大腸桿菌QseB/QseC二組分信號(hào)系統(tǒng)調(diào)控五十多種鞭毛和細(xì)胞毒性相關(guān)蛋白的表達(dá)。在本研究中,我們發(fā)現(xiàn)無(wú)論是在LB還是ABTGcasa培養(yǎng)基中,qseB基因缺失均會(huì)導(dǎo)致染色體復(fù)制起始提前；在突變體中轉(zhuǎn)入pqseB質(zhì)粒過(guò)表達(dá)QseB蛋白后,復(fù)制起始提前的現(xiàn)象均被恢復(fù)。qseB基因缺失會(huì)增加細(xì)胞中DnaA蛋白濃度；純化的QseB蛋白在體外不與oriC相互作用。在AqseC突變體細(xì)胞中也存在復(fù)制起始提前的現(xiàn)象。這些結(jié)果說(shuō)明QseB/QseC信號(hào)途徑的缺失會(huì)提高細(xì)胞中DnaA蛋白濃度,因此引起復(fù)制起始的提前。有趣的是,QseB蛋白靶基因fliM、fliQ、fliP或fliO的缺失引起復(fù)制起始延遲。我們還發(fā)現(xiàn),QseB/QseC信號(hào)途徑的缺失不影響細(xì)胞大小,而鞭毛相關(guān)基因的缺失卻會(huì)影響細(xì)胞大小,說(shuō)明細(xì)胞運(yùn)動(dòng)影響細(xì)胞分裂。熒光顯微鏡觀察發(fā)現(xiàn)QseC蛋白定位在細(xì)胞膜上,QseB蛋白定位在細(xì)胞質(zhì)中。qseB基因的缺失會(huì)大大提高細(xì)胞的運(yùn)動(dòng)能力,相反,突變體中過(guò)表達(dá)QseB蛋白使細(xì)胞運(yùn)動(dòng)能力恢復(fù)至野生型。然而,在ΔqseC和ΔflhDC突變體細(xì)胞中,細(xì)胞的運(yùn)動(dòng)能力明顯的減弱,說(shuō)明QseC信號(hào)因子、FlhD... 

【文章來(lái)源】：內(nèi)蒙古大學(xué)內(nèi)蒙古自治區(qū) 211工程院校

【文章頁(yè)數(shù)】：83 頁(yè)

【學(xué)位級(jí)別】：博士

【文章目錄】：
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



本文編號(hào)：3172673