应用16S rRNA基因V—2高变区序列进行链霉莲分子分类

用16S rRNA部分序列对Williams数值分类系统所包含的链霉菌属种或种群进行系统进货分析,以包含16S rRNA基因V－2高变区在内的120bp长核苷酸序列所作的系统进化树表明：这些链霉菌可分为34个簇,其中大簇5个（包括27－85株菌株）;中等大小簇3个（包括9－12株菌）;小簇8个（包括2－6株菌）;单成员簇19个。结果同时表明,Williams数值分析系统中根据形态、生理、生化特征进行归类而得到的种或种群内存在较大异质性。     

菌种1137116S rRNA序列分析及鉴定

通过PCR方法扩增菌种11371的16S rRNA基因并测序,将序列提交GenBank(登录号:DQ531606),并与其他链霉菌属种进行比较,通过DNAStar软件得到菌种16S rRNA基因序列进化树。同时采用插片法、显微镜观察等方法对株菌11371进行形态特征、培养特征、生理生化特征鉴定。结果表明,11371的16S rRNA序列与其他链霉菌具有一定的同源性,结合生理、生化指标鉴定结果,进一步确定菌种为不吸水链霉菌一株新亚种(Streptomyces ahygroscopicus subsp.wuzhouensis n.sub-sp.),菌株11371 16S rRNA序列为GenBank中首例Streptomyces ahygroscopicus的16S rRNA序列。     

豆豉纤溶酶产生菌分离和鉴定

从全国各地收集豆豉样品,采用不同的培养基进行富集培养,并利用纤维蛋白平板法高效获得了13株形态差异较大的产纤溶酶菌株。通过传统方法、化学方法以及16S rRNA序列分析对这13株菌进行分类鉴定,它们分属于芽孢杆菌属、链霉菌属、假单胞菌属以及节杆菌属,包括9种细菌,丰富了豆豉纤溶酶产生菌菌种资源。     

链霉菌属菌株AS4.693和AS4.702的分类学研究

链霉菌属“Setae”种群原为北里孢菌属Kitasatosporia (Omura,1982)。1992年,Wellington根据16S rRNA序列分析结果将其并入链霉菌属,并建立“Setae”种群。通过对保藏的链霉菌AS 4.693、AS 4.702进行的形态学、细胞化学、分子遗传分类研究结果表明,它们与链霉菌属“Setae”种群中的典型种——西唐链霉菌Streptomyces setae(JCM3304’)具有相似性。它们的rDNA相似性高达100％,证明它们应归属于同一种群。AS.4.693定名为西唐链霉菌不规则新亚种Streptomyces setae subsp.irregularis nov.,AS 4.702定名为西唐链霉菌波曲弗氏新亚种Streptomyces setae subsp.flexuofradiae nov.。     

药用植物内生放线菌的分离、筛选及活性菌株YIM 61470鉴定

从云南西双版纳热带雨林多种药用植物中分离到272株内生放线菌,活性筛选表明 146株菌的发酵产物具有抗菌活性,其中94株菌具有拮抗病原细菌活性,127株菌具有抑制病原真菌的功能.分离菌株YIM 61470具有广谱抗菌活性,通过形态特征、培养特征、生理生化特征、细胞化学分类特征和基于16S rRNA基因序列的相似性分析等研究,菌株YIM 61470被鉴定为链霉菌属(Streptomyces)氢化链霉菌(S.llydrogenans)的一个菌株.     

ε-聚赖氨酸产生菌TUST-2的分离鉴定

【目的】ε-聚赖氨酸是一种天然氨基酸同聚物,本研究目的为分离筛选新的ε-聚赖氨酸产生菌。【方法】采用一种新的分离方法从土壤中分离ε-PL产生菌。分离方法含3步:(1)富集培养ε-PL耐受菌;(2)通过改进的Nishikawa方法筛选;(3)挑选高浓度ε-PL耐受菌株。【结果】从海南省土样中分离获得ε-聚赖氨酸产生菌TUST-2。分类和形态特征属链霉菌属。16S rDNA序列分析比对结果表明TUST-2属淀粉酶产色链霉菌(Streptomyces diastatochromogenes)。经特征反应分析、水解物分析、红外光谱、1H NMR、13C NMR和MALDI-TOF-MS分析表明TUST-2发酵产物为ε-聚赖氨酸。【结论】根据16S rRNA基因序列比对和形态及生理生化特征表明ε-聚赖氨酸产生菌TUST-2属于淀粉酶产色链霉菌,命名为淀粉酶产色链霉菌TUST-2。     

