Molecular profiling of bacterial species in the rabbit caecum

The diversity of bacteria present in the caecum of the rabbit was investigated. Partial bacterial 16S rRNA genes from a digested sample collected from the caecum of an adult rabbit were amplified by PCR. Sequence analysis of the amplified fragments indicated highest similarity was to bacterial sequences previously described from other gut environments. However, only one sequence showed significant identity (97% threshold) to any previously described bacterial 16S rRNA genes. Furthermore, most of the sequences clustered together in groups lacking representatives from sequences already described, suggesting that the rabbit caecal flora contains organisms not previously described.     

Molecular analysis of methanogenic archaeal communities in managed and natural upland pasture soils

Grassland management influences soil archaeal communities, which appear to be dominated by nonthermophilic crenarchaeotes. To determine whether methanogenic Archaea associated with the Euryarchaeota lineage are also present in grassland soils, anaerobic microcosms containing both managed (improved) and natural (unimproved) grassland rhizosphere soils were incubated for 28 days to encourage the growth of anaerobic Archaea. The contribution of potential methanogenic organisms to the archaeal community was assessed by the molecular analysis of RNA extracted from soil, using primers targeting all Archaea and Euryarchaeota. Archaeal RT‐PCR products were obtained from all anaerobic microcosms. However, euryarchaeal RT‐PCR products (of putative methanogen origin) were obtained only from anaerobic microcosms of improved soil, their presence coinciding with detectable methane production. Sequence analysis of excised denaturing gradient gel electrophoresis (DGGE) bands revealed the presence of euryarchaeal organisms that could not be detected before anaerobic enrichment. These data indicate that nonmethanogenic Crenarchaeota dominate archaeal communities in grassland soil and suggest that management practices encourage euryarchaeal methanogenic activity.     

Bacterial and archaeal profiling of hypersaline microbial mats and endoevaporites,under natural conditions and methanogenic microcosm experiments

Extremophiles - Bacterial and archaeal community structure of five microbial communities, developing at different salinities in Baja California Sur, Mexico, were characterized by 16S rRNA...     

Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates

Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.     

Molecular identification of Pilobolus species from Yellowstone National Park

Pilobolus, a widely distributed coprophilous fungus, grows on herbivore dung. Species of Pilobolus traditionally are described with imprecise morphological characteristics potentially leading to misidentification. In this study we used PCR analysis of taxonomically informative sequences to provide more consistent species identification from isolates obtained in Yellowstone National Park. We collected Pilobolus park-wide from six taxa of herbivores over 9 y. Multiple transfers of single sporangium isolates provided pure cultures from which DNA was extracted. Sequence analysis of internal transcribed spacer (ITS) regions of DNA that code for rRNA genes were used to distinguish among similar species. We describe several species of Pilobolus associated with herbivores in various habitats, including two species not previously reported, P. heterosporus and P. sphaerosporus. Our results show that phylogenetic species identification of Pilobolus based on sequence analysis of pure culture isolates provides a more reliable means of identifying species than traditional methods.     

Molecular identification of mosquito species

Complexes of closely related cryptic species, that are indistinguishable morphologically yet have different ecology and host preferences, are widespread in mosquitoes. The advantages of DNA-based methods of identification mean that they have now largely replaced other methods of species determination for such complexes. Here we discuss the relative merits of three different approaches to species identification, all of which use amplification of the ribosomal RNA genes by the polymerase chain reaction. They include: restriction fragment length polymorphism in the Anopheles maculipennis complex from the UK; allele-specific amplification in the An. dims complex from Thailand; and single strand conformational polymorphism in the An. minimus complex from Thailand. The application of these methods is considered in the context of recent data on intraspecific genetic variation, geographic population structure and genetic introgression.     

Molecular identification of Tetrahymena species

Mitochondrial cox1 689 bp barcodes are routinely used for identification of Tetrahymena species. Here, we examine whether two shorter nuclear sequences, the 5.8S rRNA gene region and the intergenic region between H3 and H4 histone genes, might also be useful either singly or in combination with each other or cox1. We obtained sequences from ~300 wild isolates deposited at the Tetrahymena Stock Center and analyzed additional sequences obtained from GenBank. The 5.8S rRNA gene and portions of its transcribed flanks identify isolates as to their major clade and uniquely identify some, but not all, species. The ~330 bp H3/H4 intergenic region possesses low intraspecific variability and is unique for most species. However, it fails to distinguish between two pairs of common species and their rarer counterparts, and its use is complicated by the presence of duplicate genes in some species. The results show that while the cox1 sequence is the best single marker for Tetrahymena species identification, 5.8S rRNA, and the H3/H4 intergenic regions sequences are useful, singly or in combination, to confirm cox1 species assignments or as part of a preliminary survey of newly collected Tetrahymena. From our newly collected isolates, the results extend the biogeographical range of T. shanghaiensis and T. malaccensis and identify a new species, Tetrahymena arleneae n. sp. herein described.     

