荧光假单胞菌（Pseudomonas fluorescens）2P24中Fic蛋白催化及调控机制研究

发布时间：2018-02-28 01:37

  本文关键词： 毒素与抗毒素 Fic蛋白 单磷酸腺苷化 DNA促旋酶 DNA拓扑异构酶　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：细菌在进化中形成多种多样的适应性机制以应对不利的环境条件。如细菌处于抗生素逆境压力时,可通过抑制DNA复制、mRNA转录或蛋白质翻译,使生长停滞,形成存留细胞(persister cell),以适应逆境。Fic(filamentation induced by cAMP)蛋白含有保守基序 HPFx[D/E]GN[G/K]R,广泛存在于原核和真核生物中。Fic蛋白多以ATP为供体,催化转移AMP,单磷酸腺苷化修饰含三磷酸腺苷酶(ATPase)或鸟苷酶(GTPase)结构域的蛋白。动植物病原细菌Fic蛋白(效应蛋白)参与致病性,而非外泌型Fic蛋白功能未知。生防荧光假单胞菌(Pseudomonas fluorescens)2P24栖居于土壤和植物根围,生境中存在多种逆境因子,如重金属、干旱、高温及其他细菌、真菌和动植物分泌的生长抑制因子等。因此,环境适应性是其定殖和发挥生防作用的前提。2P24菌株编码3种非外泌型Fic蛋白,其作用靶标、催化机制和功能未知。研究表明,Fic-1在荧光假单胞菌或大肠杆菌中表达可抑制质粒DNA复制和细胞分裂。通过细菌双杂交系统的筛选,发现Fic-1与GyrA、GyrB、LigA、ParE、Po1B、PriA和RecX等7种蛋白互作。Fic-1单磷酸腺苷化修饰PfGyrB Tyr111和PfParE Tyr109,被修饰位点参与ATP嘌呤N3的结合,对DNA促旋酶和拓扑异构酶、Ⅳ水解ATP极为重要。Fic-1修饰GyrB,破坏其ATP水解活性,抑制DNA促旋酶负超螺旋能力,阻止DNA复制并激发SOS反应。SOS途径缺失突变体中表达Fic-1,细菌细胞伸长程度降低,说明Fic-1诱导细菌丝状化主要通过SOS反应途径,但也存在独立于SOS反应的调控途径。Fic-1蛋白能对自身进行单磷酸腺苷化修饰,修饰位点突变体Fic-1Y5A影响其自修饰和修饰GyrB活性。Fic-1与其上游AntF组成典型的Ⅱ型毒素与抗毒素对。fic-1与antF共转录,转录起始于antF起始密码子上游-25位的A碱基,其转录不依赖转录因子RpoS。AntF和Fic-1分别于指数生长前期和后期表达量增加。AntF与Fic-1结合时,抑制Fic-1自修饰和修饰GyrB的活性,阻止Fic-1对DNA复制的抑制。AntF被蛋白酶Lon降解,Fic-1与AntF结合可抑制降解。荧光假单胞菌2P24中Fic-2组成型激活突变体(Fic-2E56G)也可单磷酸腺苷化修饰ParE,但不修饰GyrB。假结核耶尔森菌(Yersiniapseudotuberculosis)和金黄色酿脓葡萄球菌(Staphylococcus aureus)Fic蛋白也能修饰ParE,但铜绿假单胞菌(P.aeruginosa)、结核分枝杆菌(Mycobacterium tuberculosis)和肺炎链球菌(Streptococcuspneumoniae)Fic蛋白既不修饰GyrB也不修饰ParE。荧光假单胞菌中诱导表达Fic-2或PfParE修饰位点突变体(ParEY109A),可抑制细菌生长,促进无DNA细胞形成。以上结果说明荧光假单胞菌Fic-1和Fic-2单磷酸腺苷化修饰DNA促旋酶和拓扑异构酶B亚基,调控DNA复制、染色体分离和细胞分裂。正常情况下Fic蛋白功能受抑制,逆境时Fic-1和Fic-2调节生防假单胞菌2P24的DNA复制、分离和细胞分裂,控制生长,增强逆境适应能力。
[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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