26S rDNA单链构象多态性分析在临床酵母菌菌种鉴定中的应用

摘要： 【目的】探讨酵母菌临床分离株26S rDNA D1/D2区序列种内相似性和种间差异性的快速检测方法,为临床酵母菌菌种鉴定方法的改进奠定基础。调查北京地区临床酵母菌的种群多样性,为国内酵母菌感染的流行病学研究提供新的基础数据。【方法】用5种常见临床酵母菌种的模式和权威菌株作为标准参考菌株,从北京四家综合性医院收集临床酵母菌260余株,PCR扩增其26S rDNA D1/D2区,对扩增产物进行单链构象多态性（Single-Strand Conformation Polymorphism,SSCP）分析和序列测定分析。【结果】常见病原酵母菌26S rDNA D1/D2区的SSCP图谱具有明显的种间差异性和种内相似性,可以通过该方法对菌株进行初步的菌种鉴定。D1/D2-SSCP和序列分析相结合,对260余株临床酵母菌进行了菌种鉴定,共鉴定有10个属20个种,优势属为念珠菌属(Candida),优势种及其所占比例分别是：C. albicans (57.7%), C. parapsilosis (10.0%), C. tropicalis (9.2%), C. glabrata (6.7%)和C. krusei (5.8%),并发现过去从未或很少报道致病的酵母菌种,愈来愈多地出现在临床分离菌株中。【结论】 26S rDNA D1/D2区的SSCP图谱分析为临床酵母菌株的快速鉴定提供了新的方法;北京地区酵母菌临床分离株呈种群多样性分布,C. albicans虽然仍占优势,但其它念珠菌种的比例已达42%。     

正己醇降解菌的分离、筛选及分类鉴定

为获得可降解正己醇的真菌菌种,分别以苹果园土壤、苹果渣、苹果酒醪和醋醅为分离源,采用富集培养和紫外线定向诱变,得到了两株能在pH3.8-4.0的条件下降解正己醇的真菌菌株TF和TM。菌株TM和TF在马铃薯葡萄糖培养液中对4.0mg/mL正己醇的降解率分别为45.60±5.43%和23.82±9.27%,与对照在α=0.01水平上差异显著。结合形态学特征及26S rDNA D1/D2区(菌株TM)和ITS区(菌株TF)序列分析,对两株菌进行了分类鉴定。结果表明:菌株TM属于地霉属Geotrichum,菌株TF为白地霉Geotrichum candidum(有性型Galactomyces geotrichum)。     

16S rRNA及rpoC1基因用于螺旋藻、节旋藻系统发育的比较北大核心CSCD

【目的】利用16S rRNA和rpoC1基因分子标记研究螺旋藻、节旋藻的系统发育关系,并对其区分能力进行比较。【方法】以84株螺旋藻、节旋藻为研究对象,对其进行16S rRNA、rpoC1基因序列的扩增、测序及分析,并对构建的系统发育树进行对比。【结果】rpoC1基因序列保守位点所占比例49.7%、平均G+C百分含量47.7%和序列相似度76%–100%明显低于16S rRNA基因序列的79.4%、55.6%和91%–100%,其变异程度高于16S rRNA基因;基于16S rRNA、rpoC1基因构建的系统发育NJ树拓扑结构基本一致,84株实验藻株分为2个属3个类群,其中仅F-351、F-904-2、F-1070和TJBC14-1藻株为螺旋藻,其余均为节旋藻;虽然2个基因都不能区分形态种和地理种,但rpoC1基因NJ树的置信度(100%)高于16S rRNA基因(99%),属内分群效果也明显优于16S rRNA基因。【结论】支持了螺旋藻、节旋藻为两个不同属的结论,且在属内分类时rpoC1基因比16S rRNA基因具有更高的区分度。     

