【作者】 于敏；

【导师】 张晓华；

【作者基本信息】 中国海洋大学 ， 微生物学， 2013， 博士

【摘要】 假交替单胞菌（Pseudoalteromonas）广泛分布于世界各地的海洋环境中，可分为产色素或不产色素两大类，其中产色素菌株的显著特征是能够形成具有多种不同生物活性的代谢产物，如抗细菌、抗真菌、溶藻等活性。目前已从该属菌株中分离纯化出多种具有抑菌活性的小分子化合物、蛋白质、多糖等物质，而对该属菌株基因组的分析也已成为研究其多种生物活性物质产生机制及对不同海洋环境适应性的重要手段。因此，对该属菌株抑菌机理的研究有助于开发新型的有益菌制剂和海洋药物，从而广泛应用于水产养殖病害、生物污损及赤潮等的防治中。本论文分离纯化了金丽假交替单胞菌（Pseudoalteromonas flavipulchra）JG1所产生的多种抑菌物质并对其抑菌机理进行了初步探讨，完成了该菌株的全基因组测序及分析，并对其适应复杂的海洋环境并形成生存优势的能力进行了阐释，为后续对该菌株及其代谢产物的开发利用奠定了理论基础。金丽假交替单胞菌JG1分离自健康大菱鲆养殖海水，对多个种属的菌株具有较强的抑菌活性，如弧菌属、芽孢杆菌属等。通过石油醚﹑乙酸乙酯﹑正丁醇对JG1发酵液上清和菌体样品进行分步萃取，发现菌株JG1发酵液的石油醚层、乙酸乙酯层萃取样品及水层样品均具有抑菌活性。这表明JG1的抑菌物质中既含有某些小分子物质如脂类、糖类、色素类化合物，也包含有大分子蛋白类物质，这两类物质协同作用产生更好的抑菌效果。采用硫酸铵沉淀法、SDS-PAGE及电洗脱法对JG1的胞外蛋白进行了分离纯化，在SDS-PAGE中可见单一条带，且胶内活性检测表明该蛋白对鳗弧菌有较强的抑菌活性。将此抑菌蛋白经De novo测序得到的两条肽段的氨基酸序列与菌株JG1的蛋白质组序列进行比对，获得其基因及氨基酸序列全长，并将其命名为PfaP。PfaP蛋白共由694个氨基酸组成，理论分子量为77.0kDa，理论等电点为4.63，Signal P预测其不含有信号肽序列。氨基酸序列比对结果显示，PfaP蛋白与被囊假交替单胞菌（P. tunicata）D2的L-赖氨酸氧化酶AlpP具有58%的一致性，与地中海海洋单胞菌（Marinomonas mediterranea）MMB-1的抗微生物蛋白marinocine具有54%的一致性。由此推测PfaP蛋白为一种L-氨基酸氧化酶，其抑菌活性是由过氧化氢介导的，过氧化氢酶能够抑制其抑菌活性的产生。对PfaP蛋白的基因序列进行克隆，并分别与表达载体pET24a(+)、pET26b(+)、pET32a(+)、pBAD/Myc-HisB及pRSFDuet-1连接，构建了重组表达菌株Escherichia coli BL21/pET24a(+)/PfaP、E. coli BL21/pET26b(+)/PfaP、E. coliBL21/pET32a(+)/PfaP、E. coli BL21/pBAD/Myc-HisB/PfaP以及E. coli BL21/pRSFDuet-1/PfaP，并在体外诱导表达。PfaP蛋白能够在大肠杆菌中得到异源表达，但可能由于在大肠杆菌中无法对该蛋白进行正确的翻译后修饰，其异源表达产物不能够形成有活性的成熟蛋白。菌株JG1的乙酸乙酯萃取物经多步硅胶柱层析及半制备反相高效液相色谱分析，共分离得到5种具有抑菌活性的单体小分子化合物，分别为对羟基苯甲酸（1）、反式桂皮酸（2）、6-溴吲哚-3-乙酸（3）、N-羟基苯并异恶唑酮（4）和2’-脱氧腺苷（5）。其中，仅带有一个溴原子的化合物3为黄褐色，稀释后呈鲜艳黄色，推测其为菌株JG1形成的一种色素分子，使菌落呈现金黄色。这5种小分子化合物对鳗弧菌均具有抑菌活性，微量稀释法检测最小抑菌浓度分别为1.25、1.25、0.25、0.25和5mg/ml。化合物3的抑菌活性最强，抑菌范围也最广，对革兰氏阳性菌和阴性菌均具有抑菌活性。由此可见，色素分子与抑菌蛋白的协同作用使菌株JG1表现出良好的抑菌活性。这5种小分子化合物均首次发现能对海水养殖病原菌产生抑制活性。采用Illumina高通量测序技术对JG1的全基因组进行了测序及分析，共预测得到4913个基因，总长度为4,828,917bp，占基因组的87.71%。同时，在JG1基因组中发现预测的104个tRNA编码基因和4个rRNA操纵子。与已知基因组序列的被囊假交替单胞菌（P. tunicata）D2、游海假交替单胞菌（P. haloplanktis）TAC125、大西洋假交替单胞菌（P. atlantica）T6c、2株假交替单胞菌TW-7和SM9913的COG注释基因分布比较显示，菌株JG1中参与次级代谢产物生物合成、转运和分解，氨基酸转运和代谢以及翻译、核糖体结构与起源的相关基因的丰度均为最高，与其能合成丰富的代谢产物的特征相一致。JG1中参与防御机制的基因丰度也较高，表明其具有良好的环境适应能力。通过对JG1基因组的分析，发现了参与合成5种抑菌小分子化合物的相关基因，并预测了这些小分子化合物在JG1中的代谢途径，而抑菌蛋白PfaP也参与了抑菌小分子物质6-溴吲哚-3-乙酸的生物合成过程，进一步表明JG1中的各种抑菌机制是紧密联系的。在JG1的基因组中还发现有大量的多肽类次级代谢产物的编码基因，几丁质酶、密度感应信号分子降解酶等与抗微生物相关的编码基因，以及对氧化压力的抵抗、重金属的抵抗、抗生素及其他药物抵抗的相关基因，表明JG1既能够形成多种拮抗机制以抑制其他微生物的生长，也能够形成良好的自我保护机制，从而适应多变的海洋环境，在微生物群落中形成生长优势。更多还原

【Abstract】 The genus Pseudoalteromonas is ubiquitous in marine environment and could bedivided clearly into pigmented and non-pigmented species clades, and the pigmentedspecies have been shown to synthesize a range of extracellular compounds withantibacterial, antifungal and algicidal activities. A great number of antimicrobialcompounds such as small molecular compounds, proteins and polysaccharides wereisolated from Pseudoalteromonas spp., and the analysis of the Pseudoalteromonasgenomes has been an important way to study the biosynthesis of its variousmetabolites and the adaption to fluctuating marine environments. Studies which focuson the antibacterial mechanisms of Pseudoalteromonas spp. might be conducive tothe discovery of new probiotics or marine drugs and provide extensively applicationson the biocontrol in aquaculture, anti-fouling and red