Analysis of Methanogen Diversity in the Rumen Using Temporal Temperature Gradient Gel Electrophoresis: Identification of Uncultured Methanogens

A temporal temperature gradient gel electrophoresis (TTGE) method was developed to determine the diversity of methanogen populations in the rumen. Tests with amplicons from genomic DNA from 12 cultured methanogens showed single bands for all strains, with only two showing apparently comigrating bands. Fingerprints of methanogen populations were analyzed from DNA extracted from rumen contents from two cattle and four sheep grazing pasture. For one sheep, dilution cultures selective for methanogens were grown and the culturable methanogens in each successive dilution examined by TTGE. A total of 66 methanogen sequences were retrieved from bands in fingerprints and analyzed to reveal the presence of methanogens belonging to the Methanobacteriales, the Methanosarcinales, and to an uncultured archaeal lineage. Twenty-four sequences were most similar to Methanobrevibacter ruminantium, five to Methanobrevibacter smithii, four to Methanosphaera stadtmanae, and for three, the nearest match was Methanimicrococcus blatticola. The remaining 30 sequences did not cluster with sequences from cultured archaea, but when combined with published novel sequences from clone libraries formed a monophyletic lineage within the Euryarchaeota, which contained two previously unrecognized clusters. The TTGE bands from this lineage showed that the uncultured methanogens had significant population densities in each of the six rumen samples examined. In cultures of dilutions from one rumen sample, TTGE examination revealed these methanogens at a level of at least 10⁵ g⁻¹. Band intensities from low-dilution cultures indicated that these methanogens were present at similar densities to Methanobrevibacter ruminantium-like methanogens, the sole culturable methanogens in high dilutions (10⁶–10⁻¹⁰ g⁻¹). It is suggested that the uncultured methanogens together with Methanobrevibacter spp. may be the predominant methanogens in the rumen. The TTGE method presented in this article provides a new opportunity for characterizing methanogen populations in the rumen microbial ecosystem.     

Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp. in Cheese by Temporal Temperature Gradient Gel Electrophoresis

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.     

Identification of the Bacterial Microflora in Dairy Products by Temporal Temperature Gradient Gel Electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, are present in cheeses, forming a complex ecosystem. Among these organisms, bacteria are responsible for most of the physicochemical and aromatic transformations that are intrinsic to the cheesemaking process. Identification of the bacteria that constitute the cheese ecosystem is essential for understanding their individual contributions to cheese production. We used temporal temperature gradient gel electrophoresis (TTGE) to identify different bacterial species present in several dairy products, including members of the genera Lactobacillus, Lactococcus, Leuconostoc, Enterococcus, Pediococcus, Streptococcus, and Staphylococcus. The TTGE technique is based on electrophoretic separation of 16S ribosomal DNA (rDNA) fragments by using a temperature gradient. It was optimized to reveal differences in the 16S rDNA V3 regions of bacteria with low-G+C-content genomes. Using multiple control strains, we first set up a species database in which each species (or group of species) was characterized by a specific TTGE fingerprint. TTGE was then applied to controlled dairy ecosystems with defined compositions, including liquid (starter), semisolid (home-made fermented milk), and solid (miniature cheese models) matrices. Finally, the potential of TTGE to describe the bacterial microflora of unknown ecosystems was tested with various commercial dairy products. Subspecies, species, or groups of species of lactic acid bacteria were distinguished in dairy samples. In conclusion, TTGE was shown to distinguish bacterial species in vitro, as well as in both liquid and solid dairy products.     

DGGE/TGGE技术及其在微生物分子生态学中的应用

变性梯度凝胶电泳(DGGE)和温度梯度凝胶电泳(TGGE)是近些年微生物分子生态学研究中的热点技术之一。由于DGGE／TGGE技术具有可靠性强、重现性高、方便快捷等优点,被广泛地应用于微生物群落多样性和动态性分析。文章对DGGE／TGGE技术原理与关键环节、局限性和应用前景进行了综述。     

利用变性梯度凝胶电泳分析微生物的多样性

综述了不依赖于培养的变性梯度凝胶电泳技术 (DGGE)分析微生物多样性的原理 ,并列举它的应用实例。DGGE和传统方法相比有很多优点 ,若将DGGE和其他方法结合起来 ,效果更好 ,应用更广泛。     

Molecular Fingerprinting of Dairy Microbial Ecosystems by Use of Temporal Temperature and Denaturing Gradient Gel Electrophoresis

Numerous microorganisms, including bacteria, yeasts, and molds, constitute the complex ecosystem present in milk and fermented dairy products. Our aim was to describe the bacterial ecosystem of various cheeses that differ by production technology and therefore by their bacterial content. For this purpose, we developed a rapid, semisystematic approach based on genetic profiling by temporal temperature gradient electrophoresis (TTGE) for bacteria with low-G+C-content genomes and denaturing gradient gel electrophoresis (DGGE) for those with medium- and high-G+C-content genomes. Bacteria in the unknown ecosystems were assigned an identity by comparison with a comprehensive bacterial reference database of ~150 species that included useful dairy microorganisms (lactic acid bacteria), spoilage bacteria (e.g., Pseudomonas and Enterobacteriaceae), and pathogenic bacteria (e.g., Listeria monocytogenes and Staphylococcus aureus). Our analyses provide a high resolution of bacteria comprising the ecosystems of different commercial cheeses and identify species that could not be discerned by conventional methods; at least two species, belonging to the Halomonas and Pseudoalteromonas genera, are identified for the first time in a dairy ecosystem. Our analyses also reveal a surprising difference in ecosystems of the cheese surface versus those of the interior; the aerobic surface bacteria are generally G+C rich and represent diverse species, while the cheese interior comprises fewer species that are generally low in G+C content. TTGE and DGGE have proven here to be powerful methods to rapidly identify a broad range of bacterial species within dairy products.     

