Heteroduplex mobility assay of the 26S rDNA D1/D2 region for differentiation of clinically relevant Candida species

The Heteroduplex Mobility Assay (HMA) method using the PCR amplified D1/D2 region of the 26S rDNA was tested for the differentiation of clinically relevant Candida species. Strains belonging to the same species are not expected to form heteroduplexes in this assay when their PCR products are mixed. D1/D2 HMA experiments between all Candida type strains tested showed heteroduplex formation, including Candida albicans and Candida dubliniensis. There was no heteroduplex formation when most clinical and non-type strains were tested against the type strain of their presumptive species, except when C. albicans WVE and C.␣dubliniensis TAI were analysed. Additional HMA experiments, phenotypic characterisation, and D1/D2 sequencing identified these isolates as Candida tropicalis and Candida parapsilosis, respectively. HMA provides a rapid and relatively simple molecular tool for the differentiation of potentially pathogenic Candida species.     

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%。     

Heteroduplex mobility assay detects DNA mutations for differentiation of closely related phytoplasma strains

We present the first use of DNA heteroduplex mobility assay (HMA) to detect the point mutations including substitutions and deletions/insertions in 16S rDNA of aster yellows phytoplasma (AY27) and to differentiate phytoplasmas collected from field samples of clover proliferation (CP) and alfalfa witches'-broom (AWB). The phytoplasmal 16S rDNA fragment was amplified from AY27 by polymerase chain reaction (PCR) and cloned into a plasmid vector. The cloned DNA fragment was subjected to in vitro mutation to produce 1- to 4-base substitutions and 1- to 3-base deletions. The mutated 16S rDNA fragments were analyzed by HMA. The results showed that a single two-base substitution or a single-base deletion/insertion in the 529 bp DNA fragment was directly detected and that a DNA divergence at a level of as low as 0.2% was detectable by HMA. Heteroduplex mobilities were affected by the number and composition of the phytoplasma DNA bases in mismatches or gaps and were proportional to the degree of DNA divergences. Gaps caused greater retardation in heteroduplex mobility than mismatches did. HMA was highly sensitive in detecting the mixed infections of phytoplasmas. In analyses of CP and AWB field samples collected in Alberta, two CP and one AWB phytoplasma isolates were differentiated from others by HMA but not by restriction fragment length polymorphism (RFLP). Therefore, HMA provides a simple, rapid, highly sensitive and analytical method to detect and estimate the genetic divergence of phytoplasmas when other methods such as RFLP are not readily applicable.     

Phylogenetic analysis of isolated biofuel yeasts based on 5.8S-ITS rDNA and D1/D2 26S rDNA sequences

The utilization of agro-industrial wastes such as whey as raw materials for the production of bio-ethanol is gaining importance as a result of the attractiveness of renewable fuel alternatives due to exhaustion of fossil fuel sources coupled with the positive impact to the environment. Here, we report the isolation of two Kluyveromyces spp. designated as BM4 and P41, able to produce ethanol as main fermentation product from fermenting whey. Three different molecular biological approaches including, the RFLP analysis of the 5.8S-ITS rDNA, the sequence of the 5.8S-ITS rDNA region and the sequence of the D1/D2 domain of the 26S rRNA gene were applied for accurate identification. While RFLP analysis of 5.8S-ITS region failed to accurate the differentiation between the two species, sequencing of this region and D1/D2 region of the 26S rRNA gene verified the identification. PCR amplification and sequence analysis of 5.8S-ITS rRNA and D1/D2 domain of the 26S rRNA genes revealed that the isolates BM4 and P41 were highly related to Kluyveromyces marxianus and Kluyveromyces lactis with homology of 99% for both. In addition, phylogenetic analysis indicated that both BM4 and P41 shared a cluster with K. marxianus and K. lactis, respectively. The fermentative performance of both strains on cheese whey to produce ethanol was evaluated at different parameters such as incubation temperature, initial pH, whey sugar concentrations, and yeast concentrations. Results show that the maximum ethanol productions achieved at pH 4.5 and 35 °C were 5.52% and 5.05% for K. marxianus and K. lactis, respectively. Our results demonstrated that K. marxianus and K. Lactis could be recommended for cheese whey bioremediation in the environment and produce renewable biofuel.     

