DegU和Spo0A影响根际促生菌Paenibacillus polymyxa SC2的抗菌物质和生物膜形成的机理研究

Paenibacillus polymyxa is widely used in agriculture, because of its promoting plant growth. But little is known about its genetic mechanism. Strain SC2 can effectively promoting many kinds of plant growth, such as pepper, garlic, cucumber, cotton and apple tree etc.. And it can great reduce soil borne diseases. Then we sequenced the wild strain whole genome, analysis results show the majority of genes involved in plant-growth promotion and synthetic antibiotic. These genes responsible for indole-3-acetic acid production, mineral phosphate and phosphonate solubilization, and synthesis of specific antimicrobial non-ribosomal peptides, involved in fusaricidin, polymyxin, iturin, lantibiotic, bacillorin, polyketides and bacitracin. In laboratory condition, we discovered and isolated two spontaneous mutants, which name is SC2-M1 and SC2-M2 based on their different colony morphology on LB agar. More importantly, both SC2-M1 and SC2-M2 lost their ability to form endospores, motility, synthesis extracellular polysaccharide, antagonistic ability and forming biofilm, seriously affect the application in agriculture. Comparative and analyses of strain SC2 and its spontaneous mutants transcriptome, gene degU and spo0A are found point mutation in the mutants. DegU can regulate the expression of about more than 170 genes in the Bacillus subtilis, and it is an important regulatory factor. Which regulate several postexponential processes, such as competence for DNA uptake, bacterial motility, and degradative enzyme synthesis. The regulatory protein Spo0A regulates spore formation and is closely related to biofilm formation in B. subtilis. So we hypothesize that DegU and Spo0A may be directly related to the formation of antifungal substances and biofilm formation in strain SC2. This study will be using complementation experimental to elucidate the important role in vivo, and to clarify the phosphorylation level of DegU and Spo0A influencing on antibiotic synthesis and biofilm forming. We will be testing whether DegU or Spo0A is combined with fusA, epsA and bslA promoters using EMSA, and detecting the fusA, epsA and bslA gene expression of recombinant strains using quantitative PCR(qPCR) method. This study will be illustrating the important role of regulator DegU and Spo0A in P. polymyxa SC2 . This is a great significance for the better utilization of SC2 strain in agriculture and the understanding of its genetic characteristics.

Paenibacillus polymyxa SC2是一株本实验室分离的PGPR，田间应用效果显著，培养中发现并分离两个自发突变株SC2-M1和SC2-M2，它们丧失了抗生素产生和生物膜形成等能力，严重影响了其应用。转录组分析发现，突变株中degU和spo0A基因发生点突变。推测DegU和Spo0A在SC2中可能与抗生素合成和生物膜形成有直接关系。本研究拟通过互补实验在Paenibacillus polymyxa SC2菌体细胞中鉴定degU 和spo0A 基因功能，阐明其磷酸化水平对抗生素合成和生物膜形成的影响；利用凝胶阻滞检测DegU与fusA，epsA 和 bslA 启动子的结合，结合荧光定量PCR检测重组菌株的fusA，epsA 和 bslA基因表达情况，探究调节因子DegU和Spo0A在SC2菌株中的调节作用。该研究结果对于更好地利用SC2菌株及了解其遗传特性具有重要意义。

本文以多粘类芽孢杆菌SC2为研究材料，利用Motif-x分析并鉴定了野生菌株SC2及其自发突变株SC2-M2的磷酸化修饰肽段的motif，确定所有鉴定位点的11％存在于共有基序xSxS中。DegU的Thr239是一个保守的功能位点和磷酸化修饰位点。DegU和AbrB3蛋白均直接结合fus启动子区域且影响其转录，DegU作为一种正调控因子，而AbrB3作为一种负调控因子来影响杀镰孢菌素的合成。首次发现DegU有两个启动子，经GFP标记，两者都能产生荧光信号且荧光强度无显著差异，两个启动子都有活性。.通过chip-seq构建了DegU调控网络图，结果表明DegU参与了多种生物过程，包括生物膜的形成，细胞运动，信号转导，遗传信息处理，非核糖体肽生物合成，杆菌肽生物合成和芽孢形成。生物学试验证明DegU与bglp1、PPSC2_RS45905和PPSC2_RS49165相互作用，负调控生物膜的形成；DegU与bglp1、PPSC2_RS45905相互作用负调控多粘菌素的合成、鞭毛的形成，正调控杀镰孢菌素的合成。同时，DegU受PPSC2_RS45905的负调控，DegU正调控yjiN的表达。.通过蛋白质组学分析，绘制了Spo0A预测调控网络图，生物学试验证明Spo0A正调控杀镰孢菌素、多糖的合成，芽孢形成和运动性。同时，Spo0A负调控AbrB、SinR和Abh的表达水平。Spo0A正调控芽孢的形成，高磷酸化水平的Spo0A促进芽孢的形成，低磷酸化水平降低芽孢的形成。Spo0A参与调控生物膜的形成，高磷酸化修饰不利于生物膜的形成，低磷酸化修饰有利于生物膜的形成。Spo0A正调控多粘菌素的合成，高磷酸化水平有利于多粘菌素产生。.该研究解析了DegU和Spo0A在分子水平上调控SC2生物膜形成、杀镰孢菌素和多粘菌素的合成机理；首次报道了多粘类芽孢杆菌的bglp1、PPSC2_RS45905和PPSC2_RS49165为细胞膜蛋白，且与DegU互作，负调控生物膜的形成，正调控杀镰孢菌素的合成。研究结果为SC2菌株能够在植物根际有效定植，并起到促生和防治植物病害发生的作用提供了理论依据。