【作者】 王峰；

【导师】 肖湘；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 深海是一个特殊的生态环境,这里有着永久的低温、高压和黑暗。生活在这些环境下的极端微生物必然有特殊的生理代谢途径来适应这种极端环境。对深海微生物的研究不仅有助于了解生命的起源,而且可以了解各种极端微生物的生活特性,有助于对深海微生物资源的开发利用。极端条件下的生命和生态现象研究将可能揭示生命适应逆境、演化自身的能力,为人类健康开辟新的途径。本文所研究的深海细菌分离自西太平洋1914米的深海沉积物中。菌种鉴定为Shewanella piezotolerans WP3,它既是耐压菌,又是低温菌,现在已经完成其全基因组序列的测定,是研究极端微生物适应极端环境的理想材料。本文用WP3作为实验材料,通过模拟深海低温高压极端生存条件,并结合各种分子生物学手段,对脂肪酸系统在深海极端环境中的作用,及其表达和调控进行了全面地分析,使我们对极端微生物的极端环境适应性机理有了更为深入的了解。此外,我们成功的从WP3中分离出了世界上第一株深海丝状噬菌体SW1,这也是由希瓦氏细菌中分离出来的第一株丝状噬菌体,在对它的性质进行研究时,我们发现了它与深海低温环境相关的一系列低温性质,这为研究深海噬菌体在深海环境中的作用奠定了一定的基础。希瓦氏细菌广泛的分布在各种环境中,它们能够合成多种低熔点脂肪酸,是研究脂肪酸系统与细胞环境适应性关系的理想材料。通过对15株已完成全基因组测序的希瓦氏细菌的基因组序列进行比较后,我们构建出了希瓦氏细菌的Ⅱ型脂肪酸合成途径和EPA合成途径。此外还发现所有的希瓦氏细菌都包含了完整的EPA合成基因簇,但是只有其中少数的希瓦氏细菌被报道能够合成EPA。我们选用Shewanella piezotolerans WP3为模型,对脂肪酸系统在希瓦氏细菌中的作用和调控进行了更为全面和深入的研究。通过GC-MS对WP3脂肪酸成分进行的分析显示,WP3能够合成支链脂肪酸,单不饱和脂肪酸和多不饱和脂肪酸EPA三种低熔点脂肪酸。随着培养温度的降低和压力的升高,其中的支链脂肪酸和多不饱和脂肪酸含量都出现了明显增加。与其它绝大多数细菌的情况不一样,单不饱和脂肪酸在低温高压环境中的含量不但没有增加,反而出现了一定程度上的下降。随后通过一系列的基因敲除实验,以及浅蓝菌素（Cerulenin）抑制单不饱和脂肪酸合成的实验,我们证明多不饱和脂肪酸EPA的缺失能够抑制WP3在低温和高压下的生长,支链脂肪酸的减少也会降低WP3在低温下的生长能力,但是单不饱和脂肪酸含量的大量降低却对WP3的低温高压生长并没有造成影响,这证明了支链脂肪酸和多不饱和脂肪酸是WP3低温高压生长所必需的,而单不饱和脂肪酸是非必需的。利用已构建好的WP3全基因组基因芯片进行芯片杂交实验,用以分析WP3在不同温度（4℃和20℃）和压力（0.1Mpa和20Mpa）条件下基因的表达差异变化。发现在低温条件下,支链氨基酸转运系统的所有基因都呈现出上调趋势,并用实时荧光定量PCR方法对芯片结果进行了验证。随后,利用DNA同源重组的方法对支链氨基酸转运系统中唯一的底物结合蛋白（swp3487）进行了缺失突变,与WP3野生菌株支链脂肪酸含量在低温和高压条件下大量增加不同,所得到的突变子WP3^△LIV-I在不同温度和压力下支链脂肪酸含量变化很小,显示了支链氨基酸转运系统对WP3支链脂肪酸的含量具有重要的调节能力。随后,通过在野生菌株和突变菌株的培养基中添加¹³C标记的外源亮氨酸,我们进一步证明了低温下支链脂肪酸含量的增加主要是由于支链氨基酸转运系统在低温下为支链脂肪酸的合成转运了更多的外源底物所致。通过对WP3全基因组序列进行分析,我们发现其中可能包含了一株具有活性的丝状噬菌体,并将其命名为SW1。SW1基因组DNA长7718bp,包含了9个开放性阅读框（ORF）,其中绝大部分ORF都与已知的一些丝状噬菌体基因显示出了最大的同源性。通过双向电泳（2D-PAGE）,我们发现噬菌体中的单链DNA结合蛋白在4℃下呈现出高表达。考虑到单链DNA结合蛋白决定了噬菌体组装的起始,因此我们判断大量成熟的SW1噬菌体在4℃被诱导合成,根据这一线索我们成功地在4℃条件下分离出了具有活性的SW1噬菌体颗粒。随后,利用希瓦氏菌属四个菌株（Shewanella oneidensis,Shewanella marinaci,Shewanellaviolacea和Shewanella sp.WP2）加上大肠杆菌共5株菌来作为测试菌,检测SW1噬菌体的宿主范围。最后发现,只有与WP3从同一地点分离出来的WP2能够被SW1侵染并形成噬菌斑。而且,只有当培养温度低于或等于15℃时才能观察到噬菌斑的形成。随后的实验证明,当温度高于15℃时噬菌体能够正常侵染WP2,但是只有当温度下降到15℃或以下时,噬菌体才在WP2中大量合成并最终导致噬菌斑的产生,这暗示了噬菌体可能存在低温诱导特性。对SW1的热稳定性分析发现,70℃处理10分钟就能让SW1噬菌体完全失活,比一般丝状噬菌体的最高耐受温度足足低了10℃,这也再一次显示了SW1的低温特性。利用实时荧光定量PCR方法对噬菌体主要基因在4℃和20℃下的转录水平进行了分析,发现在4℃下这些主要基因,包括噬菌体自身抑制子基因orf116都出现了3-12倍不同程度的高表达。随后的噬菌体滴度实验进一步发现,只有当培养温度低于或等于15℃时才能分离出具有活性的SW1噬菌体颗粒,并且随着温度的降低噬菌体的滴度不断增加。这进一步证明了低温能够诱导SW1噬菌体的合成和释放。在对SW1噬菌体低温诱导机制进行的初步研究中发现,利用紫外线（UV）和丝裂霉素C这些能够引起细胞SOS反应的因素处理WP3,同样能够诱导SW1的大量表达。这说明与其它噬菌体一样,SW1的合成也受到SOS途径的调控。但是,与低温诱导现象不同的是噬菌体的自身抑制子基因orf116在受到紫外线和丝裂霉素C处理后的表达量并没有出现增高,这说明低温诱导机制与SOS诱导机制并不完全一样,很可能在WP3中还存在着一条有别于SOS途径的噬菌体诱导途径。更多还原

