土壤及海底沉积物中可培养与未可培养粘细菌系统发育多样性研究
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摘要
粘细菌(Myxobacteria)是一类革兰氏阴性滑动细菌。粘细菌由于具有复杂的细胞社会行为和能形成多细胞聚集、形态特异的子实体结构而被视为“高等细菌”。具有鲜明色彩的子实体是在环境条件恶劣或营养条件贫瘠的情况下,由成千上万的营养细胞聚集并分化发育而成的。由于粘细菌子实体及其内含粘孢子的抗逆性,粘细菌能够广泛地分布在地球的各个地方,特别在土壤、腐木、树皮和动物粪便中最为普遍。但是粘细菌的生态学研究明显滞后于其它土壤微生物,这是因为粘细菌的许多特殊性状使得粘细菌的分离与纯化相对于其它细菌更为耗时和困难;同时,由于粘细菌子实体结构容易退化和丢失,因此传统的粘细菌分离纯化方法容易遗漏粘细菌菌株。因此,分离到的可培养粘细菌可能只是代表了自然环境中粘细菌类群的一小部分。随着不依赖培养(Cultivation Independent)方法的完善和发展,研究者已经有能力对环境中那些行使重要功能但至今无法培养的微生物进行群落组成和系统发育关系等方面的分析;同时随着公共核酸数据库GenBank中粘细菌16S rRNA基因序列的快速、大量增加也使我们通过分子生物学方法分析环境中粘细菌的系统发育多样性成为可能。在本研究中,我们利用粘细菌传统的分离纯化方法以及分子生态学方法来研究土壤及海底沉积物中粘细菌的系统发育关系及其生态多样性。
1.我们从实验室粘细菌资源菌库中选取了37株粘细菌菌株进行形态鉴定和菌株亲缘关系的分析。粘细菌的形态特征,尤其是子实体形态是粘细菌种属分类的主要依据,但由于子实体结构经过传代培养很容易退化甚至是丢失,给这些菌株的准确分类带来了很大的困难,如本研究中的孢囊杆菌亚目(Cystobacterineae)菌株0082-2、0085-4、0121-3、Myx9736和NM03以及堆囊菌亚目(Sorangineae)菌株So0007-16、So0157-52和So139-5的子实体结构都发生了不同程度的退化。通过对菌株16S rRNA基因序列的系统发育关系的分析,能够提高传统形态分类的准确性。在本研究中,通过对孢囊杆菌亚目15株菌株和堆囊菌亚目22株菌株的16S rRNA基因序列的系统发育关系的分析表明,大多数的粘细菌属在系统进化树上能形成各自独立的分支。说明在属及属以上水平,粘细菌形态分类和16S rRNA基因的系统发育关系具有较好的一致性。但在种水平上16S rRNA基因序列所揭示的系统发育关系可靠性较低。在此,我们使用了HSP60(groEL)基因序列来分析粘细菌近缘菌株之间的系统发育关系。在本研究中,我们得到了孢囊杆菌亚目5株菌株和堆囊菌亚目堆囊菌属(Sorangium)22株菌株的groEL1部分基因序列。通过分析系统发育关系表明,groEL1基因能够更为准确的揭示粘细菌种属间的系统发育关系,弥补了16S rRNA基因序列在鉴定粘细菌种属中的不足。同时发现粘细菌具有重复拷贝的HSP60基因,并扩增得到了9株堆囊菌属菌株的groEL2基因。通过分析系统发育关系发现,groEL2基因也可以作为堆囊菌属内系统进化分析的分子标记,且相同菌株不同拷贝的groEL基因序列同源性都在78%左右,这暗示着重复拷贝的groEL基因来源于同一个祖先基因,但却表现出明显不同的进化速率。
2.利用本研究得到的37个粘细菌菌株16S rRNA基因序列和70个GenBank中已公布的粘细菌16S rRNA基因序列信息来设计粘细菌亚目水平特异的寡核苷酸引物/探针,以从SDU土样中提取的总DNA为模板,建立了W1/1492R和W4/1492R两个类型文库来分别富集孢囊杆菌亚目和堆囊菌亚目的粘细菌相关序列,通过粘细菌特异探针W2和W5杂交筛选阳性克隆并测序以分析土壤中粘细菌组成多样性。在SDU土壤样品中,我们从富集平板上分离到了7株粘细菌菌株,但分析土壤16S rRNA基因序列说明土壤中可能存在着大量的粘细菌类群,能出现在富集平板上的粘细菌只代表了土壤中粘细菌类群的一小部分。在孢囊杆菌亚目,可培养粘细菌序列只能被分为4个分支。然而,我们分析土壤中未可培养粘细菌序列发现至少存在12个分支,包括上述己知的4个分支;在堆囊菌亚目,至少存在着5个分支,所有己报道的堆囊菌亚目菌株都位于分支Ⅰ内,另外4个分支都由未可培养的粘细菌序列组成。
3.通过上述环境DNA技术的方法发现土壤生境中存在大量未可培养粘细菌,但该方法是基于DNA水平,不能说明这些未可培养粘细菌的状态。因此我们又进一步从土壤RNA出发,通过建立孢囊杆菌亚目和堆囊菌亚目富集的粘细菌16S crDNA文库,并利用特异探针W2和W5进行了文库的筛选和测序分析。我们得到了83个活性粘细菌的16S crDNA序列。进一步比较可培养粘细菌、基于土壤DNA的分子生态数据和基于土壤RNA的分子生态数据显示,尽管传统分离方法只能发现7株粘细菌菌株,但环境中存在着大量具有代谢活性的未可培养粘细菌,这也说明了我们所使用的传统分离方法的局限性。而且结果显示,这些活跃代谢表达的粘细菌类群,不但与我们通过传统分离方法得到的粘细菌菌株不一致,也和我们通过DNA水平出发得到的结果有较大的出入。进一步说明了无论是分离到的粘细菌菌株还是DNA水平得到的粘细菌序列都不能真实地反映在生境中具有较高代谢活性、行使生态功能的活性粘细菌类群的组成。
4.粘细菌通常被认为是土壤微生物,但是近几年,海洋嗜盐粘细菌和海洋耐盐粘细菌都陆续地被发现。在本研究中,以来自不同海底深度的10个海底沉积物样品为研究材料,利用传统的分离纯化方法进行可培养海洋粘细菌的分离,但并没有发现可培养粘细菌的存在,说明在海底沉积物中形成子实体的可培养粘细菌非常之少。为了了解海底沉积物中细菌的群落结构以及粘细菌在细菌群落中的丰度情况,我们分别对853m和4794m深度的海底沉积物样品916-1和7K367 M2-1构建了细菌普通16S rRNA基因文库,并随机选取这两个文库的克隆子进行测序,分别得到了88个和93个序列。分析这些序列表明,位于4794m的7K367 M2-1海底沉积物中没有粘细菌相关的序列发现,说明在7K367M2-1海底沉积物中粘细菌占细菌总数的比例较低(小于1%);而位于853m的916-1海底沉积物中,发现了3个粘细菌相关序列,占细菌总数的3.4%,说明在海底沉积物916-1中粘细菌占细菌总数的比例并不像土壤中的情况那样比例较低(SDU土壤中粘细菌占细菌总数不到1%)。
