【作者】 周秀文；

【导师】 李越中；

【作者基本信息】 山东大学 ， 微生物学， 2013， 博士

【摘要】 粘细菌目（Myxococcales）分类上位于变形菌门Proteobacteria δ分支,目前分为3个亚目,7个科,20个属以及约50个种。因其能够形成对温度、干旱、辐射、盐胁迫均具有抗性的粘孢子,而具有较高的环境适应力,广泛分布于全球各地的土壤中。典型生境即为土壤,除此之外,草食动物粪便、腐败植物表面、树皮、树叶表面甚至水体中也发现粘细菌的存在。传统分离方法在分离菌株种类以及效率上的局限性,使得我们对粘细菌的生态学研究并不深入,到目前为止我们对于土壤中粘细菌的丰度以及群落结构仍然未知。不依赖于培养的分子生态学研究方法弥补了这一不足,而二代测序技术的成熟和普及使得分子生态学研究进入大数据时代,公共数据库的建立与开放更为研究者们提供了信息共享的平台。基于以上所关注的生态学问题,首先采用传统的分离纯化方法分离普通花园土中的噬细菌粘细菌,并通过定量PCR对土壤中粘细菌在细菌中的比例进行了检测,以此了解该土壤中粘细菌的大致情况。发现该土样中粘细菌比例较高,为细菌总数的2.04%-3.42%,但是分离得到的菌株种类较为单一以粘球菌属、珊瑚球菌属和孢囊杆菌属为主,并且16S rRNA基因序列与已知序列差异较小,从而反映出传统分离方法的局限性。其次利用通用引物扩增土壤中所有细菌16S rRNA基因片段,结合454高通量测序分析粘细菌在整个细菌群落中的丰度,结果显示粘细菌序列数量在总序列数量中占4.10%,以97%的序列相似度划分OTU发现粘细菌比例高达7.5%,成为第二大种类丰富的目。我们对公共数据库中分布全球的土壤高通量测序数据进行统计,粘细菌丰度在大部分土样中均保持较高的水平（>1%）,可见土壤中的粘细菌无论数量还是种类上均是一类重要的细菌,虽然我们进一步调查各土样理化性质与粘细菌丰度之间的相关性没有能够确定影响粘细菌种群数量的环境因素,但是推测以降解大分子物质为主的粘细菌的种群规模可能与更为复杂的生物因素相关。最后通过粘细菌两个亚目的特异引物进一步富集土壤中的粘细菌序列并分析这些序列特征以揭示粘细菌的群落结构和特征。种以下水平的聚类分析发现,虽然粘细菌群落结构中各科之问的相对数量依不同土样而差别较大,但即使在优势种属中种以下水平却存在大量差异较少的序列,具有相同序列的细胞均处于较低的数量水平,这说明粘细菌群落内并不存在一个优势的细胞系,而呈现为整体低丰度的多样化菌群,这构成了粘细菌群落的特征。这种群落结构意味着同一生态位中存在着较多的近缘粘细菌。那么衍生出的第二个生态学问题即是这些近缘粘细菌之间的相互关系是怎样的。粘细菌虽然为单细胞的原核生物,但是却具有复杂的群体行为,细胞间的合作贯穿并维持着粘细菌的整个生活史,无论是感应饥饿信号进行细胞聚集还是依赖菌毛pili与胞外基质结合的群体运动,均需要通过细胞间的信号或细胞表面物质的交换传递进行细胞的调度才能完成,这些精密的时间与空间上的个体协调与合作已经研究得较为深入。但是近缘粘细菌的关系则停滞于表象,研究者们仅通过混合菌株共发育的试验推测近缘粘细菌之间是相互抑制的关系。我们对分离自同土样的粘球菌进行混合培养后通过测序鉴定最后存活的菌株,进一步将优势菌株与劣势菌株之间的互作进行不同数量比例、不同时间尺度、不同共培养方式的详细分析,发现优势菌株与劣势菌株临近培养形成扩展界线,类似Dienes线而混合培养则相互抑制,但这种抑制作用并不依赖接触,可能通过生长分泌某种效应物发挥作用,并且抑制作用可能与起始菌株密度密切相关甚至可能因为起始数量的巨大差异而出现优势与劣势的地位逆转。在经过对优势菌株与劣势菌株的基本性质分析而无法得出优势菌株的优势所在后,我们转而对粘细菌中已报道的次级代谢产物Myxovirescin A进行分析发现优势菌株与劣势菌株均不能产生该抗生素,从而推测其可能不参与粘球菌近缘菌株之间的抑制。在我们发现近缘粘细菌之间的关系为相互抑制但是这种抑制作用远比预计的更为复杂,而且找寻效应物无果的情况下,我们选择在遗传背景较为清楚的模式菌株M.xanthus DK1622中继续研究其内在的机制。粘细菌的A运动和S运动的双运动系统、胞外多糖的产生与调控以及细胞进入发育阶段并分化形成子实体的信号途径的详细研究均是在该菌株中进行,并且其基因组序列已经获得注释更利于基因功能的阐明。由于近缘菌株之间能够形成扩展不融合的界线,依据该表型我们对转座子随机插入的突变株进行筛选,得到11株对亲本识别出现变异的亲缘识别（SI）突变株。这些突变株彼此之间同样产生界线,但这些突变株纯培养的基本社会学行为正常表明其相同基因型细胞之间的合作正常。将突变株与DK1622进行混合共培养发现他们存在不同的抑制等级,这种现象同野生近缘粘球菌优势菌株与劣势菌株之间的表现相同。经过插入位点定位,发现11株突变株涉及九个插入基因位点,两对插入位点距离较近,而且大部分为未知功能基因。检测其中一株突变株中其他八个基因位点的转录情况,结果表明单一位点的插入失活并未影响到其他SI位点的转录。综合以上结果,我们推测这11株SI突变株可能分别涉及多条不仅独立于已有社会学行为途径而且彼此之间相互独立的亲缘识别通路。经敲除和回补验证仅确定四株突变株中的相关基因或基因范围,分别为MXAN₀049（SI1114）、MXANN0085（SI1105）和MXAN₁307-MXAN₁309（SI1107、SI1108）,已经确定的基因之间存在一定的相关性,即序列相似度在20-30%之间,并且在DK1622之外的基因组中也发现存在类似基因,通过替换基因验证表型发现M.fulvus HW-1中的同源基因LILAB₀8510确实可以在DK1622中发挥与MXAN₀049相同的功能,从而暗示了其他菌株甚至其他非粘细菌菌株中也可能存在相同机制的异己识别。Dienes界线早在1946年就在Proteus mirabilis不同临床菌株中发现（Dienes,1946）,但是至近几年其背后的相关基因才得以揭示。与本论文思路相同,其经过筛选突变株最终确定两个基因簇Ids和Idr参与依赖Ⅵ型分泌系统但相互独立的两个途径,但是关于他们的基因功能和产物特点则没有深入研究（Gibbs et al.,2008, Wenren et al.,2013）。与本论文结果不同的是该菌产生的界线被证明均为活细胞。我们对其中一个基因MXAN₀049的进一步分析,发现该基因所编码的蛋白定位于胞内,并且该基因位于一个基因簇内,通过蛋白相互作用钓取并鉴定与MXAN₀049相互作用的蛋白发现其基因簇内基因MXAN₀50编码产物可能与其结合,暗示功能上的相关性。MXAN₀049基因的敲除导致突变株在营养生长状态以及发育生孢阶段均被野生株抑制,并且这种抑制并不依赖细胞直接接触。而Δ50与DK1622融合,同样与Δ49也融合,即该突变株与DK1622和Δ49均不相互抑制。综合以上结果,我们推测,可能编码rhs蛋白的MXAN₀050负责另一种亲缘识别的表型,该基因簇及其附近基因可能通过调控细胞的攻击与防御系统实现界线的形成。无论基于细胞之间合作的粘细菌的各项社会学行为,还是自然生境中同一生态位中不同土著粘细菌之间的共存,细胞运动或外力导致的不同粘细菌的侵入,均是以细胞之间的亲缘识别为基础的。这种能力微观上成为粘细菌各种群体行为的基础,使其具有较强的环境适应力,宏观上决定了粘细菌群落结构的组成和共进化的特点。通过本论文的研究,我们揭示了粘细菌异己识别的相关基因并对其功能和突变株性质进行了初步研究,为进一步认知亲缘识别机制提供了线索和启发。更多还原