The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene

A taxonomic study was carried out on eight strains of Saccharomyces boulardii. Morphological and physiological characteristics were consistent with those of Saccharomyces cerevisiae. Sequences of the D1/D2 domain of the 26S rDNA were identical for all strains examined and had a similarity value of 100% compared to sequences of the type strain of S. cerevisiae (CBS 1171T) and strain S288c. For all S. boulardii isolates was found the exact same ITS1-5.8S rDNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171(T) (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii isolates and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171(T) (96.6%). The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from strains of this species. The results of the present study strongly indicate a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species.     

Intraspecies diversity of the industrial yeast strains Saccharomyces cerevisiae and Saccharomyces pastorianus based on analysis of the sequences of the internal transcribed spacer (ITS) regions and the D1/D2 region of 26S rDNA

We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions.     

假丝酵母属疑难菌株大亚基rDNA D1/D2区域序列分析及其分类学意义

对根据常规形态和生理生化性状难以确定分类学地位的8株假丝酵母菌,进行了以大亚基(26S) rDNA中D1/D2区域(约500～600 bp)的碱基序列分析为依据的分子分类学研究.根据系统树上所显示的供试菌株与假丝酵母属及相关子囊菌酵母已知种的亲缘关系,以及与最近缘种模式菌株D1/D2区域序列的相似性比较,确定了各个菌株的归属.本研究也显示了DNA序列分析在假丝酵母菌快速鉴定中的优越性.     

假丝酵母属疑难菌株大亚基rDNA D1/D2区域序列分析及其分类学意义*

对根据常规形态和生理生化性状难以确定分类学地位的8株假丝酵母菌,进行了以大亚基(26S) rDNA中D1/D2区域(约500~600 bp)的碱基序列分析为依据的分子分类学研究。根据系统树上所显示的供试菌株与假丝酵母属及相关子囊菌酵母已知种的亲缘关系,以及与最近缘种模式菌株D1/D2区域序列的相似性比较,确定了各个菌株的归属。本研究也显示了DNA序列分析在假丝酵母菌快速鉴定中的优越性。     

PCR amplification of the rDNA internal transcribed spacer region for differentiation of Saccharomyces cultures

Abstract The size of the internal transcribed spacer (ITS) region as measured by gel electrophoresis of PCR products, amplified by primers ITS1 and ITS4, was over 800 bp for all Saccharomyces sensu stricto species, but yeasts belonging to other Saccharomyces species had a shorter ITS region, making this characteristic potentially useful in the identification of Saccharomyces isolates. The ITS product length was homogeneous within the species Saccharomyces cerevisiae .     

三个发表于国内的丝孢酵母种名分类地位的订正*

最新的酵母菌分类系统,丝孢酵母属Trichosporon Behrend仅限于产生节孢子的担子菌酵母的无性型。因此,以前根据该属旧的定义在国内发表的三个新种板仓丝孢酵母T. bancangense、北京丝孢酵母T. beijingense和中国丝孢酵母T. sinense的分类地位,需要进行调整。根据26S rDNA D1/D2区的序列分析和最新标准方法重新测定的生理生化反应,发现上述三个种名分别是子囊菌酵母Saccharomycopsis fibuligera, Pichia burtonii和Issatchenkia orientalis的异名。     

基于26S rDNAD1/D2区序列分析的15株白地霉分子分类学研究

采用26S rRNA基因D1/D2区系统发育分析的方法对CICC(中国工业微生物菌种保藏管理中心)保藏的15株白地霉(Geotrichum candidum)菌种进行复核鉴定。系统发育分析结果表明15株白地霉属于地霉属的成员,且形成两个系统发育分支,系统发育上最接近Galactomyces geotrichum NRRLY-17569T,与其同源性为96.3%～98.3%。15株白地霉26S rRNA基因D1/D2区序列显著不同于地霉属的模式种及其它种,可能代表地霉属的两个新种,但这一结论尚需进一步的实验去证实。     

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.     