随后,我们分别对5个不同深度的海底沉积物样品建立了W1/1492R和W4/1492R两个类型的粘细菌富集文库,但对文库进行杂交筛选并没有发现阳性信号。我们认为产生这种结果的原因可能是探针W2和W5都是从可培养陆生粘细菌的16S rRNA基因出发而设计的,而在海洋环境中存在的粘细菌可能与陆生粘细菌有很大的不同,这两个探针不能够覆盖到海洋粘细菌类群中,以至于不能通过探针W2和W5来筛选海洋粘细菌。因此,我们随机挑取了各个文库的克隆子进行测序分析。通过测序得到了68个粘细菌相关序列。分析这些序列与可培养粘细菌的代表菌株序列的系统发育关系发现,孢囊杆菌亚目的可培养粘细菌分支内并没有发现海洋粘细菌相关序列,说明在海洋环境中孢囊杆菌亚目的可培养粘细菌很少,而这个亚目菌株绝大部分都是从来源于陆地环境的样品中分离得到,说明了这类菌株比较适合在陆地环境中生存;在堆囊菌亚目中,只有来自D-030样品的克隆D030-W4-42序列位于这个分支内,但与分支内的其它序列同源性较低,只有91%左右。而未可培养海洋粘细菌主要与可培养海洋粘细菌SMP-2等处于一个分支内,来自陆地的小囊菌属(Nannocysits)内没有未可培养海洋粘细菌的发现。
5.为了进一步分析未可培养海洋粘细菌与未可培养陆生粘细菌、可培养粘细菌之间的系统发育关系,我们将土壤中未可培养粘细菌序列和所有可培养粘细菌序列与未可培养海洋粘细菌序列进行系统发育关系分析。结果显示,我们可以将所有粘细菌序列分为4个大分支,分别代表了亚目水平的孢囊杆菌亚目、堆囊菌亚目、小囊菌亚目(Nannocysfineae)和一个新发现的未知亚目。可培养陆地粘细菌分布于孢囊杆菌亚目、堆囊菌亚目和小囊菌亚目三个分支内,但它们分布的范围非常之小,只占了粘细菌类群的很小一部分,其它大部分是来自于未可培养粘细菌。我们新发现的未知亚目的大多数是由来自海洋的未可培养粘细菌组成。而小囊菌亚目的分支除了少数陆生小囊菌属菌株序列和可培养海洋粘细菌外,大多数是由我们得到的未可培养海洋粘细菌序列所组成。
本课题建立了一整套用于粘细菌系统分类和生态研究的分子生物学方法,在国际上首次利用分子生态学方法,对粘细菌在土壤生境中的生态分布和多样性进行了研究,发现了大量未可培养的粘细菌新的未知类群;同时首次利用大量海底沉积物样品来分析海洋环境中粘细菌的系统发育多样性,取得的结果填补了粘细菌相关研究领域的空白,也得到了国际上权威专家的认可,使本实验室在此领域处于国际领先水平。下一步,我们将对发现的粘细菌新类群的生理生化性质及其生态学功能等方面进行相关研究。
Myxobacteria are Gram-negative gliding bacteria.They are unique among prokaryotes for their complicated multicellular behavior,especially the morphogenesis of fruiting bodies,and therefore are considered as social bacteria. When myxobacteria faced with starvation or suffered under unfavorable environmental conditions,cells form a macroscopic fruiting body containing thousands of resting myxospores.Those myxospores are sufficiently resistant to desiccation,heat,and UV light.As a result of their specialist lifestyle,myxobacteria are found nearly everywhere.They prefer to grow in the soil,the dung of herbivores, in bark and in rotting wood.Myxobacteria are rarely mentioned in articles of ecological study on soil microbiology.The reason is that the unusual characteristics of myxobacteria make the isolation and purification of myxobacteria more difficult than other soil microorganisms,furthermore many myxobacteria strains may be omitted using traditional isolation methods because of the instability of the fruiting body structure.Therefore the cultured myxobacteria strains may be only represent a small part of myxobacteria community in environment.The advent to so called 'cultivation independent' methods has provided researchers with the ability to determine the composition of microbial communities and identify numerically important,but not yet cultured organisms.Furthermore with largely and quickly increase of the 16S rRNA gene sequences of myxobacteria which have been submitted into GenBank database library,it is feasible to study myxobacterial ecology using molecular methods based on 16S rRNA gene sequences.In this paper,we used traditional morphological methods and molecular ecological methods to study the phylogenetic relationship and ecological diversity of myxobacteria in soil and marine sediments.