基于16S rRNA和rpoB基因的分子系统发育分析在铜绿假单胞菌鉴定中的应用

为对比16S rRNA和rpo B基因分子系统发育分析与传统表型分类法对铜绿假单胞菌的鉴定,评估16S rRNA和rpo B基因序列分析在铜绿假单胞菌鉴定中的应用,用表型分类方法对临床自动微生物鉴定系统鉴定为铜绿假单胞菌的23株分离株进行再鉴定,PCR扩增23株分离株16S rRNA和rpo B基因片段,并测序进行系统发育分析。结果表明,表型再鉴定结果与自动微生物鉴定系统鉴定结果一致。基于两个基因的系统发育分析均显示分离株p22与不动杆菌属序列聚为一枝,其余22株分离株与铜绿假单胞菌序列聚为一枝。因此p22应鉴定为不动杆菌,16S rRNA和rpo B基因序列分析均能准确鉴定铜绿假单胞菌并能较好建立假单胞菌属内种间关系。     

分离自印度洋深海沉积物的部分链霉菌分离株多位点序列分析

【目的】采用多位点序列分析方法,研究印度洋3 000 m以下深海沉积物中分离得到的16S rRNA基因比对高度相似的链霉菌菌株的种间系统发育关系,同时探讨各管家基因及多基因聚类分析后的种间区分能力。【方法】以分离自印度洋深海沉积物的7株Streptomyces albidoflavus,11株Streptomyces cavourensis,16株Streptomyces pratensis为研究对象,以16S rRNA、atpD、recA和rpoB基因片段为标记,通过PCR扩增、测序,获得序列。同时从NCBI上下载5株S.pratensis上述4个基因的序列,将所有序列在MLST网站进行比对,并构建系统进化树进行比较。【结果】S.pratensis各菌株种内比较发现,16S rRNA基因构建的系统进化树中相同基因型的菌株没有聚在一起,系统进化树不稳定,区分度不高。其余3个构建的系统进化树稳定,菌株的聚类关系与MLST数据库得到的基因型一致。同时,多基因聚类分析后将菌株分为6个类群。在3个种的种间多位点序列比较中,除区分度明显增加、进化树更加稳定以外,还发现rec A基因进化上比较特殊的菌株。【结论】多位点序列分析将实验菌株分为很多不同的类型,成功地将所分离的链霉菌进行了更细的分类,同时也找到部分菌株在个别基因上差异较大。此方法可以用于相近种的快速鉴定。     

砂土法、矿油封藏法保存链霉菌17年效果的评定

本文总结了用砂土法保存130个种、14个变种、409株链霉菌以及矿油封藏法保存其中103个种、11个变种、307株菌17年的效果;其中部分菌株的抗菌素产生能力和蛋白酶活力的测定结果;经矿油封藏后,死亡菌株的矿油层,用红外光谱法检查的结果。用砂土法保存的409株菌,17年后存活384株,存活率为93.9%;在144种（包括变种）中,有101种（70%）保持着原来的形态特征。用矿油封藏法保存的307株菌,17年后存活256株,存活率为83.4%;其中169株（66%）保持着原来的形态特征。产生链霉素的灰色链霉菌（Streptomyces griscus）As 4.181、比基尼链霉菌（S.bikiniensis）AS 4.569,产氯霉素的委内瑞拉链霉菌（S.venezu（?）la（?））AS 4.223等菌株,用矿油封藏法比砂土法保存得到较好的效果。产生新霉素的弗氏链霉菌（S.fradiae）AS 4.576,用砂土法保存效果较好。矿油封藏法保存链霉菌对保持蛋白酶活力,有着良好的效果。经红外光谱法检查19株失活的菌株的矿油层,其中17个样品于1710厘米-1或1600厘米-1处有吸收带。根据红外光谱,此吸收带属于脂肪族或芳香族中（?）c=c（?）基的伸展振动,与 Arai 用此法保存诺卡氏菌,分枝杆菌测出的结果近似。故推测链霉菌经此法保存后,菌株死亡的原因是由于浸出了细胞中类脂所致。砂土法和矿油封藏法对于长期大量保存链霉菌,是两种简便而且可取的方法。     