【Abstract】 The order of Myxococcales is located in δ group of Proteobacteria, including three suborders, seven families, twenty genera and about fifty species. Due to formation of spores resistant to high heat, desiccation and UV, myxobacteria have adaptability to a wide range of environments, such as soils spreading all over the global. Besides of soil, myxobacteria have also been isolated from samples collected in dung of herbivores, rotting wood, bark, leaf surface and even ocean. Because of characteristics of myxobacteria, the traditional isolation and purification methods is time-consuming and limited to some particular population of myxobacteria, limiting the knowledge of myxobacterial ecology. Till now, we have no idea about myxobacterial abundance and community structure. As the maturity and popularity of next generation DNA sequencing techniques impelled us to a period of massive data and the establishment of public metagenomic database provided researchers a sharing platform, culture-independent molecular methods have initiated a new era of microbial ecology.Base on the above ecology issues we concerned, this study was carried out as follows:Firstly, myxobacteria strains in normal garden soil sample （named SDU soil, collected from Shandong University） were isolated by traditional methods and the percentage of myxobacteria among bacteria in this soil sample was detected by quantitative PCR. It was suggested that the proportion of myxobacteria in soil was2.04%～3.42%, but the species of culture strains were rare, predominant in Myxococcus, Corallococcus and Cystobacter with similar16S rRNA gene sequence to type strains, indicating the limitation of the culture-dependent method.Secondly, we surveyed the percentage of myxobacteria in a single soil sample via pyrosequencing on combined universal libraries of the V3-V4and V6-V8hypervariable regions of the16S rRNA gene. Surprisingly, myxobacteria accounted for4.10%of the bacterial community and7.5%of the total operational taxonomic units （OTUs） at the3%similarity level in the soil, containing almost all of the cultivated myxobacterial families or genera. To testify the appearance of myxobacteria in soil niches, we retrieved myxobacteria-related16S rRNA gene sequences of103high-throughput sequencing datasets obtained from public databases. The results indicated that myxobacteria-related sequences were among the predominant groups in these datasets, accounting for0.4%to4.5%of bacterial communities. Based on these results, we can presume that myxobacteria are a predominant terrestrial bacteria group in soil. The correlation was weak between myxobacteria percentage and environmental parameters calculated by Spearman’s rank, indicating the abundance of myxobacteria communities might be influenced by complex biological factors.At last, to conduct a sub-population census of myxobacterial community, two myxobacteria-enriched