一株分离于工业污水池的耐碱酵母

目的:从新疆温泉县一个碱性工业污水处理池中分离并鉴定耐碱酵母菌。方法:用稀释平板法分离菌种,通过形态学观察、生理生化特征及26S rDNA D1/D2区基因序列分析鉴定菌种。结果:从水样中分离得到一株耐碱酵母菌,它们能在pH3.5~11.0,12%NaCl,4~45℃生长,经形态观察及生理生化特征鉴定为酵母属,对其26S rDNA 5’端D1/D2区基因序列进行了PCR扩增并测序,GenBank注册号为DQ132884,同源序列分析结果表明该序列与酿酒酵母(Saccharomyces cerevisiae)Sb4有99.8%的同源性,因此将其命名为酿酒酵母(Saccharomyces cerevisiae)XJU-2,该菌种已保藏于中国微生物菌种保藏委员会普通微生物中心(CGM-CC),保藏号为CGMCC No.2.3095。结论:XJU-2的最高耐碱值可达pH 11.0,而且酸碱耐受范围很大,性能明显优于国内外已报道的酿酒酵母菌种。     

一株分离自酸梅酱的酵母菌的分离鉴定

目的:分离及鉴定来自于新疆塔城民间自制酸梅酱中的酵母菌。方法:用NL1/NL4引物对扩增酵母菌株的26S rDNAD1/D2区,测序结果进行序列分析,用Neighbour-joining(N-J)方法构建系统发育进化树,同时结合酵母的传统形态学鉴定对菌株进行鉴定。结果:26S rDNA D1/D2区序列分析表明菌株KKS与Metschnikowia aff.fructicola D3895相近,相似率为99.2%。在N-J法构建的系统发育进化树中,菌株KKS与Metschnikowia aff.fructicola聚类在同一分枝上。结论:从新疆塔城民间自制酸梅酱中分离得到一株酵母菌并将该菌株鉴定为Metschnikowia aff.fructicola。     

脂肪酸组分分析在不动杆菌鉴定中的应用

以不动杆菌属(Acinetobacter)中的A. baumannii、A. haemolyticus、A. johnsonii、A. junii、A. lwoffii模式菌株为参比菌株,对中国农业微生物菌种保藏管理中心(ACCC)所保藏的6株不动杆菌进行脂肪酸组分分析及16S rRNA基因系统进化分析.结果表明,利用脂肪酸分析可将6株菌完全准确地鉴定到属的水平上,这一点与16S rRNA基因分析结果一致;在种的水平上,16S rRNA基因进化分析与脂肪酸组分分析的结果可互为补充,相互印证.同时,通过对不动杆菌部分模式菌株及供试菌株的脂肪酸组分分析,报道了不动杆菌的特征性脂肪酸为C18:1 w9c、C16:00和C16:1w7c/C16:1w6c.     

拟诺卡氏菌16S rRNA,gyrB,sod和rpoB基因的系统发育分析

为了更好地了解拟诺卡氏菌属(Nocardiopsis)各物种间的系统发育关系,该属现有有效描述种的gyrB,sod和rpoB基因的部分序列被测定,结合16S rRNA基因,对拟诺卡氏菌属进行了系统发育重建。研究发现拟诺卡氏菌属gyrB,sod和rpoB基因的平均相似性分别为87.7%、87.3%和94.1%,而16S rRNA基因的平均相似性则达到96.65%,3个看家基因均比16S rRNA具有更高的分歧度。比较基于不同基因的系统树发现,由gyrB基因得到的系统树拓扑结构与16S rRNA得到的结构在亚群上基本一致。因此,gyrB基因在拟诺卡氏菌属的系统分类上比16S rRNA基因更具优越性。     