Development of a novel triplex PCR assay for the detection and differentiation of thermophilic species of Campylobacter using 16S-23S rDNA internal transcribed spacer (ITS) region

AIM: Campylobacter species are significantly implicated in human gastrointestinal infections. Of 20 species of Campylobacter, C. jejuni, C. coli and C. lari have been considered as the most important causative agents of human infections. In order to better understand the occurrence and epidemiology of these thermophilic Campylobacter species, an improved and rapid detection method is warranted. A novel triplex polymerase chain reaction (PCR) assay was developed based on the variable 16S-23S rDNA internal transcribed spacer (ITS) region to identify and discriminate between these species in water samples. METHODS AND RESULTS: Campylobacter species-specific primers for C. jejuni, C. coli and C. lari derived from highly variable sequences in the ITS region were used. Specificity of the newly designed primers and PCR conditions were verified using other species of Campylobacter as well as 31 different negative control species. The assay was further validated with 97 Campylobacter cultures from water samples. CONCLUSIONS: The assay was found to be simple, easy to perform, and had a high sensitivity, specificity and reproducibility. It enabled simultaneous detection and differentiation of multiple Campylobacter species in water samples. SIGNIFICANCE AND IMPACT OF STUDY: Use of the newly developed PCR assay, coupled with a previously developed rapid DNA template preparation step, will enable improved detection capabilities for Campylobacter species in environmental matrices.     

In Situ Accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-Labeled Oligonucleotide Probes Comprising the D1 and D2 Domains

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455^T. The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.     

In situ accessibility of Saccharomyces cerevisiae 26S rRNA to Cy3-labeled oligonucleotide probes comprising the D1 and D2 domains

Fluorescence in situ hybridization (FISH) has proven to be most useful for the identification of microorganisms. However, species-specific oligonucleotide probes often fail to give satisfactory results. Among the causes leading to low hybridization signals is the reduced accessibility of the targeted rRNA site to the oligonucleotide, mainly for structural reasons. In this study we used flow cytometry to determine whole-cell fluorescence intensities with a set of 32 Cy3-labeled oligonucleotide probes covering the full length of the D1 and D2 domains in the 26S rRNA of Saccharomyces cerevisiae PYCC 4455(T). The brightest signal was obtained with a probe complementary to positions 223 to 240. Almost half of the probes conferred a fluorescence intensity above 60% of the maximum, whereas only one probe could hardly detect the cells. The accessibility map based on the results obtained can be extrapolated to other yeasts, as shown experimentally with 27 additional species (14 ascomycetes and 13 basidiomycetes). This work contributes to a more rational design of species-specific probes for yeast identification and monitoring.     

利用26S rDNAD1/D2区序列和微卫星标记分析各种工业酿酒酵母的种内遗传差异

选取30株传统发酵工业中不同来源的酿酒酵母菌株和实验室常用酿酒酵母菌株,利用大亚基(26S)rDNA D1/D2区序列和微卫星标记分析酿酒酵母种内菌株间的遗传多样性和系统发育关系,以揭示酿酒酵母在长期的各种工业发酵环境下发生的遗传变异。结果表明:30株酿酒酵母的26S rDNA D1/D2区序列(591bp)比较保守,与测序菌株S288C相比序列相似度在99.8%–100%,说明种内菌株间存在一定的多态性,但序列差异并不十分明显,其变异情况表现在个别碱基的差异(大多由转换突变引起);通过扩增11个微卫星位点,每个菌株均有其独特的基因型,即30种基因型,共得到188个等位基因,观测杂合度平均值和期望杂合度平均值分别为0.576、0.886,多态信息含量平均值高达0.858,说明酿酒酵母种内菌株间具有较高的遗传多样性,而聚类分析表明30株酿酒酵母可以得到很好地区分,但是没有呈现与其工业来源相关的聚类。     

Heteroduplex mobility assay as a tool for predicting phylogenetic affiliation of environmental ribosomal RNA clones

Heteroduplex mobility assay (HMA) of partial 16S rRNA gene fragments was tested as a tool for predicting bacterial phylogenetic relationships. Approximately 400-bp fragments were amplified from a selection of cloned environmental DNAs representing a range of sequence identities and phylogenetic relationships. Heteroduplexes between pairs of sequences were formed by mixing equal amounts of PCR products, denaturing and annealing. Annealed mixes were separated on 8% polyacrylamide gels and silver stained. Heteroduplexes were readily distinguished from reannealed homoduplex and unannealed fragments in all sequences where percentage identity was less than 95%. The heteroduplexes showed retarded electrophoretic migration with respect to homoduplexes. The relative retardation was strongly correlated to the percentage sequence identity between the two strands. The HMA is a useful tool for screening environmental clone libraries to systematically select clones representative of the phylogenetic diversity within the sample, or to selectively retrieve members of a particular phylogenetic group for more detailed study.     