libraries of Cystobacterineae and Sorangineae suborders were constructed using myxobacteria-semi-specific primers. Although different taxa were predominant or rare, we observed a further division into many lower taxonomic units in myxobacteria-enriched libraries, resulting in low frequencies of each eco-type. These unprecedented results reveal that myxobacteria utilize an egalitarian and reciprocal strategy for community survival in the soil niche.According to the conclusion, the next ecology issue was raised:how is the relationship among the allied species? Myxobacteria are, though unicellular, famous for their complex social behaviours and the cooperation between genetic identical cells maintains throughout the entire life cycle. When cells aggregate after sensing the signal of starvation or move in a group dependent on binding of type VI pili on extracellular matrix, the exchange of signals or proteins on cell membrane is necessary to schedule preciously thousands of individuals. Though these pathways controlling the motility at time and space scales have been studied in detail, study about the interaction between different myxobacteria strains was stagnated on appearance. Exploitation had been reported might occur frequently in Myxococcus strains when mixed together, which was also observed between different Myxococcus spp. strains isolated from SDU soil. What’s more, we further analyzed the inhibition between two different strains in different mixing ratio, for different time and in different forms of co-culture. We found that when the two strains were inoculated in adjacent pair on growth medium, a clear boundary, like Dienes line in Proteus mirabilis, formed between the expanding colonies, while when they were mixed together, one strain was inhibited even killed by the other. The exploitative hierarchy could be converted, which was depended on mixing ratio but independent on direct cellular contact. The following results suggested that these strains could not product antibiotic Myxovirescin A in these strains to confirm it did not play role in the competition between Myxococcus strains.To disclose the involved mechanism of complex competition between different Myxococcus strains, we sought to identify self versus non-self discrimination factors required for boundary formation in model strain M.xanthus DK1622with clear genetic background. The pathways of myxobacterial A-motility, S-motility, production and regulation of exopolysaccharide and development were studied in this strain. We screened3,392inserted mutants generated by transposon plasmid and obtained eleven SI （self identification） mutants that recognize their neighbours as different from self, forming clearly visible boundaries between two colonies and with their parent strain DK1622as well. Though the SI mutants failed to merge with wild type strain, the social phenotypes of pure SI mutants were normal, indicating the cells from genetic identical SI mutant retain the capability to cooperate with each other. Whereas, the exploitative