一种产生纤溶酶的链霉菌C-3662的鉴定及发酵研究

对一株从土壤中分离的纤溶酶产生菌链霉菌C-3662的形态、培养、生理生化和化学分类特征进行了研究,发现其与泛温链霉菌(Streptomyces eurythermus Corbaz et al. 1968)的特征很相符。通过对链霉菌C-3662的16S rDNA序列进行测定与比对,发现它与泛温链霉菌的16S rDNA序列也有高达98.19%的同源性。根据多相分类原则,认为链霉菌C-3662属于泛温链霉菌。对链霉菌C-3662的发酵培养基组成等的研究表明,合适的发酵培养基中应含有氮源黄豆饼粉2.0%、碳源淀粉1.0%和葡萄糖2.5%以及少量的无机盐类如硫酸镁。链霉菌C3662的发酵过程研究表明,纤溶酶是在菌丝体生长停止之后才大量产生。     

一株产蓝色素链霉菌18-A-5的鉴定与系统发育分析

从原始热带雨林土壤中,分离到一株产蓝色色素菌株18-A-5,对其进行了系统分类学研究。形态学特征观察表明,在高氏合成一号培养基上初产蓝绿色色素,日久为深蓝色,基内菌丝蓝色,气生菌丝灰白色,产灰色孢子,孢子丝直或柔曲,形成长孢子链,孢子圆柱形。其DNA的G＋C摩尔分数为62.4%,16S rDNA序列分析结果(GenBank登陆号为EU054353),18-A-5与生靛链霉菌Streptomyces indigoferus ATCC23924^T 、草绿色链霉菌Streptomyces herbaricolor ATCC23924^T具有极高的同源性,达100％,聚类分析表明,18-A-5与生靛链霉菌Streptomyces indigoferus、草绿色链霉菌Streptomyces herbaricolor两株菌聚类在一起,分支置信度为74%。结合生理生化特性、细胞壁化学组成分析、脂肪酸分析等将菌株18-A-5定名为草绿色链霉菌Streptomyces herbaricolor。并对该蓝绿色可溶性色素性质进行了耐酸碱性、热稳定性、抗菌谱等初步分析。     

Phylogeny of green sulfur bacteria on the basis of gene sequences of 16S rRNA and of the Fenna-Matthews-Olson protein

The phylogeny of green sulfur bacteria was studied on the basis of gene sequences of the 16S rRNA and of the Fenna-Matthews-Olson (FMO) protein. Representative and type strains (31 strains total) of most of the known species were analyzed. On the basis of fmoA gene sequences from Chlorobium tepidum ATCC 49652(T) and Chlorobium limicola DSM 249(T) available from the EMBL database, primers were constructed that allowed sequence analysis of a major part of the fmoAgene. The largely congruent phylogenetic relationship of sequences of the fmoA gene and of 16S rDNA gives considerable support to the phylogeny of green sulfur bacteria previously suggested on the basis of 16S rDNA sequences. Distinct groups of strains were recognized on the basis of 16S rDNA and FMO sequences and supported by characteristic signature amino acids of FMO. Marine strains formed clusters separate from freshwater strains. The resulting phylogenetic grouping and relationship of the green sulfur bacteria do not correlate with their current taxonomic classification.     

Phylogenetic diversity of acidophilic sporoactinobacteria isolated from various soils

Spore forming actinobacteria (sporoactinobacteria) isolated from soils with an acidic pH in Pinus thunbergii forests and coal mine waste were subjected to taxonomic characterization. For the isolation of acidophilic actinobacteria, acidified starch casein agar (pH adjusted to 4-5) was used. The numbers of actinobacteria growing in acidic media were between 3.2 x 10(4) and 8.0 x 10(6) CFU/g soil. Forty three acidophilic actinobacterial strains were isolated and their 16S rDNA sequences were determined. The isolates were divided into eight distinctive phylogenetic clusters within the variation encompassed by the family Streptomycetaceae. Four clusters among them were assigned to the genus Streptacidiphilus, whereas the remaining four were assigned to Streptomyces. The clusters belonging to either Streptomyces or Streptacidiphilus did not form monophyletic clade. The growth pH profiles indicated that the representative isolates grew best between pH 5 and 6. It is evident from this study that acidity has played a critical role in the differentiation of the family Streptomycetaceae, and also that different mechanisms might have resulted in the evolution of two groups, Streptacidiphilus (strict acidophiles) and neutrotolerant acidophilic Streptomyces. The effect of geographic separation was clearly seen among the Streptacidiphilus isolates, which may be a key factor in speciation of the genus.     

