【作者】 李曙光；

【导师】 李越中；

【作者基本信息】 山东大学 ， 微生物学， 2014， 博士

【摘要】 粘细菌(myxobacteria)是一类能够滑动运动的革兰氏阴性杆菌,在系统进化上对应变形菌门、δ变形菌纲的粘球菌目(Myxococcales),分为3个亚目,23个属和50多个种。粘细菌能够产生丰富的次级代谢产物,被公认为是继放线菌之后的又一个重要的药源微生物新类群；因其具有复杂的多细胞群体行为和能形成多细胞聚集、形态特异的子实体结构,被认为是“社会性的细菌”。粘细菌曾被认为是一类典型的土壤微生物,近年来浅海水域分离得到嗜盐和耐盐菌株；分子生态学研究表明在海洋不同深度、不同位置的沉积物中均有粘细菌分布。此外,一些文献报道了湖泊河流中粘细菌的分离纯化,但这些菌株在形态上与陆地粘细菌高度相似,长期以来人们普遍认为水流或风力把陆地粘细菌的子实体带入淡水环境,淡水生境只是粘细菌的“暂居地”。因此,淡水环境是否为粘细菌的重要生境依然不清晰。我们在程海湖采集沉积物样品,进行菌株分离纯化,共获得113株粘细菌,绝大多数菌株隶属于Myxococcus、 Sorangium和Nannocystis,与分离自土壤的典型菌株形态高度相似,且16S rRNA基因的差距在2%以内；其中三株菌位于进化树的独立分支,与Cystobacter gracilis的相似性在92.2%与97.5%之间,可能属于新的种属。使用试剂盒提取1.0g沉积物的总DNA,并分两步对沉积物中的细菌群落进行454高通量测序。首先,使用两对通用细菌引物分别扩增16SrRNA基因的V3-V4高变区和V6-V8高变区,构建通用细菌文库UV34和UV678,分别获得25305和30604条高质量序列,包括2546和5559个种(以0.03的差异水平划分OTU)。粘细菌是沉积物细菌群落中的主要种群,占据序列数量的5.77%(在目的水平据第三位；两个文库中分别为7.25%和4.29%)和种类的7.52%(据第一位；两个文库中分别为9.62%和5.41%)；粘细菌中较多的几个属是Archangium(16.63%)、Anaeromyxobacter(9.14%)、Kofleria(8.77%)、Chondromyces(4.45%)和Phaselicystis (4.15%),其他属均低于3%。然后,分别使用Cystobacterineae和Sorangineae特异引物构建粘细菌的特异文库CV34和SV678,Cystobacterineae中较多的几个属是Anaeromyxobacter、Cystobacter和Corallococcus; Sorangineae中较多的属是Byssovorax、Sorangium和Phaselicystis。随后,我们调查了公开发表的高通量测序数据,在三个研究共39组普通淡水沉积物样品中(290658条细菌序列),粘细菌普遍存在,平均数量比例为(3.73±2.84)%,但不同样品中粘细菌的内部分类单元组成差距较大；在普通淡水水体和温泉环境中,粘细菌的比例则很低(<0.3%)。这些结果表明普通淡水沉积物是粘细菌的重要生境。土壤、海洋和淡水粘细菌的系统发育比较分析显示,陆地、海洋粘细菌在高分类水平(科)分离,即使在同一个分支,海洋粘细菌和陆地粘细菌的序列差异一般也比较大；淡水环境粘细菌一般和陆地粘细菌处于相同分支,与陆地序列亲缘关系近于海洋序列,但仍有部分序列形成了独立于陆地、海洋粘细菌的特有分支(特有的分类单元)。另一方面,粘细菌在土壤生境中分布广泛,但因其捕食特征和多细胞群体行为,而被认为是一类数量偏少的种群。一个常规土壤样品(SDU)的宏基因组测序显示,粘细菌在数量和种类上分别占4.10%和7.75%,是优势菌群。为深入理解粘细菌多样性的横向范畴(环境分布)和纵向范畴(季节演替),我们详细考察了公共数据库各类生境中粘细菌的存在及数量比例。在森林、草地、农田等普通土壤中(525组数据,6776656条细菌序列),粘细菌在细菌群落中的比例约为(3.01±±2.35)%；在海滩泥沙、极地的苔原冰川沙漠等非典型的土壤环境,粘细菌的平均数量比例也在1%以上；在普通淡水环境沉积物中,粘细菌分布广泛且数量较多(3.73%)；普通淡水水体中粘细菌分布较为普遍,存在于85.8%的样品,但数量比例较低,仅为0.23%；在温泉及海洋环境中,粘细菌数量一般小于0.2%,且存在不具有普遍性(26.7%-68.5%)。分析全球各地320组有采样时间记录的土壤样品,其中粘细菌的平均比例为3.41%(粘细菌数目与细菌数目的Pearson相关性分析,R2=0.6827,F检验P<0.001)。在不考虑样品之间差异的情况下,粘细菌的比例似乎有随季节周期性变化的趋势,在春季(3-5月)和秋季(9-11月)较多,而夏季和冬季则减少。选择一组对样品进行连续监测(5,6,7,8,10,11六个月)的测序数据(凯洛格生物站测序样品),其中粘细菌的平均比例为(2.27±0.69)%,在目的水平据第8位。三种土壤类型中粘细菌都有8月份数量、种类减少的趋势；这与整个细菌群落多样性(以Faith’s phylogenetic diversity作为指标)以及其他捕食性细菌的变化趋势均不相同。粘细菌的波动幅度最小,且数量维持在稳定的较高水平(1.2%-3.2%)。三种土壤类型的细菌群落多样性几乎都与土壤湿度和温度显著相关,而环境参数对各类捕食性细菌的数量比例影响均不明显；推测环境因素对微生物群落的影响并非具体到每一个类群,粘细菌的数量变化是多种环境参数、甚至生物因素综合作用的结果,而每种参数对其影响都不是线性的,需要结合实际情况进行分析：8月份样品采集前持续的高温干燥导致了粘细菌进入休眠状态,即形成子实体,期间大量的细胞(60-90%)发生程序性死亡致使粘细菌数量显著下降；之后经过几次降水,土壤湿度回升,10月份(秋季)样品中粘细菌数量比例上调；到了11月份,低温(促使粘细菌以形成子实体的形式休眠)逐渐成为限制因素,其数量增长变慢甚至略有下降。土壤类型对粘细菌的影响可能表现在最大值出现时间、数值波动幅度等方面。统计各样品的粘细菌内部组成并进行聚类分析,样品没有因土壤类型或采集时间聚集,说明粘细菌的内部组成也是多种因素(土壤类型、采集时间、各种环境参数、生物因素等)综合作用的结果。粘细菌各科随季节的变化趋势各不相同,甚至有此消彼长的趋势,暗示了粘细菌群落中各分类单元具有差异性,存在着种间相互作用。产自纤维堆囊菌的大环内酯类化合物埃博霉素(Epothilones),是一种作用机理类似紫杉醇的抗肿瘤药物,目前已有结构类似物商品化上市。相对于埃博霉素需求的日趋增长,通过纤维堆囊菌发酵产生的埃博霉素产量极低成为其价格高昂的主要原因。埃博霉素产生菌只占分离菌株数目的2.5%,是相对稀缺的资源菌株；纤维堆囊菌生长缓慢,遗传操作困难,而异源表达埃博霉素的产量经过发酵优化后仍然很低。因此,我们采取了菌株改良与发酵优化外的两种策略：寻找更多的埃博霉素产生菌,以期分离到具有优良性状的菌株,用于后续的基因操作,还可以对比研究埃博霉素的合成机制；利用纤维堆囊菌的共培养,验证种内相互作用是否像种间相互作用一样影响(提高)次级代谢产物的产量。结合前期工作中获得的菌株,我们发现相对于未知土壤的低埃博霉素阳性菌株比例(2.5%),在阳性土壤中进行菌株二次分离,可以获得较多的埃博霉素产生菌(25%-75%)；在一个阳性土壤附近10平方千米内的几个土样中,阳性菌株的比例也较高(27.3%)。