Systematic identification of substrates for profiling of secreted proteases from Aspergillus species

Reliable and early diagnosis of life-threatening invasive mycoses in neutropenic patients caused by fungi of the Aspergillus species remains challenging because current clinical diagnostic tools lack in sensitivity and/or specificity. During invasive growth a variety of fungal proteases are secreted into the bloodstream and protease profiling with reporter peptides might improve diagnosis of invasive aspergillosis in serum specimens. To characterise the specific protease activity of Aspergillus fumigatus and Aspergillus niger we analyzed Aspergillus culture supernatants, human serum and the mixture of both. A systematic screening for optimised protease substrates was performed using a random peptide library consisting of 360 synthetic peptides featuring fluorescence resonance energy transfer (FRET). We could identify numerous peptides that are selectively cleaved by fungus-specific proteases. These reporter peptides might be feasible for future protease profiling of serum specimens to improve diagnosis and monitoring of invasive aspergillosis.     

Molecular and morphological identification of fungal species isolated from bealmijang meju

Bealmijang is a short-term aged paste made from meju, which is a brick of fermented soybeans and other ingredients. Different types of bealmijang are available depending on the geographic region or ingredients used. However, no study has clarified the microbial diversity of these types. We identified 17 and 14 fungal species from black soybean meju (BSM) and buckwheat meju (BWM), respectively, on the basis of morphology, culture characteristics, and internal transcribed spacer and beta-tubulin gene sequencing. In both meju, Aspergillus oryzae, Rhizopus oryzae, Penicillium polonicum, P. steckii, Cladosporium tenuissimum, C. cladosporioides, C. uredinicola, and yeast species Pichia burtonii were commonly found. Moreover, A. flavus, A. niger, P. crustosum, P. citrinum, Eurotium niveoglaucum, Absidia corymbifera, Setomelanomma holmii, Cladosporium spp. and unclassified species were identified from BSM. A. clavatus, Mucor circinelloides, M. racemosus, P. brevicompactum, Davidiella tassiana, and Cladosporium spp. were isolated from BWM. Fast growing Zygomycetous fungi is considered important for the early stage of meju fermentation, and A. oryae and A. niger might play a pivotal role in meju fermentation owing to their excellent enzyme productive activities. It is supposed that Penicillium sp. and Pichia burtonii could contribute to the flavor of the final food products. Identification of this fungal diversity will be useful for understanding the microbiota that participate in meju fermentation, and these fungal isolates can be utilized in the fermented foods and biotechnology industries.     

Molecular identification of methanogenic archaea from sheep in Queensland, Australia reveal more uncultured novel archaea

Molecular diversity of rumen methanogens in sheep in Queensland, Australia was investigated using 16S rRNA gene libraries prepared from pooled rumen contents from nine merino sheep. A total of 78 clones were identified revealing 26 different sequences. Of these 26 sequences, eight sequences (15 clones) were 95-100% similar to cultivated methanogens belonging to the orders Methanobacteriales and Methanomicrobiales, and the remaining 18 phylotypes (63 clones) were 72-75% similar to Thermoplasma acidophilum and Thermoplasma volcanium. These unique sequences clustered within a distinct and strongly supported (100% bootstrap support) phylogenetic group, exclusively composed of sequences from uncharacterized archaea from very diverse anaerobic environments. Members of this unique group that were previously considered atypical for the rumen environment were the predominant clones.     

Molecular identification of Candida species from urinary infections in Honduras

Background

Candiduria is a common infection among hospitalised patients. Although the clinical relevance of yeasts in urine is not clearly defined, fungal urinary tract infections have increased significantly in the last decades, becoming a growing public health problem. Candida albicans is the most commonly reported species in urinary infections, although other species of the genus are becoming particularly important, because some of them are linked with resistance to antifungal drugs.

Molecular species identification boosts bat diversity

The lack of obvious morphological differences between species impedes the identification of species in many groups of organisms. Meanwhile, DNA-based approaches are increasingly used to survey biological diversity. In this study we show that sequencing the mitochondrial protein-coding gene NADH dehydrogenase, subunit 1 (nd1) from 534 bats of the Western Palaearctic region corroborates the promise of DNA barcodes in two major respects. First, species described with classical taxonomic tools can be genetically identified with only a few exceptions. Second, substantial sequence divergence suggests an unexpected high number of undiscovered species.     

