Fic基因对荧光假单胞菌2P24生防功能的影响及其作用机制

Protein modification after translation by Fic (Filamentation induced by cyclic AMP)-mediated NMPylation is a research hotspot in modern life sciences. Despite of a few Fic proteins involved in bacterial virulence, our understanding of Fic proteins, particularly the “housekeeping” Fic proteins that appear to conserve among diverse bacteria, is still very limited. Our previous study on disease-suppressive bacterium Pseudomonas fluorescens 2P24 revealed a Fic family member gene, fic-1, which is involved in the regulation of bacterial cell filamentation, plasmid DNA duplication and biocontrol activities against pathogenic microbes on plants. This project focuses on identification of target proteins of Fic-1, elucidation of the biochemical mechanism of Fic-1 action on its target, and discovery of downstream genes which regulated by Fic-1 and contribute to bacterial adaption and disease suppression in the changing environments. We will further establish highly efficient strategies to identify the environmental and genetic components of the signaling cascade responsible for the activation of Fic-1 protein. The predicted results not only help us to understand the Fic-1-mediated pathway that enhances the bacterial adaptive capacity upon environmental stresses, but also provide us novel cues to understand the important cell processes in basic biology, such as cell morphological changes and regulation of DNA replication.

Fic蛋白催化的单磷酸核苷化修饰是现代生物科学中蛋白翻译后加工的研究热点，但除了少数致病性相关的Fic蛋白作用机制和靶标蛋白已知外，大量Fic蛋白的功能和其发挥功能的解阻遏条件尚无研究报道。前期研究发现生防假单胞菌2P24中的fic-1基因影响菌株防病效果，在大肠杆菌和假单胞菌中表达都会造成质粒拷贝数下降和细胞形态丝状化改变。本项目拟通过遗传和生化手段阐明Fic-1的互作靶标及对靶蛋白修饰的生化机制，初步明确Fic-1调控的下游基因及其影响菌株环境适应能力和生防效果的可能途径。通过构建新的遗传筛选体系，找到可激活Fic-1功能的环境因子和调控基因。上述研究结果一方面可帮助我们明确Fic-1蛋白在菌株2P24生防功能表达和调控中的作用，加深对生防菌适应复杂环境策略的理解；另一方面，研究结果还对基础生物学中诸如细菌的形态变化机制、DNA的复制调控等研究有重要参考意义。

荧光假单胞菌2P24分离自山东小麦全蚀病自然衰退土壤，对多种病害有较好的防治作用。而环境适应性是生防菌株定殖和发挥生防作用的前提。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,如利用毒素抗毒素系统形成存留细胞(persister cell)以适应逆境。Fic (filamentation induced by cAMP)蛋白广泛存在与真核与原核生物中，多以ATP为供体，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。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。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。.本研究表明，Fic-1在2P24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蛋白互作。体外单磷酸腺苷化反应发现Fic-1修饰GyrB能力最强，ParE蛋白次之，而且其催化活性依赖于His135。Fic-1单磷酸腺苷化修饰PfGyrB的 Tyr111和PfParE 的Tyr109。Fic-1修饰GyrB，破坏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诱导细菌丝状化存在独立于SOS反应的调控途径。Fic-1蛋白的单磷酸腺苷化活性受到自修饰和上游抑制因子AntF的调控。Fic-1可以进行自修饰，而Fic-1Y5A影响自修饰和修饰GyrB活性。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。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，AntF还参与AMP-GyrB去单磷酸腺苷化过程。AntF可以被蛋白酶Lon降解，Fic-1与AntF结合可抑制降解。以上结果说明Fic-1单磷酸腺苷化修饰DNA促旋酶或拓扑异构酶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调节生防假单胞菌2P24生长，增强逆境适应能力，当逆境消失后，AntF参与去单磷酸腺苷化过程，解除对GyrB的抑制，细胞恢复生长。