1.We selected 37 myxobacteria strains from our myxobacteria strains library,and those strains were classified based on their morphological characteristics.The 16S rRNA and HSP60 gene sequences of some myxobacteria strains were amplified by PCR methods,and phylogenetically analyzed.The classification of myxobaccteria mainly based on the morphological characteristics,especially the fruiting bodies.However,the fruiting body of myxobacteria is not a stable characteristic which is easy to degenerated or even lost.For example the five Cystobacterineae strains 0082-2,0085-4,0121-3,Myx9736 and NM03,the three Sorangineae strains So0007-16,So0157-52 and So139-5 were degenerated of fruiting body structures in different extents.The phylogenetic analysis of 16S rRNA gene sequences may help to define taxonomical status of difficult to identify myxobacteria.After phylogenetic analysis of 16S rRNA gene sequences of 15 Cystobacterineae strains and 22 Sorangineae strains,the results suggested the present morphology-based classification of myxobacteria was highly consistent with the phylogenetic results of 16S rRNA gene sequences at the levels of genera or higher taxa;while after phylogenetic analysis of HSP60 gene sequences of 5 Cystobacterineae strains and 22 Sorangineae strains,the results suggested HSP60 gene sequences provided a more efficient method for identification of closely related myxobacteria species.Here we also found there were two copy of HSP60 genes in Sorangium,both two HSP60 gene sequences can be used as molecular marker for phylogenetic analysis of closely related myxobacteria species.The sequences homology within same strain between different HSP60 genes was highly consistent at about 78%homology,which probably indicates that both genes are important,but play different roles in the Sorangium cells.
2.Using the above amplified 37 16S rRNA gene sequences and seventy 16S rRNA gene sequences of cultured myxobacteria which were extracted from the GenBank to develop oligonucleotide primers/probes specific for the two suborders of myxobacteria,Cystobacterineae and Sorangineae,respectively,we constructed two different 16S rRNA gene clone library based on W1/1492R and W4/1492R primer sets from total soil DNA.After screening clone library with specific probes,Hybridization-positive clones were randomly selected for sequencing.From the soil sample in this study,we found seven species of myxobacteria on enriching plates.But analysis of the soil 16S rRNA gene sequences provided quite different data.There are potentially much more types of myxobacteria in nature.The myxobacteria appearing on enriching plates only represented a small part of the natural myxobacteria panorama.In the suborder Cystobacterineae,all the strains based on phylogenetic analysis of their 16S rRNA gene sequences can be divided into only four groups.However,our analysis from one soil niche revealed that there were at least 12 groups,including the above four known groups.In the suborder Sorangineae,phylogenetic analysis of all myxobacteria related sequences revealed that there were at least 5 groups, all the cultured strains can be located into only one group,and the other 4 groups were composed of all uncultured myxobacteria sequences.
3.Phylogenetic analysis the community of myxobacteria in a soil niche based on total soil DNA methods suggested that myxobacteria in nature are much more diverse than were ever known,even in one soil niche.However the disadvantage of DNA-based molecular methods is the lack of differentiation between active and inactive(dead or dormant)bacterial cells,and RNA-based community analysis is more suitable to describe the metabolically active members of a population because of its rapid turnover and low RNA content in dormant cells. Therefore,we constructed two different 16S crDNA clone library based on W1/1492R and W4/1492R primer sets from total soil RNA revertased product. After screening clone library with specific probes,83 Hybridization-positive clones were randomly selected for sequencing to analysis of the active myxobacteria community.Comprehensive analysis of 16S rRNA gene sequences of cultured,uncultured and active uncultured myxobacteria indicated that there were a lot of numbers of active myxobacteria in nature,while we can only isolated 7 myxobacteria strains using traditional methods.Furthermore the results based on phylogenetic analysis of 16S crDNA sequences were quite different from 16S rRNA gene sequences,it is suggested that it was more different in diversity of between composition community of myxobacteria and metabolically active community of myxobacteria in soil environment.
