The Use of Terminal Restriction Fragment Length Polymorphism (T-RFLP) for the Characterisation of Microbial Communities in Marine Sediments

Terminal Restriction Fragment Length Polymorphism (T-RFLP) of PCR amplified 16S rRNA genes was used to investigate microbial communities in the sediments of Ria Formosa, Portugal. Five replicates of surface sand sediments were collected at an artificial inlet to the sea, between June 2001 and July 2002. Restriction enzymes Msp1 and Hha1 provided 57 different terminal fragments (T-RFs). The sediments were essentially dominated by the same ribotypes throughout the year, with seasonal shifts attributed to minor ribotypes. Principal component analysis of the T-RFs profile revealed no consistent pattern of temporal variation and no consistent grouping of replicate sediment samples. The results suggest that the small-scale spatial variability outweighs the seasonal variability. Phylogenetic affiliations suggested that the dominant bacteria were representatives of the α-Proteobacteria group.     

Assessment of fecal pollution sources in a small northern-plains watershed using PCR and phylogenetic analyses of Bacteroidetes 16S rRNA gene

We evaluated the efficacy, sensitivity, host-specificity, and spatial/temporal dynamics of human- and ruminant-specific 16S rRNA gene Bacteroidetes markers used to assess the sources of fecal pollution in a fecally impacted watershed. Phylogenetic analyses of 1271 fecal and environmental 16S rRNA gene clones were also performed to study the diversity of Bacteroidetes in this watershed. The host-specific assays indicated that ruminant feces were present in 28-54% of the water samples and in all sampling seasons, with increasing frequency in downstream sites. The human-targeted assays indicated that only 3-5% of the water samples were positive for human fecal signals, although a higher percentage of human-associated signals (19-24%) were detected in sediment samples. Phylogenetic analysis indicated that 57% of all water clones clustered with yet-to-be-cultured Bacteroidetes species associated with sequences obtained from ruminant feces, further supporting the prevalence of ruminant contamination in this watershed. However, since several clusters contained sequences from multiple sources, future studies need to consider the potential cosmopolitan nature of these bacterial populations when assessing fecal pollution sources using Bacteroidetes markers. Moreover, additional data is needed in order to understand the distribution of Bacteroidetes host-specific markers and their relationship to water quality regulatory standards.     

Molecular Analysis of Deep-Sea Hydrothermal Vent Aerobic Methanotrophs by Targeting Genes of 16S rRNA and Particulate Methane Monooxygenase

Molecular diversity of deep-sea hydrothermal vent aerobic methanotrophs was studied using both 16S ribosomalDNA and pmoA encoding the subunit A of particulate methane monooxygenase (pMOA). Hydrothermal vent plume and chimney samples were collected from back-arc vent at Mid-Okinawa Trough (MOT), Japan, and the Trans-Atlantic Geotraverse (TAG) site along Mid-Atlantic Ridge, respectively. The target genes were amplified by polymerase chain reaction from the bulk DNA using specific primers and cloned. Fifty clones from each clone library were directly sequenced. The 16S rDNA sequences were grouped into 3 operational taxonomic units (OTUs), 2 from MOT and 1 from TAG. Two OTUs (1 MOT and 1 TAG) were located within the branch of type I methanotrophic ?-Proteobacteria. Another MOT OTU formed a unique phylogenetic lineage related to type I methanotrophs. Direct sequencing of 50 clones each from the MOT and TAG samples yielded 17 and 4 operational pmoA units (OPUs), respectively. The phylogenetic tree based on the pMOA amino acid sequences deduced from OPUs formed diverse phylogenetic lineages within the branch of type I methanotrophs, except for the OPU MOT-pmoA-8 related to type X methanotrophs. The deduced pMOA topologies were similar to those of all known pMOA, which may suggest that the pmoA gene is conserved through evolution. Neither the 16S rDNA nor pmoA molecular analysis could detect type II methanotrophs, which suggests the absence of type II methanotrophs in the collected vent samples.     

Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers

PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples.     

Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes

We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 x 10(-5) to 2.8 x 10(-7) g (dry weight) of feces/liter and 6.8 x 10(-7) g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.     

Molecular phylogeny of musk deer: a genomic view with mitochondrial 16S rRNA and cytochrome b gene

The phylogenetic status of the infra order Pecora is controversial, even though it is supported by paleontological, morphological, and molecular evidence. We analyzed two mitochondrial genes (i.e., 16S rRNA and cytochrome b) to resolve the phylogenetic position of pecoran species, i.e., the Bovidae, Cervidae, and Moschidae endemic to the Indian subcontinent. We used phylogenetic analysis based on different algorithms, including neighbor joining, maximum parsimony, Bayesian inference, maximum likelihood, minimum evolution, median joining network, along with multidimensional scaling, and DNA word analysis. Our results established the basal position of Tragulidae and the monophyly of the infra order Pecora within the Suborder Ruminantia. Our results also demonstrated that Bovidae, Cervidae, and Moschidae are allied with the placement of musk deer as more closely related to bovids than to cervids. Molecular dating based on sequence analysis shows that the radiation of Pecora occurred during the early Oligocene and that the majority of the pecoran families radiated and dispersed rapidly during the Oligocene/Miocene transition.     

