【作者】 李军；

【导师】 刘晓光；

【作者基本信息】 江苏大学 ， 农产品贮藏与加工， 2016， 博士

【摘要】 GacS/GacA双组分信号转导系统普遍存在于革兰氏阴性细菌中,它主要通过控制RsmB家族sRNAs的转录拮抗翻译抑制蛋白RsmA全局调控多种细菌生理活动和行为,包括运动性与生物膜形成、胞外酶、抗生素和植物生长素IAA的产生等,与细菌的生防活性或致病性,以及细菌耐药性、植物生长促进等密切相关。因此,在食品、农业、环境、医疗和生物制药相关细菌的研究中受到广泛关注。GacS/GacA系统由感应激酶GacS和反应调控子GacA组成,GacA在不同细菌中高度保守,但Gac系统调控的功能在不同细菌属种或菌株中,以及不同生长环境中表现出明显的差异。绿针假单胞菌G5是分离自香菜的内生菌,前期研究表明G5能产生蛋白酶,几丁质酶和嗜铁素；以及氢氰酸（HCN）,吩嗪和IAA等次生代谢物质,具有生防活性和植物生长促进作用。然而在菌株G5中GacA的调控作用目前尚不清楚。本研究以绿针假单胞菌G5为目标菌株,利用差异蛋白质组学方法首次全局解析了稳定生长期gacA突变对全细胞蛋白表达谱,以及外分泌蛋白表达谱的影响；并借助于生物信息学等方法对鉴定蛋白的功能进行了深入分析；进一步通过表型分析对GacA调控的关键差异蛋白点进行了验证。以期更好地了解gacA在菌株G5中的生物学功能。GacA作为全局性调控子,研究其分子功能不仅可以进一步了解其复杂的级联调控机制,也有利于从分子水平上改良G5的生防性状。旨在为研发环境友好的多功能生物农药／肥料,保护生态、实现农业可持续发展,保障食品安全和人类健康奠定基础。主要研究结果如下：1)野生菌G5和gacA突变体G5-6培养24h至稳定期后,运用比较蛋白质组学分析了gacA失活对全细胞蛋白和外分泌蛋白表达丰度的影响,质谱鉴定出244个丰度变化2倍以上的蛋白点,代表着210种蛋白。和野生菌G5相比,gacA突变体全细胞蛋白中有38个点丰度上升,89个下降；细胞外分泌蛋白中91个蛋白点丰度上升,26个蛋白点丰度下降。2)GO功能分析显示这些差异蛋白点参与多种生物学过程,包括氨基酸、糖类、脂、脂肪、核酸及无机物质的代谢,蛋白质的折叠与降解,底物转运,分泌,信号转导,防御反应和氧化还原胁迫等。在鉴定的蛋白中,39个蛋白点与胁迫反应相关,33个蛋白点与底物转运有关,29个蛋白点与氨基酸合成代谢相关,26个蛋白点则参与了碳水化合物的代谢与能量转换。KEGG分析显示多个蛋白参与了TCA循环,糖酵解,糖异生等代谢通路。主要差异蛋白点描述如下：3)GacA正调控G5菌株生防因子的产生。蛋白质组学分析表明与吩嗪抗生素生物合成相关的蛋白PhzB、PhzF、PhzD和PhzO在突变体中均不表达。碱性蛋白酶AprA,几丁质酶ChiC₁、ChiC₂以及丝氨酸蛋白酶PspA、PspB的表达水平也显著下降。表型分析表明gacA失活致使G5失去蛋白质水解能力。4) GacA正调控T6SS分泌系统的表达。gacA突变显著降低了VI型分泌系统中TssC2、效应因子Hcp、Vgr家族蛋白的表达,暗示着GacA突变可能影响细菌与细菌之间,以及与植物寄主之间的相互作用。5)GacA与细菌生态适应性密切相关。严谨饥饿蛋白SspA及超氧化物歧化酶SodB在突变体中的表达量均升高10倍以上。表型分析证实了GacA负调控超氧化物歧化酶SOD及过氧化氢酶活性,可能影响细菌的胁迫耐受性。6)四个与鞭毛合成与装配相关的蛋白FliC、FlgK、FlgL和Flil在gacA突变体中的表达水平均升高。鞭毛提取组合SDS-PAGE和质谱鉴定证实了GacA负调控鞭毛蛋白的合成。突变体中鞭毛蛋白丰度增加,而细菌运动性下降表明细菌运动性受多种因素影响,并非完全依赖于鞭毛蛋白。7) GacA突变改变了细胞外膜结构,导致了细胞壁肽聚糖合成蛋白Ddl和LPS脂多糖合成相关的LxpC蛋白表达水平升高。进一步Tricine SDS-PAGE分析证明了gacA突变体中脂多糖LPS合成增加。8) gacA突变降低了植物生长素IAA合成相关的邻氨基苯甲酸合成酶TrpE蛋白的表达水平。HPLC检测证实gacA突变后IAA的产量减少,说明GacA正调控IAA产生。相反在根际促生菌P. chlororaphis 06中GacS负调控IAA生物合成,表明Gac系统对IAA的调控具有菌株特异性,与其生态位相关。综上所述,GacA在G5菌株中全局调控细菌的多种生物学进程和生理行为,在细菌生防、营养与竞争、生态适应性、以及细菌与寄主植物间的互作等诸多方面发挥着重要作用。更多还原