分析一个阳性菌株(So0157-2)的基因组发现,埃博霉素基因簇连同上下游共计128kb的区段可能来自水平基因转移。对比三株菌的埃博霉素全基因簇,其相似性约为98.5%；埃博霉素基因簇中的酰基转移酶(AT)结构域显著区别于其他来源(非纤维堆囊菌或纤维堆囊菌中其他基因)的AT；在4个已报道埃博霉素基因簇的纤维堆囊菌中,同一模块中的AT蛋白序列差异水平都小于3.2%；14株纤维堆堆囊菌的ATModC2(epoC模块的第2个AT结构域)的蛋白序列的最大差异约为5.8%。这说明了埃博霉素基因簇的高度保守与相对多样。单个样品中获得的16S rRNA基因序列最大差异约为3.1%,与不同土壤来源的菌株序列差异相当,而埃博霉素产量更是相差2000倍以上,这说明了单一样品中纤维堆囊菌的多样化。对于遗传操作复杂的微生物,寻找更多的阳性菌株是一种可供选择的简明有效策略。在两个不同土样各选择6株纤维堆囊菌(产生埃博霉素或不产生埃博霉素)进行共培养的影响研究。菌株之间在生长上均呈现明显的相互抑制,双方生长变弱,生物量低于纯培养物对照(两个纯培养物生物量之和)的35%,推测由空间和营养的争夺所致。共培养大大影响了埃博霉素产量,45个组合平均提高倍数为2.45±3.25；33个组合的产量显著改变(提高或降低倍数在50%以上)；29个(64.4%)组合产量提高,最大提高倍数为10.4。荧光定量PCR显示,共培养物中埃博霉素的8个基因区段表达量均上调,提升倍数为2.41-7.16。可能是因为基因簇上存在不止一个启动子,或者mRNA的不同区段降解速率不同,导致了各区段检测到的表达量提升有所不同。当然,影响埃博霉素基因簇转录水平改变的深层机制还有待阐明。本工作确认了粘细菌在淡水沉积物中的普遍存在,表明淡水沉积物是粘细菌的重要生境；详细探讨了粘细菌在各类环境中的分布及多样性,发现土壤粘细菌数量比例有随季节动态变化的趋势；粘细菌内部各分类单元的季节性变化趋势各不相同。发现阳性土壤中埃博霉素产生菌株的比例大大高于未知土壤；阳性菌株似乎能够扩展到阳性土壤的邻近环境,从而在一个较大范围内形成了具有多样基因组成的埃博霉素产生菌资源库。纤维堆囊菌的共培养能够通过基因簇转录水平的变化显著地改变埃博霉素的合成能力。更多还原

【Abstract】 The Gram-negative myxobacteria are phylogenetically located in the delta division of Proteobacteria, corresponding to the order "Myxococcales", including three suborders, twenty-three genera and more than fifty species. Myxobacteria are famous for their complicated multicellular behavior and excellent production ability of secondary metabolites, and are thus among important microbial resources for drug screening and of model organisms for the studies of prokaryotic intercellular communication, multicellular morphogenesis, and biological evolution.Myxobacteria are recognized as typical soil bacteria. Recently, halophilic and halotolerant myxobacterial strains have been isolated from coastal areas; molecular surveys have indicated that myxobacteria-related16S rRNA gene sequences are also widely distributed in marine sediments at different depths and sites. Besides, several publications have reported the isolation of myxobacteria from river or lake. However, the accuracy of these early isolations was questioned because the limnetic isolates were highly similar in morphology to soil myxobacteria. It has long been accepted that these myxobacterial isolates from aquatic environments germinated from myxospores or myxospores-containing fruiting bodies that had been washed or blown into rivers or lakes. Thus, it is still unknown whether freshwater environments are also native habitats for myxobacteria.We collected mud sample from Chenghai Lake, and isolated113myxobacterial strains (87cellulolytic strains and26bacteriolytic strains), most of them belong to Myxococcus, Sorangium and Nannocystis, with high similar morphology and16S rRNA gene sequences to their soil relatives. Interestingly, three of them may belong to new genera. Then we investigated the presence of myxobacteria in lake mud using a two-step strategy by454pyrosequencing. First, we constructed two universal bacterial libraries from the V3-V4(UV34) and V6-V8(UV678) hypervariable regions of16S rRNA gene sequences. High-throughput454pyrosequencing revealed that myxobacteria were one of the major bacterial groups in the lake mud. They accounted for5.77%of the total sequences and7.52%of the total operational taxonomic