基于16S rRNA和HSP60基因序列分析粘细菌孢囊杆菌亚目形态分类的种属之间的亲缘关系

[目的]利用16S rRNA和HSP60基因分子标记分析鉴定形态分类特征不稳定的粘细菌种属.[方法]利用粘细菌的传统分离纯化方法从土壤中分离粘细菌,根据菌株的形态特征进行分类,PCR方法扩增菌株的16S rRNA和HSP60基因序列并进行系统发育关系分析.[结果]根据形态特征,分离得到的15株粘细菌菌株归入孢囊杆菌亚目(Cystobacterineae)的2个科3个属.其中11株粘细菌具有典型的所在种属的子实体结构,而菌株0085-4、0121-3、NM03和Myx9736的子实体结构发生了不同程度退化.15株粘细菌的16S rRNA基因序列的相似性在95.4%到99.5%之间.而HSP60基因序列差异较大.[结论]在属水平上,粘细菌形态分类特征和16S rRNA基因系统进化关系具有很好的一致性;在揭示粘细菌种间系统发育关系中,HSP60基因序列更为适用.     

表型特征结合基因组分析鉴定不同菌落形态Bacillus velezensis ACCC 19742

在中国农业微生物菌种中心(ACCC)菌株的定期转接保藏过程中,发现解淀粉芽孢杆菌ACCC 19742在同一培养基上出现两种不同的菌落形态,将这两个不同形态的菌株编号为19742-1和19742-2。通过形态学、生理生化及基因组分析相结合鉴定不同菌落形态ACCC 19742,并进一步确定该菌株的分类地位。首先将菌株进行分离与纯化,其次将纯化后的菌株进行16S rRNA及gyrB基因扩增及序列分析,通过MEGA 7.0软件构建系统发育树;API 20NE、BIOLOG及脂肪酸等分析菌株的生理生化特性;全基因组分析菌株的ANI和DDH值。两株菌在API 20NE中,仅葡萄糖酸盐同化反应存在差异;脂肪酸检测中主要组成相同,仅是百分含量方面略有差别;两株菌的16S rRNA基因相似性为100%,gyrB基因的相似性为99.4%;全基因组测序表明,两株菌的ANI值为99.95%,DDH值为99.62%。综合遗传学特征和表型特征,证实两者为来源于同一菌株不同的形态变异型,而并非污染所致。同时,19742-1和19742-2与Bacillus velezensis NRRL_B 41580~T的ANI及DDH值最高,分别为97%和77%,且16S rRNA和gyrB系统进化分析也表明,该菌株在分类地位上属于贝莱斯芽孢杆菌(Bacillus velezensis),而非解淀粉芽孢杆菌。这为菌株的保藏提供了一定的参考价值。     

第六次北极科学考察海洋沉积物可培养细菌的多样性分析

【目的】研究北极海洋沉积物可培养细菌的菌种资源多样性。【方法】采用海水Zobell2216E培养基和涂布平板法对第六次北极科学考察获得的海洋沉积物开展细菌分离培养,通过16S rRNA基因系统发育分析了解可培养细菌的多样性。【结果】根据菌落形态特征,从40个站位的北极海洋沉积物样品中共分离并获得16S rRNA基因有效序列的细菌达445株;基于16S rRNA基因的相似性分析与系统发育研究结果表明,分离获得的细菌分属于细菌域的4个门、6个纲、13个目、28个科、49个属、91个种,其中γ-Proteobacteria占大多数;有12株与模式菌株的16S rRNA基因序列相似性小于97%,可能代表了6个潜在的细菌新物种;此次获得的细菌种类组成与以往第五次北极科考获得的相比,在属水平上差异较大。【结论】北极海洋沉积物中存在着丰富的微生物菌种资源,具有很多新型微生物仍未被发现,是亟待开发的微生物资源宝库。     

青海东部土壤中酵母物种多样性研究

从青海的互助、民和、门源等10个州县收集土样分离得到98株酵母菌, 利用26S rDNA D1/D2区域序列分析并结合形态学和生理生化特性对这些菌株进行了分类学研究, 探讨了青海东部土壤中酵母的物种多样性及其分布。共鉴定出10属13种(其中有两个疑似新种), 其中 Galactomyces geotrichum和Rhodotorula mucilaginosa为该地的优势种。     