【Abstract】 GacS/GacA two-component signal transduction system is widespread in Gram negative bacteria.Gac system globally regulates bacterial physiology and behaviours via activating the transcription of small RNAs in RsmB family which can sequester the RNA-bingding protein RsmA/CsrA to release their translational repression.These phenotypes regulated by the Gac/Rsm system including cell motility, biofilmformation, production of exoenzymes, antibiotics and indole-3-acetic acid （IAA） are closely related to biocontrol activity or pathogenicity, drug resistance, plant growth promotion. Thereby, the GacS/GacA system has been extensively studied in bacteria related to food safety, environment, medicine, pharmaceutical industry and agricultural production. GacS/GacA is comprised of a sensor kinase GacS and a response regulator GacA. GacA as a global regulator is highly conserved among Gram negative bacteria, but functions differentially depending on strains or their habitats. Endophytic Psuedomonas.chlororaphis G5 was isolated from the stems of Chinese parsley（Coriandrum sativum L.）, previous studies displayed that G5 can produce a variety of secondary metabolites such as hydrogen cyanide （HCN）, phenazine and its derivatives, indole-3-acetic acid （IAA）, as well as siderophores and protease, which contribute to biocontrol of many plant diseases and plant growth promotion in strain G5. However, the specific role of GacA playes in G5 remains to be investigated.In the study, the endophytic starin G5 was used as a bacterial model to determine the global influence of gacA inactivation during stationary phase on the expression profiles of the cellular and extracellular proteins, respectively through comparative proteomics strategy. The identified proteins differentially expressed were annotated by bioinformatics technique for functional analysis. Furthermore, some key protein spots differentially expressed were confirmed by phenotypic analysis with a view to get more insights into the biological function of GacA in G5, which will pave the way for developing environmentally friendly biopesticides/biofertilizer to conserve ecological environment and realize sustainable agriculture, especially for food security and human health.The results obtained in this experiment were shown as follows:1)Wild type G5 and the gacA mutant were grown for 24 hours entry into stationary phase, comparative proteomic was performed to identify the differential protein expression profiles in cellular and extracellular proteomes, respectively. The total of 244 protein spots more than two-fold change in abundance were identified by MALDI-TOF mass spectrometry representing two hundreds and ten of unique proteins. Comparison with wild type G5, the expression of 38,91 protein spots were up-regulated and 89,26 protein spots were down-regulated in cellular and extracellular components by gacA mutation, respectively.2) Function analysis by GO annotation showed these proteins were involved in a wide range of biological processes including biosynthesis and metabolism of amino acid, polysaccharide, lipid, fatty acid, nucleotide and inorganic substrate, transport, secretion, protein folding and degradation, defense response, signal transduction, oxidation-reduction stress etc.. Among identified proteins,39,33,29 and 26 spots are related to stress response, substrate transport, amino acid biosynthesis and metabolism, carbohydrate metabolism and energy conversion, respectively. KEGG analysis indicated many proteins are involved into metabolism pathway such as tricarboxylic acid cycle （TCA）, glycolysis and gluconeogenesis.3) gacA mutation had a profound influence on biocontrol activity of strain G5. The expressions of identified proteins of PhzB, PhzF, PhzD and PhzO were abolished in the gacA mutant, which are required for biosynthesis of the orange pigment phenazine. In addition, the expression of alkaline metalloprotease AprA, chitinase ChiC₁, ChiC₂ and serine protease PspA, PspB were dramatically decreased. Further protease activity assay verified that gacA inactivation resulted in the loss of proteolytic activity.4) GacA also positively regulated type Ⅵ secretion system. gacA inactivation reduced the abundance of TssC2, effector Hcp and Vgr family proteins.It suggested that gacA mutation had an influence on the interaction mediated by type VI secretion system between strain G5 and plant hosts.5) GacA is closely related to ecology fitness in strain G5. The expression of stringent starvation protein SspA and superoxide dismutase SodB was increased （> 10-fold in abundance） in the gacA mutant. The SOD and catalase activities were measured, in agreement with the data from proteomics showing GacA negatively regulate the expression of sodB gene, hence enhanced tolerance to oxidation stress.6) Four proteins of FliC,F1gK,F1gL and FliI were identified by MALDI-TOF/MS and significantly enhanced in abundance in gacA mutant, which are required for flagellar biosynthesis and assembly, Flagellin shearing was done, followed by SDS-PAGE and MS analysis. The results verified that GacA negatively regulated the biosynthesis of flagellin FliC. The relation between the increase of flagellar biosynthesis and the decrease of motility ability suggested that flagellin is not a limitation factor for motility in G5.7) gacA mutation altered the structure of cell envelope. The expression of protein Ddl required for peptidoglycan biosynthesis of cell wall and LpxC responsible for lipid A biosynthesis were induced.Tricine SDS-PAGE analysis revealed that gacA mutation resulted in enhanced rough-type LPS production.8) Unlike in P. chlororaphis 06, gacA inactivation in strain G5 inhibited the expression of anthranilate synthase TrpE responsible for IAA biosynthesis. HPLC analysis verified that IAA production was significantly lower in the gacA mutant than the wild type G5 implying that the GacA regulation of IAA biosynthesis shows niche specificity.Taken together, the results presented here revealed that GacA is involved in global regulation of a diversity of biological processes including primary and secondary metabolism, and plays a central role in biological control, plant growth promotion, stress resistance, competitive fitness and the interactions between bacteria and plant hosts.更多还原