units (OTUs) at a phylogenetic distance of0.03. The community composition and taxonomic structure of the mud myxobacterial community were further analysed using myxobacteria enriched libraries targeting the V34and V678regions, which were amplified with Cystobacterineae and Sorangineae-specific primer pairs respectively.In addition, we surveyed several published data in GenBank, which showed that myxobacteria are widely distributed in normal limnetic sediments, and accounted for3.73%of total bacteria sequences. However, the compositions of myxobacteria varied among different samples. In normal limnetic water body and hotspring environments, myxobacteria have a very low proportion (<0.3%) among bacteria. These results indicated that normal limnetic sediments are primary habitats for myxobacteria. Comparatively phylogenetic analysis showed that the limnetic myxobacteria exhibited closer relationships to their soil than their marine relatives, but there were also exclusive taxa of limnetic myxobacteria detected.The concept that myxobacteria are widely distributed is commonly held beliefs, however, as their multicellular behavior and microbial-predation characteristics, myxobacteria are recognized as minor bacteria group. To understand the diversity of myxobacteria in levels of space and time, we surveyed public databases. For normal soil samples in forest, grassland and agricultural lands, myxobacteria have a proportion of3.01%; surprisingly, myxobacteria account for more than1%in beach sands and deserts, glacier foreland, tundra of high latitude region; myxobacteria are widely distributed in normal limnetic environments, but have two distinct proportions among sediments (3.73%) and water body (0.25%); hotspring and ocean are not preferred habitats for myxobacteria, where they had lower distribution frequencies (26.7%-68.5%) and quantitative proportions (<0.1%).Then we selected320global soil samples that have clear collection times, which had an average proportion of3.41%. Taking the heterogeneity of different samples out of consideration, myxobacteria population seems have a seasonal changing, i.e., a larger numbers in spring (February to May) and autumn (September to November), and reduced in summer and winter. Afterwards, we surveyed a set of pyrosequencing data with samples collected from May to November. In three soil types, a-Diversity of bacteria community was significantly correlated with soil moisture and temperature. However, neither of the predatory bacteria was significantly correlated with single environmental factor, and their changing tendencies differed with each other and total bacteria. The amount of myxobacteria reduced in August, which may be resulted from long time high temperature without of rainfall, but other complicated factors (environmental or biotic factors) effect collectively. The changing tendencies of different myxobacteria families are not identical, which indicated that myxobacteria are not a homogeneous community, and complicated interactions works.Sorangium producing epothilones are16-membered macrolides that mimic taxol-induced microtubule stabilization, thus leading to