基于16S rRNA基因测序技术分析猪链球菌2型感染BALB/c小鼠粪便样本中微生物的变化

【目的】通过研究粪便样本的微生物特点间接探讨猪链球菌2型感染BALB/c小鼠后肠道微生物的变化。【方法】本研究采用IlluminaMiSeq测序技术,测定了健康小鼠和猪链球菌2型感染小鼠粪便中微生物16S rRNA V3-V4区序列,并对群落结构和多样性进行了比较分析。【结果】多样性分析表明,感染组和对照组小鼠的粪便中微生物多样性和群落组成均不相同,存在较大的差异,感染组展示了较高的物种丰度和种群差异性。在门水平上,相对于对照组,感染组增加厚壁菌门和拟杆菌门等有益微生物比例,以提高机体免疫力,但也同时增加了变形杆菌等条件致病菌的比例,增加了疾病发生的可能性。在科水平上,感染组和对照组优势菌均含有瘤胃菌科,但其所占细菌总序列的比例存在较大的差异,分别占感染组和对照组细菌总序列的36.58%和11.02%。在属水平上,感染组和对照组的优势菌属类别及占总微生物比例存在显著的差异。感染组主要以瘤胃菌科UCG-002和乳酸杆菌属为优势菌属,对照组以螺旋体科GWE2-31-10和密螺旋体属2为优势菌属。综上,BALB/c小鼠感染猪链球菌2型后存在肠道微生态失调。【结论】通过本研究,不仅对猪链球菌2...     

Molecular phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial inoculants

Nitrogen is often a limiting nutrient, therefore the sustainability of food crops, forages and green manure legumes is mainly associated with their ability to establish symbiotic associations with stem and root-nodulating N2-fixing rhizobia. The selection, identification and maintenance of elite strains for each host are critical. Decades of research in Brazil resulted in a list of strains officially recommended for several legumes, but their genetic diversity is poorly known. This study aimed at gaining a better understanding of phylogenetic relationships of 68 rhizobial strains recommended for 64 legumes, based on the sequencing of the 16S rRNA genes. The strains were isolated from a wide range of legumes, including all three subfamilies and 17 tribes. Nine main phylogenetic branches were defined, seven of them related to the rhizobial species: Bradyrhizobium japonicum, B. elkanii, Rhizobium tropici, R. leguminosarum, Sinorhizobium meliloti/S. fredii, Mesorhizobium ciceri/M. loti, and Azorhizobium caulinodans. However, some strains differed by up to 35 nucleotides from the type strains, which suggests that they may represent new species. Two other clusters included bacteria showing similarity with the genera Methylobacterium and Burkholderia, and amplification with primers for nifH and/or nodC regions was achieved with these strains. Host specificity of several strains was very low, as they were capable of nodulating legumes of different tribes and subfamilies. Furthermore, host specificity was not related to 16S rRNA, therefore evolution of ribosomal and symbiotic genes may have been diverse. Finally, the great diversity observed in this study emphasizes that tropics are an important reservoir of N2-fixation genes.     

Comparative phylogenetic analyses of Halomonas variabilis and related organisms based on 16S rRNA, gyrB and ectBC gene sequences

Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids. Ten strains were selected for physiological and phylogenetic characterization in detail. All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant. Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments. The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC). The phylogenetic relationships of H. variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB. The ectC-based tree was inconsistent with the other phylogenetic trees. For that reason, ectC was inferred to derive from horizontal transfer.     

Sequence of the gene encoding the 16S rRNA of the beer spoilage organismMegasphaera cerevisiae

The 16S ribosomal RNA gene from the beer-spoilage organism,Megasphaera cerevisiae was polymerase chain reaction (PCR)-amplified and sequenced. Analysis confirmed the phylogenetic position ofM. cerevisiae as a sister taxon ofMegasphaera elsdenii, within the obligately anaerobic, Gram-negative cocci. The sequence obtained should facilitate the development of DNA probes for early detection of this spoilage organism.     