tides. In this study, theantibacterial components have been isolated and purified from Pseudoalteromonasflavipulchra, and their activities also have been explored. Furthermore, we havesequenced and analyzed the genome of P. flavipulchra and expounded the geneticcapabilities which enable the bacterium to reveal a survival advantage in variedmarine environments. This could provide further insight into the synthesis andapplications of the bacterium or its antimicrobial agents.The marine antagonistic bacterium Pseudoalteromonas flavipulchra JG1with agolden-yellow color was isolated from rearing water of healthy turbot (Scophthalmusmaximus) in Qingdao, China. JG1was confirmed to show excellent antibacterialactivities against many fish pathogens, such as Vibrio spp.. After the extraction withpetroleum ether, EtOAc and n-BuOH, the supernatant and the cells of JG1wasseparated into four fractions, petroleum ether, EtOAc, n-BuOH and water extracts.The antibacterial activities against Vibrio anguillarum were observed in the extractsof petroleum ether, EtOAc and water, indicating that the antibacterial substances ofJG1may include small molecular compounds and macromolecules such asantibacterial protein. The antibacterial protein was purified from the extracellular products (ECPs) ofJG1through ammonium sulfate precipitation, SDS-PAGE and electroelution. Afterelectroelution of the gel band with antibacterial activity, the protein was purified toapparent homogeneity, and the antibacterial activity of the single band was alsoobserved by the in-gel antibacterial assay against V. anguillarum. Sequences of twopeptide-fragments from the antibacterial protein were obtained through De novopeptide sequencing. After sequence alignment with the proteome sequence of JG1, acomplete protein sequence and an open reading frame (ORF) were achieved. The ORF,named pfaP, was2,085bases in length. The deduced translation product of pfaP wasa protein of694amino acids with a predicted molecular mass of77.0kDa and atheoretical pI value of4.63. No putative signal peptide was detected with either theSignal P server or the Secretome P server. The PfaP protein was shown to be58%identical to L-lysine oxidase AlpP of Pseudoalteromonas tunicata D2(GenBankAAP73876.1) and54%identical to marinocine antimicrobial protein of Marinomonasmediterranea MMB-1(GenBank AAY33849.1). The inhibitory activity ofextracellular proteins of JG1against V. anguillarum could be abolished in thepresence of catalase, suggesting that the inhibitory effect was mediated by the actionof hydrogen peroxide.The gene pfaP was cloned from the genomic DNA of JG1, inserted into prokaryoticexpression plasmids pET24a(+), pET26b(+), pET32a(+), pBAD/Myc-HisB andpRSFDuet-1, and expressed in Escherichia coli BL21(DE3). However, theantibacterial activities were not observed, which may attribute to the absence ofposttranslational modification of the protein in E. coli BL21(DE3).The EtOAc extract of the P. flavipulchra JG1was subjected to repeated columnchromatography over silica gel, Sephadex LH-20and RP-18to yield five knowncompounds (15). They were identified as p-hydroxybenzoic acid (1), trans-cinnamicacid (2),6-bromoindolyl-3-acetic acid (3), N-hydroxybenzo isoxazolone (4) and2′-deoxyadenosine (5), respectively, by detailed spectroscopic analysis and these datacomparing with the literatures. The antibacterial activities against V. anguillarum ofthe five compounds were measured through TLC bioautography overlay assay.Inhibition zone of each compound was observed, which showed different effectagainst the target strain. MIC values of compounds15were1.25,1.25,0.25,0.25and5mg/ml, respectively. Compound3isolated from JG1was the only brominated compound with a brown color while the other four compounds were non-halogenatedand almost achromatous. In addition, compound3exhibited a yellow color when itwas diluted, so it probably contributes to the yellow colonies of JG1on marine agar2216plate. Compound3showed the greatest antibacterial activity against both Grampositive and Gram negative bacteria, indicating that this pigment could be associatedwith the antibacterial activity of JG1.The genome of P. flavipulchra JG1was sequenced using the Illumina HiSeq2000,and a total of4,913protein encoding genes (4,828,917bp),104tRNA-encoding genesand4rRNA operons were predicted in the draft genome of JG1. Comparing with thegenomes of P. tunicata D2, P. haloplanktis TAC125, P. atlantica T6c and twoPseudoalteromonas strains TW-7and SM9913, a number of COG categories that areoverrepresented in P. flavipulchra JG1, including those for secondary metabolitesbiosynthesis, transport and catabolism, amino acid transport and metabolism as wellas translation, ribosomal structure and biogenesis. The abundance of genes involvedin expression and transport of potential primary and secondary metabolites could beconsistent with the capability of P. flavipulchra to produce various bioactivecompounds. P. flavipulchra shows the second highest proportion of genes assigned todefense mechanisms among Pseudoalteromonas species, giving a hint that JG1could better adapt to the marine environment fluctuation.The genome of P. flavipulchra JG1unveils significant genetic advantages againstother microorganisms, encoding antimicrobial agents as well as abilities to adapt tovarious adverse environments. The antibacterial protein PfaP not only catalyticallyproduces hydrogen peroxide as a bacteriostat but likely also participates in thebiosynthesis of small molecular antibacterial compound (6-bromoindolyl-3-aceticacid). Both the macromolecule and small molecules contribute to the antibacterialactivities of JG1. Besides these already identified chemical structures produced bystrain JG1, a large number of peptide-based secondary metabolites encoded in thegenome still awaits discovery. The identification of various antimicrobial enzymessuch as chitinases, AHL acylases enriches the antagonistic mechanisms of P.flavipulchra JG1and affords several admissible biocontrol procedures in aquaculture.Furthermore, JG1also evolved a range of mechanisms such as genes involved inoxidative and heavy metal stress, antibiotics and other drug resistances to adapt theadverse marine environments or multidrug rearing conditions. The analysis of the genome of P. flavipulchra JG1presented here provides a better understanding of itssurvival advantages against other microorganisms and also an extensive applicationprospect.更多还原