mitotic arrest at the G2-M transition and cytotoxicity in proliferating cells. Four epothilone modified derivatives has been approved for clinical use by the U.S. Food and Drug Administration. However, in contrast to increasing progress in their applications, the low production of epothilones in Sorangium strains makes the price very expensive. Epothilone producing strains account only a small proportion (about2.5%) of total S. cellulosum strains; Sorangium cells grow slowly and hard to be genetically manipulated, however, epothilone productions are very low among heterologous hosts even in optimized ferment conditions. In this study, we take two additional strategies rather than stains or culture conditions optimized:First, searching for more epothilone producers to obtain potentially suitable characteristics for further genetic modification, and comparative analysis the biosynthetic mechanisms; Second, culture Sorangium strains pairwise to learn whether intraspecies interactions affect the production of anti-fungal epothilones.Compared with the<2.5%positive strains collected from different places, epothilone producers comprised25.0-75.0%of the Sorangium isolates in positive soil samples. These sympatric epothilone producers differed not only in their16S rRNA gene sequences and morphologies but also in their production of epothilones and biosynthesis genes. A further exploration of14soil samples collected from a larger area around a positive site showed a similar high positive ratio of epothilone producers among the Sorangium isolates. Thus, in an area containing epothilone producers, the long-term genetic variations and refinements resulting from selective pressure form a large reservoir of epothilone producing Sorangium strains with diverse genetic compositions.Co-cultivation on filter papers showed that different Sorangium strains inhibited one another’s growth, whereas epothilone production by the producing strains changed markedly for most (73%) pairwise mixtures. Using a quantitative polymerase chain reaction, we demonstrated that the expression of epothilone biosynthetic genes in the epothilone producers typically changed significantly when these bacteria were mixed with non-producing strains. The results indicated that intraspecies interactions between different S. cellulosum strains not only inhibited the growth of partners, but also could change epothilone production. Future studies are needed to determine the factors involved in the expression level changes of the epothilone biosynthetic clusters.To summarize, we demonstrated that myxobacteria are widely distributed in limnetic sediments as a predominant group, and limnetic mud is a primary habitat for myxobacteria; then we descripted the distribution of myxobacteria in various environments and seasonal succession of the community and sub-group. Epothilone producing strains are more likely appeared in positive places and neighborhood than unknown samples; co-cultivation of different Sorangium strains could markedly changing epothilone production, as a result of the changed expression level of biosynthetic genes in the epothilone producers.更多还原