Specific detection of an oil-degrading bacterium, Corynebacterium sp. IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences

A species-specific PCR technique to detect an oil-degrading bacterium, Corynebacterium sp. IC10, released into sand microcosms is described. PCR primers, specific to strain IC10, were designed based on 16S rRNA gene sequences and tested against both closely and distantly related bacterial strains using four primer combinations involving two forward and two reverse primers. Two sets of them were specific to the strain IC10 and Corynebacterium variabilis and one set was selected for further analysis. The PCR amplification was able to detect 1 pg template DNA of strain IC10 and 1.2×10⁴ c.f.u. of IC10 ml wet sand^–1 in the presence of 3×10⁸ Escherichia coli cells. In non-sterile sand microcosms seeded with the strain IC10, the sensitivity of detection decreased to 9.6×10⁵ c.f.u. ml wet sand^–1. The detection sensitivity thus depends on the complexity of background heterogeneous DNA of environmental samples. The assay is suitable for detection of Corynebacterium sp. IC10 in laboratory microcosms, however, cross reaction with non-oil degrading coryneforms may prohibit its use in uncharacterized systems.     

Molecular diversity of 16S rRNA and gyrB genes in copper mines

The molecular diversities of the microbial communities from four sites impacted by acid mine drainage (AMD) at Dexing Copper Mine in Jiangxi province of China were studied using 16S rRNA sequences and gyrB sequences. Of the four sampled sites, each habitat exhibited distinct geochemical characteristics and the sites were linked geographically allowing us to correlate microbial community structure to geochemical characteristics. In the present study, we examined the molecular diversity of 16S rRNA and gyrB genes from water at these sites using a PCR-based cloning approach. We found that the microbial community appears to be composed primarily of Proteobacteria, Acidobacteria, Actinobacteria, Nitrospira, Firmicutes, Chlorella and unknown phylotypes. Of clones affiliated with Nitrospira, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and Leptospirillum group III were all detected. Principal-component analysis (PCA) revealed that the distribution of the microbial communities was influenced greatly by geochemical characteristics. The overall PCA profiles showed that the sites with similar geochemical characteristics had more similar microbial community structures. Moreover, our results also indicated that gyrB sequence analysis may be very useful for differentiating very closely related species in the study of microbial communities. H. Yin and L. Cao contributed equally to this work.     

Molecular Phylogeny of Coral-Reef Sea Cucumbers (Holothuriidae: Aspidochirotida) Based on 16S Mitochondrial Ribosomal DNA Sequence

Members of the Holothuriidae, found globally at low to middle latitudes, are often a dominant component of Indo–West Pacific coral reefs. We present the first phylogeny of the group, using 8 species from the 5 currently recognized genera and based on approximately 540 nucleotides from a polymerase chain reaction–amplified and conserved 3′ section of 16S mitochondrial ribosomal DNA. Parsimony and likelihood analyses returned identical topologies, permitting several robust inferences to be drawn. Several points corroborated the Linnean classification. Actinopyga and Bohadschia each appear monophyletic and Pearsonothuria is sister to Bohadschia. Other aspects of our phylogeny, however, were not in accord with the taxonomy of Holothuriidae or previous speculations about the group’s evolutionary history. Most notably, the genus Holothuria appears paraphyletic. Actinopyga and Bohadschia, sometimes held to be closely related to one another because of certain morphologic similarities, are only distantly related. The morphologically distinct Labidodemas, even thought to warrant separation at the family level, is nested well within Holothuria. A maximum parsimony reconstruction of ancestral ossicle form on the phylogeny indicated that, in addition to a probable bout of elaboration in ossicle form (the modification of rods or rosettes to holothuriid-type buttons), at least 2 rounds of ossicle simplification also transpired in which buttons reverted to rods or rosettes. Cuvierian tubules, defensive organs unique to numerous members of Holothuriidae, were probably present before the initial radiation of the family, but the reconstruction is ambiguous as to their ancestral function.     

Identification of Comamonas species using 16S rRNA gene sequence

A bacterial strain Bz02 was isolated from a water sample collected from river Gomti at the Indian city of Lucknow. We characterized the strain using 16S rRNA sequence. Phylogenetic analysis showed that the strain formed a monophyletic clade with members of the genus Comamonas. The closest phylogenetic relative was Comamonas testosteroni with 95% 16S rRNA gene sequence similarity. It is proposed that the identified strain Bz02 be assigned as the type strain of a species of the genus Comamonas (Comamonas sp Bz02) based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phylogenetic tree analysis. The sequence is deposted in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ211417","term_id":"209164434"}}FJ211417.     

Detection and Characterization of a Dehalogenating Microorganism by Terminal Restriction Fragment Length Polymorphism Fingerprinting of 16S rRNA in a Sulfidogenic, 2-Bromophenol-Utilizing Enrichment

Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a second Desulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.     