产甲烷条件下岩溶湿地沉积物中古菌群落的变化规律

【背景】桂林会仙湿地位于喀斯特峰丛洼地和峰丛平原的过渡区,主要由岩溶地下河补给,水质呈现富钙偏碱特征,是一处典型的岩溶湿地环境,湿地沉积物以厌氧环境为主,独特的环境特征使之成为研究新型厌氧微生物的理想场所。氢型产甲烷菌和乙酸型产甲烷菌是环境中有机质厌氧降解的重要参与者,但目前会仙湿地产甲烷菌生理生态功能的研究十分有限。【目的】揭示乙酸盐营养型产甲烷条件和氢气营养型产甲烷条件下岩溶湿地环境古菌群落结构的变化规律和产甲烷菌的主要类群,探讨岩溶环境古菌的功能和相互关系。【方法】以会仙岩溶湿地沉积物为研究材料,分别以乙酸盐和氢气为唯一能源物进行富集培养,结合未添加任何能源底物的对照处理,动态监测产甲烷通量,分别构建了3个共包含146条古菌16S rRNA基因全长序列的文库,以及3个共包含138条甲基辅酶M还原酶基因mcr A基因序列的文库,通过构建系统发育树比较分析不同产甲烷底物培养条件下典型岩溶湿地古菌群落的变化规律。【结果】从乙酸型和氢型产甲烷条件培养物顶空气中都检测到了几乎等浓度的甲烷产生,甲烷八叠球菌是mcr A文库中主要的古菌类群,其中包含了Methanosarcina属古菌、Zoige Cluster I (ZC-I)和ANME-2d Cluster (AD)类群古菌,以及两个相对独立且不包含任何参考序列的分支KT-I和KT-II,可能代表产甲烷菌的新类群;经过两种产甲烷条件的富集培养后,ZC-I、AD和KT-II类群古菌的序列数占比有较为显著的增加(45%–155%);深古菌(Bathyarchaeota)是所有古菌16Sr RNA基因文库的优势类群,序列占比为88%–100%,其中MCG-11亚群最为丰富,占所有深古菌的84%,并且在乙酸盐产甲烷条件下增加了17%。【结论】会仙湿地沉积物中蕴涵着丰富的新型古菌序列,沉积物中主要的氢型产甲烷菌和乙酸型产甲烷菌都来自甲烷八叠球菌目,深古菌在岩溶环境和乙酸型产甲烷条件下可能都发挥着重要的作用。     

Molecular identification of Anisakis species from Pleuronectiformes off the Portuguese coast

Anisakid nematodes belonging to the Anisakis simplex complex are highly prevalent in several fish species off the coast of Portugal and are an important zoonotic problem in the Iberian Peninsula. Two reproductively isolated sibling species of the Anisakis simplex complex were identified from Pleuronectiformes inhabiting the Portuguese coast using restriction fragment length polymorphism (RFLP). Recombinant genotypes corresponding to presumptive Anisakis simplex sensu stricto and Anisakis pegreffii hybrids were also detected by this technique, as well as the species Anisakis typica. Although 25 species of Pleuronectiformes were investigated, Anisakis spp. larvae were only found in seven: Arnoglossus imperialis, Arnoglossus laterna, Lepidorhombus boscii, Citharus linguatula, Platichthys flesus, Dicologlossa cuneata and Solea senegalensis. The occurrence of hybrids in relatively sedentary fishes such as the Pleuronectiformes suggests that the Portuguese coast may constitute an area of hybridization and, therefore, is of particular interest for the study of the process of hybridization and speciation for these anisakids.     

Molecular forensic profiling of Cryptosporidium species and genotypes in raw water

The emerging concept of host specificity of Cryptosporidium spp. was exploited to characterize sources of fecal contamination in a watershed. A method of molecular forensic profiling of Cryptosporidium oocysts on microscope slides prepared from raw water samples processed by U.S. Environmental Protection Agency Method 1623 was developed. The method was based on a repetitive nested PCR-restriction fragment length polymorphism-DNA sequencing approach that permitted the resolution of multiple species/genotypes of Cryptosporidium in a single water sample.     

Structural, mass and elemental analyses of storage granules in methanogenic archaeal cells

Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granule-like inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility.     

Molecular identification of co-occurring Cortinarius and Dermocybe species from southeastern Australian sclerophyll forests

 The ability of restriction fragment length polymorphism (RFLP) analysis of the rDNA internal transcribed spacer (ITS) region to discriminate 10 co-occurring Cortinarius and Dermocybe species at a southeastern Australian sclerophyll forest site was assessed. Using the basidiomycete-specific primers ITS1F and ITS4B, some taxa were separated on the basis of individual RFLP patterns derived using the restriction endonucleases Hae III or Hinf I. Combined data from both endonucleases were, however, required to separate all taxa [Dermocybe austro-veneta Clel. (Moser & Horak), C. rotundisporus Clel. & Cheel, C. archeri Berk., C. sinapicolor Clel., C. violaceus (L.: Fr.) S.F.Gray, C. radicatus Clel. and four morphologically-distinct, but unidentified Cortinarius spp.]. ITS sequence comparisons confirmed that D. austro-veneta belongs in Dermocybe, that C. rotundisporus is correctly placed in subgenus Phlegmacium, and suggest that Australian C. violaceus collections are not conspecific with northern hemisphere C. violaceus. Accepted: 4 March 1999     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.