4.The myxobacteria are considered to be the typical soil microorganisms,however in recent years,some halophilic and halotolerant myxobacteria strains also have been found.In this paper,we isolated myxobacteria strain from 10 different depths marine sediments using traditional myxobacteria isolation methods, however there was no cultured myxobacteria strain found.This suggested there was little number of fruiting myxobacteria in marine sediment environments.For comprehensive analysis of bacteria community of marine sediment 916-1 and 7K367 M2-1,we constructed two universal bacteria 16S rRNA gene clone library. After randomly sequencing 88 and 93 clones of marine sediment 916-1 and 7K367 M2-1,we found there was no myxobacteria related sequences in marine sediment 7K367 M2-1,this may suggested myxobacteria are not a dominant group in microbial community of sediment 7K367 M2-1;and also found 3 myxobacteria related sequences in marine sediment 916-1,counted up to 3.4%of total bacteria clones,high than those in a soil niche.
After construction of two types of 16S rRNA clone library with W1/1492R and W4/1492R primer sets from 5 different marine sediments,we used specific probes to screen the myxobacteria related clones.Unfortunately,there was no hybridization positive clone found.The reason maybe is too narrow specificity of probes,because the probe W2 and W5 were designed based on the 16S rRNA gene sequences of territorial cultured myxobacteria,and those marine myxobacteria may have high sequences discrepancy compared with territorial myxobacteria.So we randomly selected clones to sequence,and obtained 68 myxobacteria related sequences.After phylogenetic analysis of those marine myxobacteria related sequences and all cultured myxobacteria sequences,we found there was no marine sequence in cultured Cystobacterineae Group.This may suggested suborder Cystobacterineae strains may more suitable to survive in territorial environment,this result also consisted with the experiments since most of the Cystobacterineae strains were isolated from territorial environments.In suborder Sorangineae,there was only one clone D030-W4-42 belongs to this suborder;however have low sequence homology of 91%with the other members of this suborder.Some uncultured marine myxobacteria sequences and marine myxobacteria strains SMP-2 belonged to the same group and some groups only were composed of all uncultured marine myxobacteria sequences.The group which the genus Nannocysits belong to had no uncultured marine myxobacteria sequence.
5.Furthermore we analyzed the phylogenetic relationship of uncultured marine myxobacteria sequences,uncultured territorial myxobacteria sequences and all cultured myxobacteria sequences.The result showed myxobacteria can be divided into 4 major phylogenetic clades which represented Cystobacterineae, Sorangineae,Nannocystineae and a new unknown suborder.All cultured myxobacteria were located into three known suborders Cystobacterineae, Sorangineae and Nannocystineae,but they only represented a very small part of total myxobacteria panorama.The new unknown suborder was composed of uncultured marine myxobacteria sequences.In suborder Nannocystineae,there were a lot of numbers of uncultured marine myxobacteria sequences besides cultured genus Nannocystis and marine myxobacteria strains.