【Abstract】 Deep-sea is a special ecosystem for its permanent coldness, high pressure and darkness. The study towards the deep-sea microbes will be helpful to the understanding of the origin of life, the character of biodiversity and to the exploitation of microbial resources in deep sea. The metabolic and ecological study of life on extreme conditions will give some insights on evolution and adaptation mechanism of extremophiles in response to stress conditions and will pave a new way for the development of anti-stress agricultural and animal husbandry products and for health of human beings.The deep sea bacteria used in this study was isolated from deep-sea sediments of 1914m depth in west Pacific. The phylogenetic analysis and molecular study shows that the bacterium is a psychrophilic and piezotolerant microbe with the term as Shewanella piezotolerans WP3, which will be an ideal material to study the adaptation mechanism of extremophiles to the extreme environments. The aim of this study is to investigate the role and regulation of fatty acid system in Shewanella response to different growth conditions. Additionally, we have isolated and identified a novel filamentous phage SW1 from WP3, which is the first filamentous phage isolated from Shewanella genus. Furthermore, we have found this phage can be induced by low temperature and some other low temperature related characters, which will give the basis for investigating the role of phage in deep-see environment.Shewanella inhibits in various environments and it is the most microbes isolated from deep sea. The Shewanella genus is capable of synthesis various types of low melting fatty acids, so it is the idea material for investigating the role and regulation of fatty acid system in adaptation of deep-sea environment. The genome sequences of 15 Shewanella strains were searched and compared for genes involved in fatty acid synthesis. All genes supposed to be involved in typical typeⅡfatty acid biosynthesis pathway could be easily identified from the genomes and the typeⅡfatty acid biosynthesis pathway of Shewanella genus was constructed. Complete EPA synthesis gene cluster was found in all of the Shewanella genomes although only few of them were found to produce EPA. The roles and regulation of fatty acids synthesis in Shewanella was further elucidated in Shewanella piezotolerans WP3 response to different temperatures and pressures. WP3 increased significantly the contents of EPA and BCFA when grown under low temperature and /or high pressure. EPA, but not MUFA was determined to be crucial for its growth at low temperature and high pressure.The microarray of WP3 was used to do the transcriptome analysis of WP3 in response to different growth temperatures （4℃and 20℃） and pressures （0.1Mpa and 20Mpa）. Among the genes involved in the fatty acid synthesis, only a gene cluster for branched chain amino acid ABC transporter （swp3487-3492, LIV-I） was up regulated by low temperature. Quantitative RT-PCR clearly verified the induction of these transporter genes by low temperature. To determine if this gene cluster is responsible for the BCFA increment at low temperature, mutations were constructed on orfswp3487, which encodes the only putative substrate-binding protein of this ABC transporter as described in the materials and methods. The LIV-I deficient mutant was named WP3^ΔLIV. The fatty