hierarchy was also found in SI mutants when different SI mutants were mixed and inoculated on growth plate, just like different wild Myxococcus strains. Mapping the insertion loci of transposon in eleven SI mutants showed nine loci scattered throughout DK1622genome and none of the inserted genes were reported involved in known pathway or system. The results of quantitative RT-PCR showed that the expressions of other eight SI loci were normal in SI1103（inserted in upstream of MXAN₅062）. These results indicated that there were several independent pathways involved kin discrimination and independent on other social behaviour systems. Three gene loci were identified to be responsible for formation of boundary by deletion and ectopic complementation, ie., MXAN₀049（SI11114）, MXAN₀085（SI1105） and MXAN₁307-MXAN₁309（SI1107, SI1108）. It is interesting that these genes share a sequence similarity with20-30%, and it also seems likely that other species of bacteria have homologous genes encoding self-recognition, which had been verified by replacing MXAN₀049with its homologous LILAB₀8510from M.fulvus HW-1.The Dienes mutual inhibition test has been used as an epidemiological tool to characterize isolates of Proteus mirabilis and Pseudomonas aeruginosa. Recently, by screening the mutants in the same way with us, Gibbs found that two loci were essential for the behaviour, and the two independent pathways were linked by a shared machinery, might type VI secretion system, for export of encoded self-recognition elements. But the biochemical mechanism remained to be identified.The next we focused on gene MXAN₀049for further analyze. As for the function of49, the sequence analysis might give a clue. MXAN₀049had30%identity with IpaC, which has been shown to associate with IpaB to form a translocation pore that enables the secretion of effector proteins by Shigella directly into target cells. However, we found that MXAN₀049was located in cytoplasm. RT-PCR showed that the MXAN₀049gene is part of an operon spanning MXAN₀046to MXAN₀050. What’s more, protein MXAN₀050might interact with MXAN₀049by his-tag pull down. The inhibition also occurred between deletion mutant of MXAN₀049and wild type strain DK1622both on CTT plate and TPM plate, resulting in disappearance or induction of△49. But deletion mutant of MXAN₀050swarmed merging with△49and DK1622. Therefore, we proposed that MXAN₀049and MXAN₀050which shared a similarity of26%with rhs protein were responsible for boundary formation by controlling attack and defense system.Intraspecies territoriality separates incompatible groups of single-celled bacterial species to prevent harm, usually mutual harm, from occurring. We hypothesized that in natural environment after adaptive evolution, different strains of one myxobacterial species localize to separate mini-territories to avoid cannibalism resulting from mixing and thus survive. Accordingly, kin discrimination is important not only for Myxococcus sociality, but also for their ecological functions, as well as speciation.更多还原