In this study,we established series of molecular methods for analysis of myxobacteria systemic classification and ecological diversity.We used molecular ecological methods to study ecological distribution and phylogenetic diversity of myxobacteria, and found there were potentially much more new types of myxobacteria in nature. Meanwhile,we used molecular methods to comprehensive analysis of myxobacteria community in marine sediments which have not been published before,and the results showed great existing information of myxobacteria ecology and evolution.However we are still wondering that what characteristic of those uncultured myxobacteria have? How to live so many uncultured myxobacteria in nature?
1.我们从实验室粘细菌资源菌库中选取了37株粘细菌菌株进行形态鉴定和菌株亲缘关系的分析。粘细菌的形态特征,尤其是子实体形态是粘细菌种属分类的主要依据,但由于子实体结构经过传代培养很容易退化甚至是丢失,给这些菌株的准确分类带来了很大的困难,如本研究中的孢囊杆菌亚目(Cystobacterineae)菌株0082-2、0085-4、0121-3、Myx9736和NM03以及堆囊菌亚目(Sorangineae)菌株So0007-16、So0157-52和So139-5的子实体结构都发生了不同程度的退化。通过对菌株16S rRNA基因序列的系统发育关系的分析,能够提高传统形态分类的准确性。在本研究中,通过对孢囊杆菌亚目15株菌株和堆囊菌亚目22株菌株的16S rRNA基因序列的系统发育关系的分析表明,大多数的粘细菌属在系统进化树上能形成各自独立的分支。说明在属及属以上水平,粘细菌形态分类和16S rRNA基因的系统发育关系具有较好的一致性。但在种水平上16S rRNA基因序列所揭示的系统发育关系可靠性较低。在此,我们使用了HSP60(groEL)基因序列来分析粘细菌近缘菌株之间的系统发育关系。在本研究中,我们得到了孢囊杆菌亚目5株菌株和堆囊菌亚目堆囊菌属(Sorangium)22株菌株的groEL1部分基因序列。通过分析系统发育关系表明,groEL1基因能够更为准确的揭示粘细菌种属间的系统发育关系,弥补了16S rRNA基因序列在鉴定粘细菌种属中的不足。同时发现粘细菌具有重复拷贝的HSP60基因,并扩增得到了9株堆囊菌属菌株的groEL2基因。通过分析系统发育关系发现,groEL2基因也可以作为堆囊菌属内系统进化分析的分子标记,且相同菌株不同拷贝的groEL基因序列同源性都在78%左右,这暗示着重复拷贝的groEL基因来源于同一个祖先基因,但却表现出明显不同的进化速率。
2.利用本研究得到的37个粘细菌菌株16S rRNA基因序列和70个GenBank中已公布的粘细菌16S rRNA基因序列信息来设计粘细菌亚目水平特异的寡核苷酸引物/探针,以从SDU土样中提取的总DNA为模板,建立了W1/1492R和W4/1492R两个类型文库来分别富集孢囊杆菌亚目和堆囊菌亚目的粘细菌相关序列,通过粘细菌特异探针W2和W5杂交筛选阳性克隆并测序以分析土壤中粘细菌组成多样性。在SDU土壤样品中,我们从富集平板上分离到了7株粘细菌菌株,但分析土壤16S rRNA基因序列说明土壤中可能存在着大量的粘细菌类群,能出现在富集平板上的粘细菌只代表了土壤中粘细菌类群的一小部分。在孢囊杆菌亚目,可培养粘细菌序列只能被分为4个分支。然而,我们分析土壤中未可培养粘细菌序列发现至少存在12个分支,包括上述己知的4个分支;在堆囊菌亚目,至少存在着5个分支,所有己报道的堆囊菌亚目菌株都位于分支Ⅰ内,另外4个分支都由未可培养的粘细菌序列组成。
3.通过上述环境DNA技术的方法发现土壤生境中存在大量未可培养粘细菌,但该方法是基于DNA水平,不能说明这些未可培养粘细菌的状态。因此我们又进一步从土壤RNA出发,通过建立孢囊杆菌亚目和堆囊菌亚目富集的粘细菌16S crDNA文库,并利用特异探针W2和W5进行了文库的筛选和测序分析。我们得到了83个活性粘细菌的16S crDNA序列。进一步比较可培养粘细菌、基于土壤DNA的分子生态数据和基于土壤RNA的分子生态数据显示,尽管传统分离方法只能发现7株粘细菌菌株,但环境中存在着大量具有代谢活性的未可培养粘细菌,这也说明了我们所使用的传统分离方法的局限性。而且结果显示,这些活跃代谢表达的粘细菌类群,不但与我们通过传统分离方法得到的粘细菌菌株不一致,也和我们通过DNA水平出发得到的结果有较大的出入。进一步说明了无论是分离到的粘细菌菌株还是DNA水平得到的粘细菌序列都不能真实地反映在生境中具有较高代谢活性、行使生态功能的活性粘细菌类群的组成。
4.粘细菌通常被认为是土壤微生物,但是近几年,海洋嗜盐粘细菌和海洋耐盐粘细菌都陆续地被发现。在本研究中,以来自不同海底深度的10个海底沉积物样品为研究材料,利用传统的分离纯化方法进行可培养海洋粘细菌的分离,但并没有发现可培养粘细菌的存在,说明在海底沉积物中形成子实体的可培养粘细菌非常之少。为了了解海底沉积物中细菌的群落结构以及粘细菌在细菌群落中的丰度情况,我们分别对853m和4794m深度的海底沉积物样品916-1和7K367 M2-1构建了细菌普通16S rRNA基因文库,并随机选取这两个文库的克隆子进行测序,分别得到了88个和93个序列。分析这些序列表明,位于4794m的7K367 M2-1海底沉积物中没有粘细菌相关的序列发现,说明在7K367M2-1海底沉积物中粘细菌占细菌总数的比例较低(小于1%);而位于853m的916-1海底沉积物中,发现了3个粘细菌相关序列,占细菌总数的3.4%,说明在海底沉积物916-1中粘细菌占细菌总数的比例并不像土壤中的情况那样比例较低(SDU土壤中粘细菌占细菌总数不到1%)。
随后,我们分别对5个不同深度的海底沉积物样品建立了W1/1492R和W4/1492R两个类型的粘细菌富集文库,但对文库进行杂交筛选并没有发现阳性信号。我们认为产生这种结果的原因可能是探针W2和W5都是从可培养陆生粘细菌的16S rRNA基因出发而设计的,而在海洋环境中存在的粘细菌可能与陆生粘细菌有很大的不同,这两个探针不能够覆盖到海洋粘细菌类群中,以至于不能通过探针W2和W5来筛选海洋粘细菌。因此,我们随机挑取了各个文库的克隆子进行测序分析。通过测序得到了68个粘细菌相关序列。分析这些序列与可培养粘细菌的代表菌株序列的系统发育关系发现,孢囊杆菌亚目的可培养粘细菌分支内并没有发现海洋粘细菌相关序列,说明在海洋环境中孢囊杆菌亚目的可培养粘细菌很少,而这个亚目菌株绝大部分都是从来源于陆地环境的样品中分离得到,说明了这类菌株比较适合在陆地环境中生存;在堆囊菌亚目中,只有来自D-030样品的克隆D030-W4-42序列位于这个分支内,但与分支内的其它序列同源性较低,只有91%左右。而未可培养海洋粘细菌主要与可培养海洋粘细菌SMP-2等处于一个分支内,来自陆地的小囊菌属(Nannocysits)内没有未可培养海洋粘细菌的发现。
5.为了进一步分析未可培养海洋粘细菌与未可培养陆生粘细菌、可培养粘细菌之间的系统发育关系,我们将土壤中未可培养粘细菌序列和所有可培养粘细菌序列与未可培养海洋粘细菌序列进行系统发育关系分析。结果显示,我们可以将所有粘细菌序列分为4个大分支,分别代表了亚目水平的孢囊杆菌亚目、堆囊菌亚目、小囊菌亚目(Nannocysfineae)和一个新发现的未知亚目。可培养陆地粘细菌分布于孢囊杆菌亚目、堆囊菌亚目和小囊菌亚目三个分支内,但它们分布的范围非常之小,只占了粘细菌类群的很小一部分,其它大部分是来自于未可培养粘细菌。我们新发现的未知亚目的大多数是由来自海洋的未可培养粘细菌组成。而小囊菌亚目的分支除了少数陆生小囊菌属菌株序列和可培养海洋粘细菌外,大多数是由我们得到的未可培养海洋粘细菌序列所组成。
本课题建立了一整套用于粘细菌系统分类和生态研究的分子生物学方法,在国际上首次利用分子生态学方法,对粘细菌在土壤生境中的生态分布和多样性进行了研究,发现了大量未可培养的粘细菌新的未知类群;同时首次利用大量海底沉积物样品来分析海洋环境中粘细菌的系统发育多样性,取得的结果填补了粘细菌相关研究领域的空白,也得到了国际上权威专家的认可,使本实验室在此领域处于国际领先水平。下一步,我们将对发现的粘细菌新类群的生理生化性质及其生态学功能等方面进行相关研究。
