大肠杆菌QseB/QseC信号途径影响染色体复制起始和细胞运动

发布时间:2021-05-06 21:51
  大肠杆菌QseB/QseC二组分信号系统调控五十多种鞭毛和细胞毒性相关蛋白的表达。在本研究中,我们发现无论是在LB还是ABTGcasa培养基中,qseB基因缺失均会导致染色体复制起始提前;在突变体中转入pqseB质粒过表达QseB蛋白后,复制起始提前的现象均被恢复。qseB基因缺失会增加细胞中DnaA蛋白浓度;纯化的QseB蛋白在体外不与oriC相互作用。在AqseC突变体细胞中也存在复制起始提前的现象。这些结果说明QseB/QseC信号途径的缺失会提高细胞中DnaA蛋白浓度,因此引起复制起始的提前。有趣的是,QseB蛋白靶基因fliM、fliQ、fliP或fliO的缺失引起复制起始延迟。我们还发现,QseB/QseC信号途径的缺失不影响细胞大小,而鞭毛相关基因的缺失却会影响细胞大小,说明细胞运动影响细胞分裂。荧光显微镜观察发现QseC蛋白定位在细胞膜上,QseB蛋白定位在细胞质中。qseB基因的缺失会大大提高细胞的运动能力,相反,突变体中过表达QseB蛋白使细胞运动能力恢复至野生型。然而,在ΔqseC和ΔflhDC突变体细胞中,细胞的运动能力明显的减弱,说明QseC信号因子、FlhD... 

【文章来源】:内蒙古大学内蒙古自治区 211工程院校

【文章页数】:83 页

【学位级别】:博士

【文章目录】:
Abstract
摘要
Prior publications
List of abbreviations
Chapter one The Escherichia coli DNA replication and two-component signaling systems/Literature review/
    1.1 The Escherichia coli K-12 bacterium
    1.2 Initiation of DNA replication
    1.3 Bacterial signaling systems
    1.4 Quorum sensing system
    1.5 Two-component system
    1.6 Signal transduction
    1.7 The QseB/QseC two-component system
    1.8 The flagella regulon
Chapter two The QseB/QseC signaling affects initiation of DNA replication and cell motility inEscherichia coli
    2.1 Overall objective
    2.2 Introduction
    2.3 Materials and methods
        2.3.1 Bacterial strains and plasmids
        2.3.2 Primers
        2.3.3 Medium
        2.3.4 Buffer and stock solutions
        2.3.5 Antibiotics
        2.3.6 Enzymes
        2.3.7 Recombinant DNA techniques
        2.3.8 Determination of doubling time
        2.3.9 Agarose gel electrophoresis
        2.3.10 Preparation competent cell
        2.3.11 Transformation
        2.3.12 Measurement of cell size
        2.3.13 Polymerase chain reaction
        2.3.14 Flow cytometry
        2.3.15 Motility assay
        2.3.16 Subcellular localization of protein
        2.3.17 Purification of QseB
        2.3.18 Bacterial two-hybrid assay
        2.3.19 P1 transduction
        2.3.20 Western blotting
    2.4 Results
        2.4.1 The QseB/QseC signaling influences initiation of DNA replication
            2.4.1.1 Absence of QseB/QseC leads to early initiation of DNA replication
            2.4.1.2 Over-expression of qseB delays initiation of DNA replication
            2.4.1.3 The QseB protein does not directly interact with oriC region
            2.4.1.4 Absence of QseB enhances concentration of the DnaA protein
            2.4.1.5 Absence of the fliM, fliQ, fliP or fliO gene delays initiation of DNA replication
        2.4.2 The QseB/QseC signaling affects cell motility but not cell size
            2.4.2.1 Absence of the qseB gene increases cell motility
            2.4.2.2 Flagelar proteins are required for cell motility
            2.4.2.3 Absence of QseB/QseC signaling does not but of flagelar genes influences cells size
        2.4.3 The QseC protein localizes at cell membrane while QseB in cytosol
        2.4.4 The QseB protein interact with the DnaK and FtsZ proteins
            2.4.4.1 Several proteins are co-immunoprecipitated with QseB-GFP protein
            2.4.4.2 The QseB protein to directly interact with DnaK and FtsZ proteins in vivo
            2.4.4.3 Absence of the DnaK or FtsZ protein decreases cell motility
    2.5 Discussion
References
Acknowlodgements



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