八门湾红树林土壤芽胞杆菌分离与多样性分析

【目的】了解八门湾红树林海漆林区土壤中可培养芽胞杆菌资源的多样性。【方法】采用水浴处理与直接涂布相结合的方法选择性分离土壤中的芽胞杆菌;利用16S rDNA PCR-RFLP与16S rDNA序列分析技术研究可培养芽胞杆菌资源的遗传多样性和系统发育关系。【结果】16S rDNA PCR-RFLP酶切图谱UPGMA聚类分析表明,在100%的相似性水平上,分离的155株芽胞杆菌分属21个遗传类群,显示了较为丰富的遗传多样性;由21种遗传类型代表菌株的16S rDNA序列分析结果得知,这些芽胞杆菌主要分布在Bacillaceae和Paenibacillaceae科下的Bacillus、Halobacillus、Virgibacillus和Paenibacillus 4个属,其中Bacillus为优势属;有8株芽胞杆菌的16S rDNA序列与数据库中相应模式菌株的最大相似性在95.1%-99.0%之间。【结论】八门湾红树林土壤可培养芽胞杆菌有着较为丰富的遗传多样性,并存在新的芽胞杆菌物种资源。     

链霉菌的rep-PCR基因指纹分析

对重复片段PCR(repPCR)基因指纹分析应用于链霉菌分子分型进行研究,结果表明repPCR基因指纹分析具有分辨率高、稳定、重现性好、简便易行等特点,在一定程度上与16S rDNA 序列比较结果相一致,是一种快速而有效的DNA指纹技术,能反映出链霉菌种和菌株水平的基因型、系统发育和分类学关系,可应用于种及以下水平的分类和快速鉴定,尤其适用于分析大量的菌株或分离株。     

Intrageneric structure of the genus Gluconobacter analyzed by the 16S rRNA gene and 16S-23S rRNA gene internal transcribed spacer sequences

Forty-nine strains belonging to the genus Gluconobacter were re-examined with respect to their species identification based on the sequences of the 16S rDNA and 16S-23S rDNA internal transcribed spacer regions (ITS). A phylogenetic tree constructed from the 16S rDNA sequences indicated the presence of five clusters corresponding, respectively, to the major five species of the genus Gluconobacter, namely G. albidus, G. cerinus, G. frateurii, G. oxydans (type species), and G. thailandicus. The type strain of G. asaii, NBRC 3276T (T=type strain) was included in the G. cerinus cluster, which is consistent with the report that G. asaii is a junior subjective synonym of G. cerinus. Existence of the G. albidus, G. cerinus, G. frateurii, G. oxydans, and G. thailandicus clusters was also recognized by the ITS sequence analysis. Both sequence analyses revealed that the G. cerinus and G. frateurii clusters were heterogeneous. The G. cerinus cluster comprised three strains of G. cerinus and one strain of G. frateurii, while the G. frateurii cluster included ten strains of G. frateurii, three of G. cerinus, and eleven of G. oxydans. These results suggest that phenotypic differences among Gluconobacter species are ambiguous and the species definition must be re-evaluated. The 16S rDNA and ITS sequences determined in this study are valuable for the identification and phylogenetic analysis of Gluconobacter species.     

Bacterial phylogenetic clusters revealed by genome structure.