Sequence analysis in the 23S rDNA region of Mycobacterium tuberculosis and related species

We sequenced a 516 base pair segment in the 23S rRNA gene of 54 Mycobacterium tuberculosis isolates, 52 of which were clinical isolates from Ethiopia. Sequence polymorphism was observed with 19 of the 54 strains; the polymorphic sites occurred in less than 2% (9/516) of the total sequence positions. The sequence variations represented base pair substitutions (14/23), insertions (9/23) or both (1/23). Insertions occurred at one site only, whereas substitutions were observed in various regions of the gene. There was no relation between mutational sites and drug susceptibility. However, using information from the GenBank database, comparison between the 23S rDNA sequences of M. tuberculosis and the corresponding sequences of other mycobacteria and of related non-mycobacterial species revealed considerable variation, suggesting that this region may provide a target for rapid detection and identification of mycobacteria both at the genus or species level.     

An evaluation of LSU rDNA D1-D2 sequences for their use in species identification

Background

The Radical Pair model proposes that magnetoreception is a light-dependent process. Under low monochromatic light from the short-wavelength part of the visual spectrum, migratory birds show orientation in their migratory direction. Under monochromatic light of higher intensity, however, they showed unusual preferences for other directions or axial preferences. To determine whether or not these responses are still controlled by the respective light regimes, European robins, Erithacus rubecula, were tested under UV, Blue, Turquoise and Green light at increasing intensities, with orientation in migratory direction serving as a criterion whether or not magnetoreception works in the normal way.

Results

The birds were well oriented in their seasonally appropriate migratory direction under 424 nm Blue, 502 nm Turquoise and 565 nm Green light of low intensity with a quantal flux of 8·10¹⁵ quanta s^-1 m^-2, indicating unimpaired magnetoreception. Under 373 nm UV of the same quantal flux, they were not oriented in migratory direction, showing a preference for the east-west axis instead, but they were well oriented in migratory direction under UV of lower intensity. Intensities of above 36·10¹⁵ quanta s^-1 m^-2 of Blue, Turquoise and Green light elicited a variety of responses: disorientation, headings along the east-west axis, headings along the north-south axis or 'fixed' direction tendencies. These responses changed as the intensity was increased from 36·10¹⁵ quanta s^-1 m^-2 to 54 and 72·10¹⁵ quanta s^-1 m^-2.

Conclusion

The specific manifestation of responses in directions other than the migratory direction clearly depends on the ambient light regime. This implies that even when the mechanisms normally providing magnetic compass information seem disrupted, processes that are activated by light still control the behavior. It suggests complex interactions between different types of receptors, magnetic and visual. The nature of the receptors involved and details of their connections are not yet known; however, a role of the color cones in the processes mediating magnetic input is suggested.     

15 Phylogeny of the chlorophyta: inferences from 18S and 26S rDNA

Recent studies of the Chlorophyceae using 18S and 26S rDNA data in meta‐analysis have demonstrated the power of combining these two sets of rDNA data. Furthermore, the 26S rDNA data complement the more conserved 18S gene for many chlorophycean lineages. Consequently, this data approach was pursued in an expanded taxon‐sampling scheme for the Chlorophyta, with special reference to the classes Chlorophyceae and Trebouxiophyceae. Results of these new phylogenetic analyses identify Microspora sp. (UTEX LB 472) and Radiofilum transversale (UTEX LB 1252) as sister taxa which, in turn, form a basal clade in the Cylindrocapsa alliance (Treubaria, Trochiscia, Elakatothrix). The relative position of the “Cylindrocapsa” clade within the Chlorophyceae remains uncertain. The enhanced taxon‐sampling has not resolved the relative positions of the Oedogoniales, Chaetophorales or Chaetopeltidales. Furthermore, the Sphaeropleaceae are supported as members of the Sphaeropleales in only some analyses, raising concerns about the status of the order. Although based on a limited set of taxa (currently <10), a combined data approach reveals support for a monophyletic Trebouxiophyceae that includes the distinctive organisms, Geminella and Eremosphaera. The goal of a well‐resolved phylogeny for the Chlorophyta remains just that, a goal. Achieving that goal obviously will require additional taxon sampling in the Prasinophyceae and Ulvophyceae, as well as, the Trebouxiophyceae. Moreover, it is clear that other genes (e.g., cp‐atpB, cp‐rbcL, cp‐16S, mt‐nad5) will be needed to help address problems of resolution based on the rDNA data alone. Supported by NSF DEB 9726588 and DEB 0129030.