Myxobacteria are Gram-negative gliding bacteria.They are unique among prokaryotes for their complicated multicellular behavior,especially the morphogenesis of fruiting bodies,and therefore are considered as social bacteria. When myxobacteria faced with starvation or suffered under unfavorable environmental conditions,cells form a macroscopic fruiting body containing thousands of resting myxospores.Those myxospores are sufficiently resistant to desiccation,heat,and UV light.As a result of their specialist lifestyle,myxobacteria are found nearly everywhere.They prefer to grow in the soil,the dung of herbivores, in bark and in rotting wood.Myxobacteria are rarely mentioned in articles of ecological study on soil microbiology.The reason is that the unusual characteristics of myxobacteria make the isolation and purification of myxobacteria more difficult than other soil microorganisms,furthermore many myxobacteria strains may be omitted using traditional isolation methods because of the instability of the fruiting body structure.Therefore the cultured myxobacteria strains may be only represent a small part of myxobacteria community in environment.The advent to so called 'cultivation independent' methods has provided researchers with the ability to determine the composition of microbial communities and identify numerically important,but not yet cultured organisms.Furthermore with largely and quickly increase of the 16S rRNA gene sequences of myxobacteria which have been submitted into GenBank database library,it is feasible to study myxobacterial ecology using molecular methods based on 16S rRNA gene sequences.In this paper,we used traditional morphological methods and molecular ecological methods to study the phylogenetic relationship and ecological diversity of myxobacteria in soil and marine sediments.
1.We selected 37 myxobacteria strains from our myxobacteria strains library,and those strains were classified based on their morphological characteristics.The 16S rRNA and HSP60 gene sequences of some myxobacteria strains were amplified by PCR methods,and phylogenetically analyzed.The classification of myxobaccteria mainly based on the morphological characteristics,especially the fruiting bodies.However,the fruiting body of myxobacteria is not a stable characteristic which is easy to degenerated or even lost.For example the five Cystobacterineae strains 0082-2,0085-4,0121-3,Myx9736 and NM03,the three Sorangineae strains So0007-16,So0157-52 and So139-5 were degenerated of fruiting body structures in different extents.The phylogenetic analysis of 16S rRNA gene sequences may help to define taxonomical status of difficult to identify myxobacteria.After phylogenetic analysis of 16S rRNA gene sequences of 15 Cystobacterineae strains and 22 Sorangineae strains,the results suggested the present morphology-based classification of myxobacteria was highly consistent with the phylogenetic results of 16S rRNA gene sequences at the levels of genera or higher taxa;while after phylogenetic analysis of HSP60 gene sequences of 5 Cystobacterineae strains and 22 Sorangineae strains,the results suggested HSP60 gene sequences provided a more efficient method for identification of closely related myxobacteria species.Here we also found there were two copy of HSP60 genes in Sorangium,both two HSP60 gene sequences can be used as molecular marker for phylogenetic analysis of closely related myxobacteria species.The sequences homology within same strain between different HSP60 genes was highly consistent at about 78%homology,which probably indicates that both genes are important,but play different roles in the Sorangium cells.