Current bacterial taxonomy is mostly based on phenotypic criteria, which may yield misleading interpretations in classification and identification. As a result, bacteria not closely related may be grouped together as a genus or species. For pathogenic bacteria, incorrect classification or misidentification could be disastrous. There is therefore an urgent need for appropriate methodologies to classify bacteria according to phylogeny and corresponding new approaches that permit their rapid and accurate identification. For this purpose, we have devised a strategy enabling us to resolve phylogenetic clusters of bacteria by comparing their genome structures. These structures were revealed by cleaving genomic DNA with the endonuclease I-CeuI, which cuts within the 23S ribosomal DNA (rDNA) sequences, and by mapping the resulting large DNA fragments with pulsed-field gel electrophoresis. We tested this experimental system on two representative bacterial genera: Salmonella and Pasteurella. Among Salmonella spp., I-CeuI mapping revealed virtually indistinguishable genome structures, demonstrating a high degree of structural conservation. Consistent with this, 16S rDNA sequences are also highly conserved among the Salmonella spp. In marked contrast, the Pasteurella strains have very different genome structures among and even within individual species. The divergence of Pasteurella was also reflected in 16S rDNA sequences and far exceeded that seen between Escherichia and Salmonella. Based on this diversity, the Pasteurella haemolytica strains we analyzed could be divided into 14 phylogenetic groups and the Pasteurella multocida strains could be divided into 9 groups. If criteria for defining bacterial species or genera similar to those used for Salmonella and Escherichia coli were applied, the striking phylogenetic diversity would allow bacteria in the currently recognized species of P. multocida and P. haemolytica to be divided into different species, genera, or even higher ranks. On the other hand, strains of Pasteurella ureae and Pasteurella pneumotropica are very similar to those of P. multocida in both genome structure and 16S rDNA sequence and should be regarded as strains within this species. We conclude that large-scale genome structure can be a sensitive indicator of phylogenetic relationships and that, therefore, I-CeuI-based genomic mapping is an efficient tool for probing the phylogenetic status of bacteria.     

Application of the hypervariable region of the 16S rDNA sequence as an index for the rapid identification of species in the genus Alicyclobacillus

A comparison of the 16S rRNA gene (rDNA) sequences of seven type strains belonging to different Alicyclobacillus species (i.e., five validated species, one proposed species and one genomic species) suggested that the 5' end hypervariable region (259-273 bases in length) of 16S rDNA was specific for the respective type strains. Further phylogenetic analysis based on DNA sequences of the hypervariable region using 24 Alicyclobacillus strains revealed that the strains could be categorized into five species and the A. acidocaldarius-Alicyclobacillus genomic species 1 group. The hypervariable region was highly conserved among the five species: A. acidiphilus, A. acidoterrestris, A. cycloheptanicus, A. herbarius, and A. hesperidum. The strains in the A. acidocaldarius-Alicyclobacillus genomic species 1 group were subdivided into two clusters (Clusters I and II) based on DNA sequences in the hypervariable region. On the basis of phenotypic characteristics, chemotaxonomic and phylogenetic analyses, and DNA-DNA hybridization data, strains in Cluster I were grouped as Alicyclobacillus genomic species 1 and strains in Cluster II were re-identified as A. acidocaldarius, thereby demonstrating that the hypervariable regions were also highly conserved within these two species. These results suggest that as is the case with Bacillus, the hypervariable region is significantly species-specific in the genus Alicyclobacillus to distinguish Alicyclobacillus species by DNA sequence comparison of the hypervariable region.     

Differentiation of Micromonospora isolates from a coastal sediment in Wales on the basis of Fourier transform infrared spectroscopy, 16S rRNA sequence analysis, and the amplified fragment length polymorphism technique

A number of actinomycetes isolates were recovered from coastal sediments in Aberystwyth (Wales, United Kingdom) with standard isolation techniques. Most of them were putatively assigned to the genera Streptomyces and Micromonospora on the basis of their morphological characteristics, and there appeared to be no difference whether the isolation media contained distilled water or seawater. A group of 20 Micromonospora isolates was selected to undergo further polyphasic taxonomic investigation. Three approaches were used to analyze the diversity of these isolates, 16S rDNA sequencing, fluorescent amplified fragment length polymorphism (AFLP), and Fourier transform infrared spectroscopy (FT-IR). The 16S rDNA sequence analysis confirmed that all of these isolates should be classified to the genus Micromonospora, and they were analyzed with a group of other Micromonospora 16S rDNA sequences available from the Ribosomal Database Project. The relationships of the 20 isolates were observed after hierarchical clustering, and almost identical clusters were obtained with these three techniques. This has obvious implications for high-throughput screening for novel actinomycetes because FT-IR spectroscopy, which is a rapid and reliable whole-organism fingerprinting method, can be applied as a very useful dereplication tool to indicate which environmental isolates have been cultured previously.