抗生素单一及联合暴露对RP4质粒介导的抗性基因水平转移的hormesis效应研究

抗生素滥用导致的抗性基因(ARGs)泛滥问题引起人们的日益关注。目前关于抗性基因的研究主要集中在环境监测方面,但是关于抗生素对ARGs传播的影响研究还相对缺乏。质粒是ARGs传播的重要载体,因此以基于RP4质粒的大肠杆菌(E.coli)接合转移体系为研究对象,探讨了盐酸四环素、甲氧苄啶、磺胺氯哒嗪、磺胺异唑在单一暴露条件下对RP4质粒接合转移的影响。根据单一暴露的结果设计混合抗生素的浓度比,探究抗生素联合暴露实验对质粒接合转移的影响。结果表明:在单一暴露条件下,只有盐酸四环素对含有四环素抗性的RP4质粒的接合转移频率有低促高抑的hormesis效应;在联合暴露条件下,对RP4质粒接合转移无促进作用的抗生素会抑制四环素的hormesis效应,这一定程度上降低了抗生素的环境风险。并且用受体理论解释了抗生素对质粒接合转移的hormesis效应产生机制,为目前尚无定论的hormesis受体理论机制提供了证据支持。     

苯甲酸类化合物的微生物降解研究

用富集培养法从工业污水中分离到5个能以苯甲酸为唯一碳源和能源而生长的细苗菌株：不动杆菌（Acinetobactersp.）BJ1,无色杆菌（Achromobactersp.）BY1,假单胞苗（Pseudomonasspp.）SJ1、SY1和SH1、测定了这5个菌株的底物特异性和抗菌素抗性其中BJ1菌株含有1个大质粒和2个小质位在最适培养条件下BJ1菌株对苯甲酸的降解率达98％以上．     

牛蛙生长激素基因在COS7细胞中的转染表达

应用RT-PCR技术克隆牛蛙生长激素(BfGH)的cDNA,T-A克隆法构建反向插入的pMD18-T/BfGH重组质粒,回收BamHⅠ酶切的BfGH cDNA片段,并与去磷酸化处理的真核表达载体VR1020/BamHⅠ,酶切鉴定方法筛选BfGH正向插入的重组真核表达质粒VBfGH.脂质体法介导质粒VBfGH转染哺乳动物细胞COS7,对转染后的COS7细胞进行RT-PCR、ELISA和免疫荧光检测,分别在转录和翻译水平证实BfGH基因在COS7细胞中得到了正确的转染表达.图4表1参17     

离子液体[BMIm][PF6]增强抗性质粒RP4介导的接合转移基因mRNA的表达水平

环境中广泛存在的抗生素和抗生素抗性基因会导致很严重的人类健康风险。在我们前期研究中发现,离子液体1-丁基-3-甲基咪唑六氟磷酸盐([BMIm][PF6])作为环境选择性压力可以促进抗生素抗性基因的水平转移。本研究以E.coli DH5α(RP4)和同属的E.coli HB101,以及E.coli DH5α(RP4)和跨属的Salmonella enterica之间的纯菌接合转移体系为考察对象,从mRNA基因表达调控水平角度阐明离子液体[BMIm][PF6](0.001~2.5 g·L-1)影响质粒RP4接合转移的机理。结果表明,离子液体[BMIm][PF6]通过抑制基因kor A,kor B和trb A的mRNA表达水平来提高接合和跨膜转运基因trb Bp和trf Ap的表达;增强水平转移基因tra F的mRNA表达水平,并通过增强负调控基因kil A和kil B的mRNA表达水平抑制质粒的垂直传递过程;通过抑制基因trb K的mRNA表达水平降低接合转移体系的排斥效果,从而促进质粒RP4的接合转移。该结果有助于为抗生素抗性基因在环境中水平转移扩散的机理研究提供理论依据。     

营养物质对细菌质粒pEA9：：Km在土壤中拼命转移频率的影响

用成团肠杆菌（Ｅｍｔｅｒｏｂａｃｔｅｒ ａｇｇｌｏｍｅｒａｎｓ）Ｔ７（ｐＥＡ９：：Ｋｍ）为供体菌,以Ｅ．ａ．３３９（ｐＥＡ９＾－）为受体菌在天然土壤中进行接合实验,测定各种营养物质对重组质粒ｐＥＡ９：：Ｋｍ同源接合转移频率的影响。实验发现,如果在土壤中加入生理盐水或无机盐培养基,就观察不到细菌数量的增加和质粒的转移;只有加入碳源物质后才检测到细菌数量的增加和质粒的接合转移,接合转移频率的大小位于１     