Screening of micro-organisms for decolorization of melanins produced by bluestain fungi

A total of 17 fungi and four bacteria were screened for their ability to decolorize melanin, using isolated extracellular melanin of the bluestain fungus Aureobasidium pullulans as substrate. On agar media, decolorization was observed by four fungal strains: Bjerkandera adusta VTT-D-99746, Galactomyces geotrichum VTT-D-84228, Trametes hirsuta VTT-D-95443 and Trametes versicolor VTT-D-99747. The four fungi were more efficient on nitrogen-limited medium than on complete medium. The melanin-decolorizing activity of G. geotrichum appeared to be located on the mycelium and could be liberated into the medium enzymatically.     

Fungi isolated from olive ecosystems and screening of their potential biotechnological use

This study investigated the fungi diversity of fresh olive (Olea europaea L.) fruits, olive paste (crushed olives) and olive pomace (solid waste) and screened and quantified enzymatic activities with biotechnological applications. Fungi were randomly isolated from olive cultivars from Castilla La Mancha region (Spain). Identification included comparison of their polymerase chain reaction (PCR) amplicons of the ITS1-5.8S-ITS2 ribosomal DNA region, followed by nucleotide sequence analysis. Fourteen different species with DNA sequences of different similarities were identified, belonging to seven different genera (Aspergillus, Penicillium, Rhizomucor, Mucor, Rhizopus, Lichtheimia and Galactomyces). Aspergillus fumigatus, followed by Galactomyces geotrichum, Penicillium commune and Rhizomucor variabilis var. regularior were the most frequent species. Specific enzyme screening was assayed on agar plates, using cellobiose, carboxymethylcellulose (CMC), polygalacturonic acid and CaCl(2)/Tween 80 as substrates for β-glucosidase, carboxymethylcellulase (CMCase), polygalacturonase and lipase, respectively. Species exhibiting the best activities were: Aspergillus fumigatus (for β-glucosidase, CMCase and lipase); Rhizopus oryzae (for β-glucosidase and lipase); Rhizomucor variabilis (for β-glucosidase, CMCase and polygalacturonase); Mucor fragilis (β-glucosidase, CMCase and lipase); Galactomyces geotrichum (for β-glucosidase, polygalacturonase and lipase) and Penicillium commune and Penicillium crustosum (for lipase). The species that had shown the best enzymatic activities were grown on hemicellulose, cellulose and pectin and some activities were quantified (xylanase, cellulase, β-glucosidase and pectinase). An isolate of A. fumigatus and one of A. niger showed the best cellulase and xylanase activities, while no species presented good pectinase and β-glucosidase activities. The selected species with potential enzymatic activities could be used for future applications of industrial interest.     

Galactomyces geotrichum Y25产脂肪酶条件的优化

应用响应面法对Galactomyces geotrichumY25液体发酵产脂肪酶的条件进行了优化。首先采用Plackett-Burman设计对影响产酶因素的效应进行评价,筛选出黄豆粉、玉米浆和发酵时间3个对产酶影响显著的因素。用最陡爬坡路径逼近最大产酶区域后,利用响应面设计对显著因素进行优化,得出黄豆粉、玉米浆最佳质量分数分别为2.51%、2.12%,最佳发酵时间101.95 h。优化后液体发酵液中脂肪酶活力提高到34.65 U/mL,比初始酶活力9.6 U/mL提高了3.61倍。表明响应面法可显著优化Galactomyces geotrichumY25液体发酵产脂肪酶条件。     

Commercial ripening starter microorganisms inoculated into cheese milk do not successfully establish themselves in the resident microbial ripening consortia of a South german red smear cheese

