【作者】 吴大强；

【导师】 许煜泉； 张雪洪；

【作者基本信息】 上海交通大学 ， 微生物学， 2012， 博士

【摘要】 假单胞菌M18是本实验室1996年在上海郊区甜瓜根际分离的高产两种抗生素吩嗪-1-羧酸和藤黄绿菌素的植物生防菌，其产品申嗪霉素已广泛应用于我国的植物保护工作中，对于我国粮食生产安全具有重要的保障作用。而本课题组前期研究发现M18菌株与条件性致病菌铜绿假单胞菌遗传背景最为接近，但该菌株又与铜绿假单胞菌在表型和基因调控方式上存在巨大差异，因此阐明假单胞菌M18的遗传组成，对研究其生防机理具有重要意义。本论文从基因组、表型、转录组和基因组岛功能等角度，分析了M18菌株特殊基因组的特点，对于环境响应的转录组变化和特定基因组岛的功能，以期发掘该菌株的优良生防功能和独特基因组结构产生的原因，主要结果如下：第一、利用下一代高通量测序技术，本文全基因组测序M18菌株基因组，发现M18基因组由一条长6327754bp的环状染色体构成，含有5684个编码基因和80个RNA基因。序列分析显示M18菌株基因组序列与已测序铜绿假单胞菌基因组同源性最高，达到90%以上，并且全基因组比对、进化树分析和COG基因分类比较均显示相同现象，因此将该生防菌株定名为Pseudomonas aeruginosa M18。基因组水平分析发现，相比其它人体分离的铜绿假单胞菌基因组，根际分离的M18菌株基因组具有多个特点，包括含有最多数量的CRISPR和最少的IS遗传元件，有助于维持其基因组稳定。此外，M18菌株附加基因组不含任何临床分离株的致病性基因组岛，由5个基因组岛和2个原噬菌体岛组成，其中含有限制性修饰系统、抗生素合成、糖基化修饰、营养物分解等功能基因，有助于M18菌株的根际环境适应。同时，M18菌株基因组携带5类次生生防相关代谢产物的合成基因簇，也对菌株的生防作用起到重要作用。第二、本文通过研究铜绿假单胞菌M18和近源高致病性菌株的致病性基因和耐药性基因，发现两个菌株存在致病基因上存在多处基因差异，在核心基因组中的耐药性基因上差异较小。之后，开展小鼠肺部急性感染竞争和抗菌剂抗性试验，结果显示M18菌株株易于清除，且对庆大霉素和卡那霉素等较为敏感。因此，本章结果显示和耐药性均弱于近源高致病性菌株，提示不同环境进化压力对于铜绿假单胞不同菌株遗传组成和表型的巨大作用。第三、采用定制DNA芯片技术，本文检测了根际分离株M18和模式临床分离株PAO1，在各自典型生境温度下（根际温度28℃、人体温度37℃）的转录组变化情况。比较分析发现不同环境条件对于铜绿假单胞菌转录组影响明显大于基因组，不同菌株在适应非原环境条件时，调动更多基因表达以适应新环境条件。此外，结果还显示，根际来源菌株M18在土壤温度下，22个基因组岛中基因表达上调，但在人体环境温度下，却没有发现任何基因组岛中基因被诱导表达，提示附加基因组的表达对于细菌的环境适应性具有重要影响。第四、序列分析发现，根际分离铜绿假单胞菌株M18附加基因组由5个基因岛和两个原噬菌体组成,缺少决定人体致病性辅基因岛和噬菌体。M18菌株特有基因组岛MGI-I和MGI-II分别含有一套含完整亚基的限制性修饰（RM）系统，在近源株没有发现完整RM系统，并且两个菌株的附加基因组具有明显差异。因此，本文首先试验发现两个基因组岛是可以环切出M18染色体，结合GC含量差异暗示这两个基因组岛为新近引入M18基因组中。随后，敲除基因岛上RM系统限制亚基基因后发现这些系统具有限制外源DNA通过转化和接合方式进入细胞的作用，证明这些RM系统具有限制外源遗传物质进入的功能。这些结果提示RM基因组岛对于M18菌株基因组结构形成的重要功能，提示基因组岛在M18菌株维持基因组稳定，以适应环境中各种压力胁迫的重要作用。综上所述，本文系统的研究了根际分离铜绿假单胞菌生防菌株的基因组、温控转录组和相关表型，并与其它临床菌株深入比较分析，进一步发现M18菌株特异性基因组岛在其基因组形成中起到的重要作用。结果表明，在根际环境中选择压力下，M18菌株进化出独特的基因组结构和基因表达模式以适应特定环境生存的需要。更多还原