假单胞菌P12对菲的降解

从武汉石油化工厂的活性污泥中,分离到一株可以以菲为唯一碳源和能源的假单胞菌P_(12)该菌经靛蓝产生法测定有双加氧酶,在菲无机盐培养基中生长时,有3-菲酚物质积累,经检测有一条质粒带。用丝裂霉素C消除质粒后的菌落,失去了降解菲的能力,说明P_(12)对菲的降解功能是由质粒控制的。     

湖流域土壤微生物基因组总DNA分离纯化及其质粒文库的初步构建

采用研磨／冻融和SDV蛋白酶K热处理以及CTAB处理等理化方法，直接从太湖流域土壤样品中抽提得到微生物总基因组DNA，所得粗DNA用透析袋法以及Nucleaotrap suspension DNA纯化试剂盒进行了纯化，纯化过的DNA能够满是常用限制性内切酶酶切、细菌16S rDNA通用引物和随机引物进行PCR扩增的要求，将所得纯品DNA经过EcoRI部分酶切之后与酶切并脱磷酸的pSK^ 空载体连接，再以大肠杆菌JM109为宿主细胞初步构建了土壤微生物基因组文库，图6参7     

Location and PCR analysis of catabolic genes in a novel Streptomyces sp. DUT AHX capable of degrading nitrobenzene

A novel strain of Streptomyces sp.DUT_AHX was isolated from sludge contaminated with nitrobenzene and identified on the basis of physiological and biochemical tests and 16S ribosomal DNA (rDNA) sequence analysis.The optimal degradation conditions were as follows:temperature 30℃,pH 7.0-8.0,shaking speed 150-180 r/min,and inocula 10%(V/V).The strain,which possessed a partial reductive pathway with the release of ammonia,was also able to grow on mineral salts basal (MSB) medium plates with 2-aminophenol, ph...     

萘降解质粒pND6-1中萘趋化基因nahY的克隆和核苷酸序列分析

假单胞菌（Pseudomonas）ND6菌株的萘降解基因位于102kb的质粒pND6-1上。以pUC18质粒为载体，制备了含有1.5-3.0kbpND6-1DNA随机片段的基因文库。通过DNA测序和DNA序列的同源性分析，从基因文库中筛选出含有萘趋化基因nahY的克隆。NahY基因的大小为1617bp，编码的萘趋化蛋白由538个氨基酸组成，与假单胞菌G7菌株的nahY基因相比，核苷酸序列的同源性是96.7％，编码的氨基酸序列的同源性是95％。图4参7     

Promotion mechanism of self-transmissible degradative plasmid transfer in maize rhizosphere and its application in naphthalene degradation in soil

Rhizospheres can promote self-transmissible plasmid transfer, however, the corresponding mechanism has not received much attention. Plant-microbe remediation is an effective way to promote pollutant biodegradation; however, some pollutants, such as naphthalene, are harmful to plants and result in inefficient plant-microbe remediation. In this study, transfer of a TOL-like plasmid, a self-transmissible plasmid loaded with genetic determinants for pollutant degradation, among different bacteria was examined in bulk and rhizosphere soils as well as addition of maize root exudate and its artificial root exudate (ARE). The results showed that the numbers of transconjugants and recipients as well as bacterial metabolic activities, such as xylE mRNA expression levels and catechol 2,3-dioxygenase (C23O) activities of bacteria, remained high in rhizosphere soils, when compared with bulk soils. The number of transconjugants and bacterial metabolic activities increased with the increasing exudate and ARE concentrations, whereas the populations of donor and recipient bacteria were substantially unaltered at all concentrations. All the experiments consistently showed that a certain number of bacteria is required for self-transmissible plasmid transfer, and that the increased plasmid transfer might predominantly be owing to bacterial metabolic activity stimulated by root exudates and ARE. Furthermore, ARE addition increased naphthalene degradation by transconjugants in both culture medium and soil. Thus, the combined action of a wide variety of components in ARE might contribute to the increased plasmid transfer and naphthalene degradation. These findings suggest that ARE could be an effectively alternative for plant-microbe remediation of pollutants in environments where plants cannot survive.