体ｐＵＣ１１９的多克隆位点，构成了ｐＫＡＯ质粒，此质粒经ＨｐａＩ，ＥｃｏＲＩ酶切获得的５．１ｋｂ片段亚屯隆到ｐＵＣ１１９中，衍生出ｐＮＮ１质粒，绘制出内切酶图谱。从ｐＮＮＩ质粒上切下含目的基因ｎａｈＩ、ｎａｈＮ和ｎａｈＬ的ｋｐｎＩ－ｋｐｎＩ片段（２．３ｋｂ）．正向插入载体ＢｌｕｅｓｃｒｉｐｔⅡＳＫ＋中获得重组质粒ｐＮＮ２，反向插入为ｐＮＮ３，将其转化到大肠杆菌ＸＬ１－Ｂｌｕｅ中，目的基因得以大量增殖。     

SPF级实验小鼠4种致病菌多重PCR方法的建立及优化

目的 建立一种可同时检测4种SPF级屏障环境常见致病菌的多重PCR方法。方法 本研究针对鼠伤寒沙门氏菌invA基因、肺炎克雷伯杆菌khe基因、嗜肺巴斯德菌16SrRNA基因、铜绿假单胞菌ecfX基因序列分别设计合成特异性引物,构建可同时鉴别4种菌的多重PCR反应体系,优化后测定其特异性、灵敏性并人工模拟感染样本。结果 建立的4重PCR方法能对同一样品中的4种致病菌模板进行特异性扩增,无交叉反应;对4种目的菌的检测灵敏度为10-3ng/μL;也可从混合感染的病料中特异性地检测出4种致病菌。结论 本试验建立的多重PCR方法具有快速、简便、灵敏的特点,为4种致病菌的快速诊断和监控提供了有效的技术支持。     

紫外诱变选育优势铜绿假单胞菌菌株

采用紫外照射的方法对铜绿假单胞菌菌株进行诱变,从中筛选出一株优势正突变菌菌株B2,并对其形态,生长条件及产鼠李糖脂能力进行了研究。研究发现,相对于亲本B0,菌株B2产鼠李糖脂产量大幅度增高,从0.82 g/L增加到2.71 g/L。菌株形态为杆状菌。最佳生长条件为:温度为30℃,甘油30 g/L,酵母膏2 g/L,微量元素5 m L/L。研究结果表明从重金属含量高的制革污泥中筛选得到的铜绿假单胞菌经紫外诱变后可显著提高产鼠李糖脂产量,并进一步确定诱变后的菌株产鼠李糖脂的最佳发酵条件,为鼠李糖脂的量产提供理论基础。     

Bioleaching of copper oxide ore by Pseudomonas aeruginosa

Bioleaching is an environmentally friendly method for extraction of metal from ores. In this study, bioleaching of copper oxide ore by Pseudomonas aeruginosa was investigated. Pseudomonas aeruginosa is a heterotrophic bactermm that can produce various organic acids in an appropriate culture medium, and these acids can operate as leaching agents. The parameters, such as particle size, glucose percentage in the culture medium, bioleaching time, and solid/liquid ratio were optimized. Optimum bioleaching conditions were found as follows： particle size of 150-177 μm, glucose percentage of 6%, bioleaching time of 8 d, and solid/liquid ratio of 1：80. Under these conditions, 53% of copper was extracted.     

脱氮假单胞菌发酵生产维生素B12高产菌株的选育

以维生素B12生产菌株脱氮假单胞菌05-04为出发菌株,采用紫外线和5-澳尿嘧啶复合诱变处理,选育到一株抗5-澳尿嘧啶突变株5-BU-3-5,该菌株经摇瓶发酵,发酵单位较出发菌株提高了18.0%.该菌株特性优良,经57m3发酵罐试验,平均发酵单位提高8.9%,是一株性状较稳定可深入开发研究的优良菌株.