Turkey fecal microbial community structure and functional gene diversity revealed by 16S rRNA gene and metagenomic sequences

The primary goal of this study was to better understand the microbial composition and functional genetic diversity associated with turkey fecal communities. To achieve this, 16S rRNA gene and metagenomic clone libraries were sequenced from turkey fecal samples. The analysis of 382 16S rRNA gene sequences showed that the most abundant bacteria were closely related to Lactobacillales (47%), Bacillales (31%), and Clostridiales (11%). Actinomycetales, Enterobacteriales, and Bacteroidales sequences were also identified, but represented a smaller part of the community. The analysis of 379 metagenomic sequences showed that most clones were similar to bacterial protein sequences (58%). Bacteriophage (10%) and avian viruses (3%) sequences were also represented. Of all metagenomic clones potentially encoding for bacterial proteins, most were similar to low G+C Gram-positive bacterial proteins, particularly from Lactobacillales (50%), Bacillales (11%), and Clostridiales (8%). Bioinformatic analyses suggested the presence of genes encoding for membrane proteins, lipoproteins, hydrolases, and functional genes associated with the metabolism of nitrogen and sulfur containing compounds. The results from this study further confirmed the predominance of Firmicutes in the avian gut and highlight the value of coupling 16S rRNA gene and metagenomic sequencing data analysis to study the microbial composition of avian fecal microbial communities.     

Molecular diversity of Bacteroides spp. in human fecal microbiota as determined by group-specific 16S rRNA gene clone library analysis

Bacteroides spp. represent a prominent bacterial group in human intestinal microbiota with roles in symbiosis and pathogenicity; however, the detailed composition of this group in human feces has yet to be comprehensively characterized. In this study, the molecular diversity of Bacteroides spp. in human fecal microbiota was analyzed from a seven-member, four-generation Chinese family using Bacteroides spp. group-specific 16S rRNA gene clone library analysis. A total of 549 partial 16S rRNA sequences amplified by Bacteroides spp.-specific primers were classified into 52 operational taxonomic units (OTUs) with a 99% sequence identity cut-off. Twenty-three OTUs, representing 83% of all clones, were related to 11 validly described Bacteroides species, dominated by Bacteroides coprocola, B. uniformis, and B. vulgatus. Most of the OTUs did not correspond to known species and represented hitherto uncharacterized bacteria. Relative to 16S rRNA gene universal libraries, the diversity of Bacteroides spp. detected by the group-specific libraries was much higher than previously described. Remarkable inter-individual differences were also observed in the composition of Bacteroides spp. in this family cohort. The comprehensive observation of molecular diversity of Bacteroides spp. provides new insights into potential contributions of various species in this group to human health and disease.     

基于16S rDNA扩增子测序技术揭示真胃左方变位对奶牛粪便微生物的影响

[目的]本试验旨在测定产后健康奶牛和罹患真胃左方变位(left displacement of the abomasum, LDA)奶牛粪便中微生物菌群的变化,以期探讨LDA发生与菌群的关联性,评估其对机体代谢的潜在影响.[方法]采用16S rDNA高通量测序技术,测定10头健康奶牛(Health)、10头真胃左方变位...     

Molecular phylogeny of the crab genus Brachynotus (Brachyura: Varunidae) based on the 16S rRNA gene

The crab genus Brachynotus de Haan, 1833 is restricted to the intertidal and shallow subtidal of the Mediterranean and northeastern Atlantic. It is presently recognized to consist of four species, of which three (B. foresti, B. gemmellari and B. sexdentatus) are endemic to the Mediterranean. The fourth species, B. atlanticus, is found along the Atlantic coasts of northern Africa and southern Europe, but also extends into the western Mediterranean. This high level of endemism suggests that speciation within Brachynotus is strongly correlated with the geography and geology of the Mediterranean Sea. A molecular phylogeny based on the mitochondrial large subunit (16S) rRNA gene indicates that the four species of Brachynotus form a monophyletic group within Atlantic Varunidae. The DNA sequence data also show that the genus Brachynotus can be subdivided into two species groups, one comprising B. atlanticus and B. foresti, and the other one B. gemmellari and B. sexdentatus. While B. atlanticus and B. foresti are clearly genetically distinct, B. gemmellari and B. sexdentatus are identical in the studied region of the 16S rRNA gene, suggesting a recent separation or continuing gene flow.     

七种蝽mtDNA-16S rRNA基因序列多态性的研究(半翅目:蝽科)

测定蝽亚科2族4属7个种(宽碧蝽,辉蝽,凹肩辉蝽,角肩真蝽,褐真蝽,斑真蝽,全蝽)9个个体线粒体DNA(mtDNA)的16SrRNA基因片段,分析了其遗传多态性。通过测定该基因片段的序列发现,不同种群存在丰富的DNA序列多态性,同一种的不同个体差异较小,9个个体具有9种基因型。在扩增的长为400bp的基因片段中,通过排序,有338个碱基可用于这9个个体的比较。在这一基因片段中,共检测到122个多态性核苷酸位点(约36．1％)。NJ法构建的分子系统树表明碧蝽属归于短中片族,全蝽属的分化较其它属要早。