Production of smear-ripened cheese critically depends on the surface growth of multispecies microbial consortia comprising bacteria and yeasts. These microorganisms often originate from the cheese-making facility and, over many years, have developed into rather stable, dairy-specific associations. While commercial smear starters are frequently used, it is unclear to what degree these are able to establish successfully within the resident microbial consortia. Thus, the fate of the smear starters of a German Limburger cheese subjected to the "old-young" smearing technique was investigated during ripening. The cheese milk was supplemented with a commercial smear starter culture containing Debaryomyces hansenii, Galactomyces geotrichum, Arthrobacter arilaitensis, and Brevibacterium aurantiacum. Additionally, the cheese surface was inoculated with an extremely stable in-house microbial consortium. A total of 1,114 yeast and 1,201 bacterial isolates were identified and differentiated by Fourier transform infrared spectroscopy. Furthermore, mitochondrial DNA restriction fragment length polymorphism, random amplified polymorphic DNA, repetitive PCR, and pulsed field gel electrophoresis analyses were used to type selected isolates below the species level. The D. hansenii starter strain was primarily found early in the ripening process. The G. geotrichum starter strain in particular established itself after relocation to a new ripening room. Otherwise, it occurred at low frequencies. The bacterial smear starters could not be reisolated from the cheese surface at all. It is concluded that none of the smear starter strains were able to compete significantly and in a stable fashion against the resident microbial consortia, a result which might have been linked to the method of application. This finding raises the issue of whether addition of starter microorganisms during production of this type of cheese is actually necessary.     

Purification and characterization of an extracellular lipase of Galactomyces geotrichum

Summary Galactomyces geotrichum secretes an extracellular lipase with a relative molecular weight of 57±2 kDa in the unglycosylated state and 62±2 kDa when glycosylated. The enzyme has optimal activity at pH 7.75 and 30°C and has a pI of 5.2. The specificity of the lipase is similar to lipase A of Geotrichum candidum, but the amino acid composition of the lipases are different.     

Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal DNA D1/D2 domain sequence analysis

The nucleotide sequences of the D1/D2 domains of large subunit (26S) ribosomal DNA for 76 strains of 46 species of pathogenic dematiaceous fungi and related taxa were determined. Intra-species sequence diversity of medically important dematiaceous fungi including Phialophora verrucosa, Fonsecaea pedrosoi, Fonsecaea compacta, Cladophialophora carrionii, Cladophialophora bantiana, Exophiala dermatitidis, Exophiala jeanselmei, Exophiala spinifera, Exophiala moniliae, and Hortaea werneckii were extremely small; as few as 0 changes were detected in C. bantiana, Fonsecaea and Exophiala species, 1 bp in C. carrionii and H. werneckii, and 2 bp in P. verrucosa. Inter-species nucleotide diversity between most species was higher. These data suggested that the D1/D2 domain is sufficiently variable for identification of pathogenic dematiaceous fungi and relevant species. The phylogenetic trees constructed from the sequence data revealed that most human pathogenic species formed a single cluster and that Cladosporium and Phialophora species were distributed polyphyletically into several clusters.     

The D1/D2 domain of the large-subunit rDNA of the yeast species Clavispora lusitaniae is unusually polymorphic

Ten different versions of the D1/D2 divergent domain of the large-subunit ribosomal DNA were identified among interbreeding members of the yeast species Clavispora lusitaniae. One major polymorphism, located in a 90-bp structural motif of the D2 domain, exists in two versions that differ by 32 base substitutions. Three other polymorphisms consist of a two-base substitution, a two-base deletion, and a single-base deletion, respectively. The polymorphisms are independent of one another and of the two mating types, indicating that the strains studied belong to a single, sexually active Mendelian population. Several strains were heterogeneous for one or more of the polymorphisms, and one strain was found to be automictic and capable of producing asci on its own by isogamous conjugation or by bud-parent autogamy. These observations suggest circumspection in the use of sequence divergence as the principal criterion for delimiting yeast species.