acid profiles of WP3^ΔLIV grown under different conditions were characterized. Interestingly, its fatty acid profile was very similar with that of the wild type strain when they grown at 20℃and 0.1Mpa. However, the mutant strain WP3^ΔLIV almost lost the low-temperature and high-pressure dependent BCFA regulation ability. By using the isotopic tracer method, we further verified that the regulation of BCFA synthesis is controlled by the transporter efficiency.Genome sequencing of the deep-sea bacterium Shewanella piezotolerans WP3 suggests that it may harbor a functional filamentous phage （named as SW1）. SW1 has 7718 nucleotides, contains 9 open reading frames （ORFs） encoding putative proteins which are homologous to that of previously known filamentous phages. Interestingly, a putative ssDNA binding protein （ORF104） of the phage, which is determinant protein for phage particle assembly, was found over-expressed at 4℃versus 25℃by two-dimensional polyacrylamide gel electrophoresis （2D-PAGE）. According to this clue, the active phage particles were isolated from WP3; it has a single-stranded DNA （ssDNA） genome, able to integrate into the chromosome with a single copy, and also exists as extra chromosomal plasmid. The host range of SW1 was tested using Shewanella oneidensis, Shewanella marinintestina, Shewanella violacea, Shewanella psychrophila WP2 and E. coli. SW1 can only infect WP2 and form phage plaques on plates when culture temperature was equal to or lower than 15℃. It implied that the SW1 production may be regulated by temperature. To testing its temperature stability, 70℃about 10min will totally inactivate its activity, which is 10℃lower than other filamentous phage’s maximum temperature. Reverse-transcription quantitative PCR （RT-QPCR） analysis further revealed that the transcription of the key genes of SW1 including the putative repressor gene orf116 was significantly induced （3-12 times） at low temperature （4℃）. Followed, phage particles were isolated from WP3 cultures incubated at different temperatures （4℃, 10℃, 15℃, 20℃, and 25℃）. The quantity of the phage particles were enumerated by counting the phage plaques formed on WP2 cell lawns on the agar plates. No or few active phage particles were isolated from cultures of 20, 25℃as no phage plague was observed. Active phage particles were isolated from the cultures of WP3 at 4℃, 10℃and 15℃. The WP3 cell culture at 4℃yields higher number of phage particles than that from 10℃and 15℃. This data clearly demonstrated that the phage production is temperature regulated, and low temperature can induce the phage production in WP3.SW1 is also induced by Mitomycin C and UV irradiation, indicating that the production of the phage depends on the cellular SOS response. However, the transcription of orf116 is not induced by MMC and UV, which is different from that at low temperature, implying that the mechanism of low temperature inducement may be not the same as the SOS response inducement.更多还原