2.Using the above amplified 37 16S rRNA gene sequences and seventy 16S rRNA gene sequences of cultured myxobacteria which were extracted from the GenBank to develop oligonucleotide primers/probes specific for the two suborders of myxobacteria,Cystobacterineae and Sorangineae,respectively,we constructed two different 16S rRNA gene clone library based on W1/1492R and W4/1492R primer sets from total soil DNA.After screening clone library with specific probes,Hybridization-positive clones were randomly selected for sequencing.From the soil sample in this study,we found seven species of myxobacteria on enriching plates.But analysis of the soil 16S rRNA gene sequences provided quite different data.There are potentially much more types of myxobacteria in nature.The myxobacteria appearing on enriching plates only represented a small part of the natural myxobacteria panorama.In the suborder Cystobacterineae,all the strains based on phylogenetic analysis of their 16S rRNA gene sequences can be divided into only four groups.However,our analysis from one soil niche revealed that there were at least 12 groups,including the above four known groups.In the suborder Sorangineae,phylogenetic analysis of all myxobacteria related sequences revealed that there were at least 5 groups, all the cultured strains can be located into only one group,and the other 4 groups were composed of all uncultured myxobacteria sequences.
3.Phylogenetic analysis the community of myxobacteria in a soil niche based on total soil DNA methods suggested that myxobacteria in nature are much more diverse than were ever known,even in one soil niche.However the disadvantage of DNA-based molecular methods is the lack of differentiation between active and inactive(dead or dormant)bacterial cells,and RNA-based community analysis is more suitable to describe the metabolically active members of a population because of its rapid turnover and low RNA content in dormant cells. Therefore,we constructed two different 16S crDNA clone library based on W1/1492R and W4/1492R primer sets from total soil RNA revertased product. After screening clone library with specific probes,83 Hybridization-positive clones were randomly selected for sequencing to analysis of the active myxobacteria community.Comprehensive analysis of 16S rRNA gene sequences of cultured,uncultured and active uncultured myxobacteria indicated that there were a lot of numbers of active myxobacteria in nature,while we can only isolated 7 myxobacteria strains using traditional methods.Furthermore the results based on phylogenetic analysis of 16S crDNA sequences were quite different from 16S rRNA gene sequences,it is suggested that it was more different in diversity of between composition community of myxobacteria and metabolically active community of myxobacteria in soil environment.
4.The myxobacteria are considered to be the typical soil microorganisms,however in recent years,some halophilic and halotolerant myxobacteria strains also have been found.In this paper,we isolated myxobacteria strain from 10 different depths marine sediments using traditional myxobacteria isolation methods, however there was no cultured myxobacteria strain found.This suggested there was little number of fruiting myxobacteria in marine sediment environments.For comprehensive analysis of bacteria community of marine sediment 916-1 and 7K367 M2-1,we constructed two universal bacteria 16S rRNA gene clone library. After randomly sequencing 88 and 93 clones of marine sediment 916-1 and 7K367 M2-1,we found there was no myxobacteria related sequences in marine sediment 7K367 M2-1,this may suggested myxobacteria are not a dominant group in microbial community of sediment 7K367 M2-1;and also found 3 myxobacteria related sequences in marine sediment 916-1,counted up to 3.4%of total bacteria clones,high than those in a soil niche.
After construction of two types of 16S rRNA clone library with W1/1492R and W4/1492R primer sets from 5 different marine sediments,we used specific probes to screen the myxobacteria related clones.Unfortunately,there was no hybridization positive clone found.The reason maybe is too narrow specificity of probes,because the probe W2 and W5 were designed based on the 16S rRNA gene sequences of territorial cultured myxobacteria,and those marine myxobacteria may have high sequences discrepancy compared with territorial myxobacteria.So we randomly selected clones to sequence,and obtained 68 myxobacteria related sequences.After phylogenetic analysis of those marine myxobacteria related sequences and all cultured myxobacteria sequences,we found there was no marine sequence in cultured Cystobacterineae Group.This may suggested suborder Cystobacterineae strains may more suitable to survive in territorial environment,this result also consisted with the experiments since most of the Cystobacterineae strains were isolated from territorial environments.In suborder Sorangineae,there was only one clone D030-W4-42 belongs to this suborder;however have low sequence homology of 91%with the other members of this suborder.Some uncultured marine myxobacteria sequences and marine myxobacteria strains SMP-2 belonged to the same group and some groups only were composed of all uncultured marine myxobacteria sequences.The group which the genus Nannocysits belong to had no uncultured marine myxobacteria sequence.
5.Furthermore we analyzed the phylogenetic relationship of uncultured marine myxobacteria sequences,uncultured territorial myxobacteria sequences and all cultured myxobacteria sequences.The result showed myxobacteria can be divided into 4 major phylogenetic clades which represented Cystobacterineae, Sorangineae,Nannocystineae and a new unknown suborder.All cultured myxobacteria were located into three known suborders Cystobacterineae, Sorangineae and Nannocystineae,but they only represented a very small part of total myxobacteria panorama.The new unknown suborder was composed of uncultured marine myxobacteria sequences.In suborder Nannocystineae,there were a lot of numbers of uncultured marine myxobacteria sequences besides cultured genus Nannocystis and marine myxobacteria strains.