【Abstract】 Pseudomonas sp. M18was first isolated from sweet melon rhizosphere in Shanghaisuburb in1996, and has effective biocontrol ability with highly yield ofphenazine-1-carboxylic acid and pyoluteorin. Shengqin antibiotic produced by the strainM18has been applied in Chinese plant protection for helping to ensure the food safety ofChina. Previous works from our group found that the genetic background of strain M18ismost close to Pseudomonas aeruginosa, but also with several unique phenotype and generegulation pattern. Therefore, it is important to clarify the genetic composition thebiocontrol strain Pseudomoans sp. M18.Through the prospects of genome, phenotype, transcriptome and function of genomicisland, we analyzed the genomic features of strain M18, the modification of transcriptomein response to the environmental stress and the function of specific genomic island (GI), toexplore the reasons of the excellent biocontrol ability and unique genome structure of thestrain M18. The results are as follows:Firstly, the complete genome of strain M18was sequenced using next generationsequencing technology. The M18genome is composed of a single chromosome of6,327,754bp containing5684coding genes and80RNA genes. Based on the higher than90%of genome similarity with the available genomes of P. aeruginosa, the strain M18isnow designated as Pseudomonas aeruginosa strain M18. Compared with other P.aeruginosa strains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18genome maycontribute to the relative genome stability. Furthermore, the accessory genome of strainM18including two novel prophages and five specific non-phage islands were identified. Itcontains genes responsible for restriction-modification systems, pyoluteorin biosynthesis,glycosylation modification and environmental substance degradation. The M18genomealso carries biosynthesis gene clusters of five types of biocontrol related secondarymetabolites.Secondly, comparing the sequence of virulence gene and antibiotic resistance gene inP. aeruginosa M18and its close related hyper virulence strain LESB58, we found that thedifference in several virulence genes between two strains, but less difference in theantibiotic resistance genes located in core genome between two strains. After competitiveactivity in mouse lung model and MIC tests, we found that the strain M18is moresusceptible to several antimicrobial agents and easier to be erased in a mouse acute lunginfection model than the strain LESB58. The results suggested that environmental selectivepressure has a huge influence on the genotype and phenotype of different P. aeruginosastrains.Thirdly, using customer-desigened DNA chip technology, this study measured thetranscriptome modifactions of rhizosphere strain M18and model clinical isolate PAO1atdifferent typical niche temperatures (28℃for rhizosphere,37℃for human body). Thecomparative results showed that the transcriptome at two typical temperatures showed thesignificant temperature-dependent differences compared with that of the genome structure.The specific induced genes at non-originating growth temperature of the each strain areevidently more than those of the induced genes at originating growth temperature.Furthermore, we found that22genes up-regulated at28°C in three GIs and one prophagebut none at37°C of rhizosphere strain M18, suggesting that the expression of accessorygenome is important in environmental adaption of bacteria. Fourthly, rhizosphere originated P. aeruginosa strain M18carries two full subunitrestriction and modification system located in two specific genomic island MGI-I andMGI-II. But, there is no complete RM system in close related strain. Therefore, this studyfirstly demonstrated that the two GI are newly introduced in M18genome. Then, after geneknock out experiment, the results showed that RM system can restrict exogenous DNAentering host cell via transformation and conjunction to prevent the foreign geneticmaterial enter bacteria cell. The results indicted the important function of RM genomicisland in shaping the genome structure of strain M18to stable the genome under theenvironmental selective pressure.In conclusion, this study systematically analyzed the genome, temperature-dependenttranscriptome, related phenotype, and further investigated the important role of M18strainspecific GI in shaping the genome structure. The results indicated that the P. aeruginosastrain M18has evolved its specific genomic structures and temperature dependentexpression patterns to meet the requirement of fitness and competitiveness under selectivepressures imposed on the strain in rhizosphere niche.更多还原