本文關(guān)鍵詞： 毒素與抗毒素 Fic蛋白 單磷酸腺苷化 DNA促旋酶 DNA拓?fù)洚悩?gòu)酶　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：細(xì)菌在進(jìn)化中形成多種多樣的適應(yīng)性機(jī)制以應(yīng)對(duì)不利的環(huán)境條件。如細(xì)菌處于抗生素逆境壓力時(shí),可通過(guò)抑制DNA復(fù)制、mRNA轉(zhuǎn)錄或蛋白質(zhì)翻譯,使生長(zhǎng)停滯,形成存留細(xì)胞(persister cell),以適應(yīng)逆境。Fic(filamentation induced by cAMP)蛋白含有保守基序 HPFx[D/E]GN[G/K]R,廣泛存在于原核和真核生物中。Fic蛋白多以ATP為供體,催化轉(zhuǎn)移AMP,單磷酸腺苷化修飾含三磷酸腺苷酶(ATPase)或鳥苷酶(GTPase)結(jié)構(gòu)域的蛋白。動(dòng)植物病原細(xì)菌Fic蛋白(效應(yīng)蛋白)參與致病性,而非外泌型Fic蛋白功能未知。生防熒光假單胞菌(Pseudomonas fluorescens)2P24棲居于土壤和植物根圍,生境中存在多種逆境因子,如重金屬、干旱、高溫及其他細(xì)菌、真菌和動(dòng)植物分泌的生長(zhǎng)抑制因子等。因此,環(huán)境適應(yīng)性是其定殖和發(fā)揮生防作用的前提。2P24菌株編碼3種非外泌型Fic蛋白,其作用靶標(biāo)、催化機(jī)制和功能未知。研究表明,Fic-1在熒光假單胞菌或大腸桿菌中表達(dá)可抑制質(zhì)粒DNA復(fù)制和細(xì)胞分裂。通過(guò)細(xì)菌雙雜交系統(tǒng)的篩選,發(fā)現(xiàn)Fic-1與GyrA、GyrB、LigA、ParE、Po1B、PriA和RecX等7種蛋白互作。Fic-1單磷酸腺苷化修飾PfGyrB Tyr111和PfParE Tyr109,被修飾位點(diǎn)參與ATP嘌呤N3的結(jié)合,對(duì)DNA促旋酶和拓?fù)洚悩?gòu)酶、Ⅳ水解ATP極為重要。Fic-1修飾GyrB,破壞其ATP水解活性,抑制DNA促旋酶負(fù)超螺旋能力,阻止DNA復(fù)制并激發(fā)SOS反應(yīng)。SOS途徑缺失突變體中表達(dá)Fic-1,細(xì)菌細(xì)胞伸長(zhǎng)程度降低,說(shuō)明Fic-1誘導(dǎo)細(xì)菌絲狀化主要通過(guò)SOS反應(yīng)途徑,但也存在獨(dú)立于SOS反應(yīng)的調(diào)控途徑。Fic-1蛋白能對(duì)自身進(jìn)行單磷酸腺苷化修飾,修飾位點(diǎn)突變體Fic-1Y5A影響其自修飾和修飾GyrB活性。Fic-1與其上游AntF組成典型的Ⅱ型毒素與抗毒素對(duì)。fic-1與antF共轉(zhuǎn)錄,轉(zhuǎn)錄起始于antF起始密碼子上游-25位的A堿基,其轉(zhuǎn)錄不依賴轉(zhuǎn)錄因子RpoS。AntF和Fic-1分別于指數(shù)生長(zhǎng)前期和后期表達(dá)量增加。AntF與Fic-1結(jié)合時(shí),抑制Fic-1自修飾和修飾GyrB的活性,阻止Fic-1對(duì)DNA復(fù)制的抑制。AntF被蛋白酶Lon降解,Fic-1與AntF結(jié)合可抑制降解。熒光假單胞菌2P24中Fic-2組成型激活突變體(Fic-2E56G)也可單磷酸腺苷化修飾ParE,但不修飾GyrB。假結(jié)核耶爾森菌(Yersiniapseudotuberculosis)和金黃色釀膿葡萄球菌(Staphylococcus aureus)Fic蛋白也能修飾ParE,但銅綠假單胞菌(P.aeruginosa)、結(jié)核分枝桿菌(Mycobacterium tuberculosis)和肺炎鏈球菌(Streptococcuspneumoniae)Fic蛋白既不修飾GyrB也不修飾ParE。熒光假單胞菌中誘導(dǎo)表達(dá)Fic-2或PfParE修飾位點(diǎn)突變體(ParEY109A),可抑制細(xì)菌生長(zhǎng),促進(jìn)無(wú)DNA細(xì)胞形成。以上結(jié)果說(shuō)明熒光假單胞菌Fic-1和Fic-2單磷酸腺苷化修飾DNA促旋酶和拓?fù)洚悩?gòu)酶B亞基,調(diào)控DNA復(fù)制、染色體分離和細(xì)胞分裂。正常情況下Fic蛋白功能受抑制,逆境時(shí)Fic-1和Fic-2調(diào)節(jié)生防假單胞菌2P24的DNA復(fù)制、分離和細(xì)胞分裂,控制生長(zhǎng),增強(qiáng)逆境適應(yīng)能力。
[Abstract]:Bacteria evolved into a variety of adaptive mechanisms to cope with adverse environmental conditions. For example, bacteria under antibiotic stress can arrest growth by inhibiting DNA replication, transcription or protein translation. The conserved motif HPFx [D / E] GN [G / K] R was formed to adapt to stress. ATP was widely used in prokaryotes and eukaryotes. Catalytic transfer of AMP, adenosine monophosphate monophosphate modified proteins containing the domain of adenosine triphosphatase (ATP) or guanosine monophosphate (GTPase). The Fic protein (effector protein) of plant and animal pathogenic bacteria is involved in pathogenicity. But the function of non-exocrine Fic protein is unknown. Pseudomonas fluorescens)2P24 inhabit soil and plant root environs. There are many stress factors in habitat, such as heavy metal, drought, high temperature and other bacteria. Therefore, environmental adaptability is the premise of colonization and biocontrol. 2P24 strains encode three kinds of non-exocrine Fic proteins. The catalytic mechanism and function are unknown. The expression of Fic-1 in Pseudomonas fluorescein or Escherichia coli can inhibit plasmid DNA replication and cell division. It was found that Fic-1 interacted with PfGyrB Tyr111 and PfParE Tyr109 proteins, such as PfGyrB Tyr111 and PfParE Tyr109. The modified sites were involved in the binding of ATP purine N3 to DNA and topoisomerase, 鈪，

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