In this study,we established series of molecular methods for analysis of myxobacteria systemic classification and ecological diversity.We used molecular ecological methods to study ecological distribution and phylogenetic diversity of myxobacteria, and found there were potentially much more new types of myxobacteria in nature. Meanwhile,we used molecular methods to comprehensive analysis of myxobacteria community in marine sediments which have not been published before,and the results showed great existing information of myxobacteria ecology and evolution.However we are still wondering that what characteristic of those uncultured myxobacteria have? How to live so many uncultured myxobacteria in nature?
引文
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[46]Bagwell,C.E.& Lovell,C.R.(2000).Persistence of selected Spartina alterniflora rhizoplane diazotrophs exposed to natural and manipulated environmental variability.Appl Environ Microbiol 66,4625-4633.
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[64]Longato,S.& Bonfante,P.(1997).Molecular identification of mycorrhizal fungi by direct amplification of microsatellite regions.Mycol Res 101,425-432.
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[66]Karlin,S.& Brocchieri,L.(2000).Heat shock protein 60 sequence comparisons:duplications,lateral transfer,and mitochondrial evolution.Proc Natl Acad Sci USA 97.11348-11353.
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[78]Wang,Q.,Garrity,G.M.,Tiedje,J.M.& Cole,J.R.(2007).Naive Bayesian Classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Appl Environ Microbiol 73,5261-5267
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[50]MacNanghton,S.J.,Stephen,J.R.,Venosa,A.D.,Davis,G.A.,Chang,Y.J.& White,D.C.(1999).Microbial population changes during bioremediation of an experimental oil spill.Appl Environ Microbiol 65.3566-3574.
[51]Maarit-Niemi,R.,Heiskanen,I.,Wallenius,K.& Lindstrom,K.(2001).Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia.J Microbiol Methods 45,155-165.
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[53]Fjeilbirkeland,A.,Torsvik,V.& Ovreas,L.(2001).Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmuA,mxaF and 16S rDNA sequences.Antonie Van Leeuwenhoek 79,209-217.
[54]Knief,C.,Lipski,A.& Dunfield,P.F.(2003).Diversity.and activity of methanotrophic bacteria in different upland soils.Appl Environ Microbiol 69,6703-6714.
[55]Orita,M.,Suzuki,Y.,Sekiya,T.& Hayashi,K.(1989).A rapid and sensitive detection of point mutations and genetic polymorphisms using polymerase chain reaction.Genomics 5,874-879
[56]Peters,S.,Koschinsky,S.,Schwieger,F.& Tebbe,C.C.(2000).Succession of microbial communities during hot composting as detected by PCR single strand conformation polymorphismbased genetic profiles of small-subunit rRNA genes.Appl Environ Microbiol 66,930-936.
[57]Zumstein,E.,Moletta,R.& Godon,J.J.(2000).Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction(PCR)single-strand conformation polymorphism analysis.Environ Mwrobiol 2,69-78.
[58]Dunbar,J.,Ticknor,L.O.& Kuske,C.R.(2000).Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis.Appl Environ Mirobiol 66,2943-2950.
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[61]Clement,B.G.,Kehl,L.E.,DeBord,K.L.& Kitts,C.L.(1998).Terminal restriction fragment patterns(TRFPs),a rapid,PCR-based method for comparison of complex bacterial communities.J Microbiol Methods 31,135-142.
[62]Fisher,M.M.& Triplett,E.W.(1999).Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities Appl Environ Microbiol 65.4630-4636.
[63]Zeze,A.,Hosny,M.,Gianinazzi-Pearson,V.& Dulieu,H.(1996).Characterization of a highly repeated DNA sequence(SC1)from the arbuscular mycorrhizal fungus Scutellospora castanea and its detection in planta.Appl Environ Microbiol 62,248-2443.
[64]Longato,S.& Bonfante,P.(1997).Molecular identification of mycorrhizal fungi by direct amplification of microsatellite regions.Mycol Res 101,425-432.
[65]Lin,Z.& Rye,H.S.(2006).GroEL-mediated protein folding:making the impossible.possible.Crit Rev Biochem Mol Biol 41,211-239.
[66]Karlin,S.& Brocchieri,L.(2000).Heat shock protein 60 sequence comparisons:duplications,lateral transfer,and mitochondrial evolution.Proc Natl Acad Sci USA 97.11348-11353.
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[72]Kumar,S.,Tamura,K.& Nei,M.(2004).MEGA3:Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment.Briefings Bioinformat 5,150-163
[73]Zhou,J.Z.,Bruns,M.A.A.& Tiedje,M.J.(1996).DNA Recovery from Soils of Diverse Composition.Appl Environ Microbl 62,316-322
[74]Brosius,J.,Palmer,M.L.,Kennedy,P.J.& Noller,H.F.(1978).Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli.Proc Natl Acad Sci USA 75,4801-4805.
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[76]Gray J.P.& Herwig R.P.(1996).Phylogenetic analysis of the bacterial communities in marine sediments.Appl Environ Microbiol 62,4049-4059
[77]Stevens,H.,Stubner,M.,Simon,M.& Brinkhlff,T.(2005).Phylogeny of Proteobacteria and Bacteroidetes from oxic habitats of a tidal flat ecosystem.FEMS Microbiol Ecol 54,351-365
[78]Wang,Q.,Garrity,G.M.,Tiedje,J.M.& Cole,J.R.(2007).Naive Bayesian Classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy.Appl Environ Microbiol 73,5261-5267
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