Evaluation of fungal aerosols using Temporal Temperature Gradient Electrophoresis (TTGE) and comparison with culture

Information obtained from fungal air samples can assist in the assessment of health hazards and can be useful in proactive indoor air quality monitoring. The objective of the present study was to evaluate the PCR-TTGE technique for the analysis of fungal diversity in the air. Eleven air samples were collected in five different sites using the bioimpactor CIP 10-M (Arelco). After a 2 hours sampling period, the collection liquid was recovered for subsequent cultivation and PCR-TTGE. A set of three fungi-specific primers (Fungcont 1, Fungcont 2+GC and Fungcont 3) was designed for the partial amplification of the 18S rRNA gene. The amplification was obtained in a single reaction tube by a semi-nested PCR. For identification, the TTGE bands were extracted and sequenced. PCR-TTGE allowed the clear separation of amplicons corresponding to distinct fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The number of fungal taxa detected after culture was systematically higher than the number of taxa found using PCR-TTGE. However, few fungal species were detected by PCR-TTGE and not by cultivation, suggesting that the combination of these approaches may provide a better analysis of fungal diversity in air samples than either method alone.     

变性梯度凝胶电泳技术在微生物多样性研究中的应用

变性梯度凝胶电泳是不依赖于培养的、依据DNA分子的大小和所带电荷分析微生物多样性和动态变化的分子生物学技术,具有检测极限低、分析速度快及重复性好等优点。主要对变性梯度凝胶电泳原理、特点及其在微生物多样性应用方面进行综述。     

温度梯度凝胶电泳技术及应用

温度梯度凝胶电泳（TGGE）是一种用于检测核酸序列变异和点突变的电泳方法.该法利用不同构象的核酸分子具有不同的变性温度（T_m)来进行分离.TGGE方法具有分辨能力高、重复性好和节省时间的特点,可广泛应用于分子生物学研究领域.     

Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE). Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074^T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.     

Analysis of Fungal Diversity in the Wheat Rhizosphere by Sequencing of Cloned PCR-Amplified Genes Encoding 18S rRNA and Temperature Gradient Gel Electrophoresis

Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.     

Genetic Diversity of cry Gene Sequences of Bacillus thuringiensis Strains Analyzed by Denaturing Gradient Gel Electrophoresis

PCR has been widely used to identify cry-type genes, to determine their distribution, to detect new such genes and to predict insecticidal activities. We describe here a molecular approach to analyze the genetic diversity of B. thuringiensis cry-like genes based on denaturing gradient gel electrophoresis (DGGE). This analysis demonstrated that different B. thuringiensis isolates can be distinguished according to its PCR-DGGE profile of cry-like genes. Identification of the resolvable DNA fragments was easy to accomplish by DNA sequencing, which was confirmed in this work. Importantly, the strategy allowed the identification of unknown B. thuringiensis cry-like sequences present in a single strain that remained cryptic after PCR analysis using degenerate primers. The method developed in this work contributes to the availability of molecular techniques for both B. thuringiensis strains and cry-like genes identification and discovery.     

Assessment of Genotypic Diversity of Antibiotic-Producing Pseudomonas Species in the Rhizosphere by Denaturing Gradient Gel Electrophoresis

The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing gradient gel electrophoresis (DGGE) of 350-bp fragments of phlD, a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD⁺ reference strains and indigenous isolates. DGGE analysis of the phlD fragments provided a level of discrimination between phlD⁺ genotypes that was higher than the level obtained by currently used techniques and enabled detection of specific phlD⁺ genotypes directly in rhizosphere samples with a detection limit of approximately 5 × 10³ CFU/g of root. DGGE also allowed simultaneous detection of multiple phlD⁺ genotypes present in mixtures in rhizosphere samples. DGGE analysis of 184 indigenous phlD⁺ isolates obtained from the rhizospheres of wheat, sugar beet, and potato plants resulted in the identification of seven phlD⁺ genotypes, five of which were not described previously based on sequence and phylogenetic analyses. Subsequent bioassays demonstrated that eight genotypically different phlD⁺ genotypes differed substantially in the ability to colonize the rhizosphere of sugar beet seedlings. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.     

Diversity and Seasonal Variability of β-Proteobacteria in Biofilms of Polluted Rivers: Analysis by Temperature Gradient Gel Electrophoresis and Cloning

The β-subgroup of the Proteobacteria has been shown to be important in aquatic habitats and was investigated in depth here by molecular 16S rRNA techniques in biofilms of the Elbe River and its polluted tributary, the Spittelwasser River. The bacterial 16S rRNA genes were cloned from each site, screened for β-proteobacterial clones and sequenced. River biofilm clones from both rivers grouped into 9 clusters (RBFs). RBFs 1, 2, and 3 fell into the recently described βI cluster of cosmopolitan freshwater bacteria, where they represented new species related to Rhodoferax, Aquaspirillum, and Hydrogenophaga. RBFs 4 to 7 affiliated with Aquabacterium commune, Ideonella dechloratans, and Sphaerotilus natans, respectively. The two remaining RBFs were uncultivated clusters, one of them being distantly related to Gallionella ferruginea. Seasonal changes in the relative intensity of the β-proteobacterial 16S rRNA genes of biofilms harvested monthly for 1 year were determined by specific amplification and separation by temperature gradient gel electrophoresis (TGGE). Bands were identified by comparison of clones to community fingerprints by TGGE. Eight of 13 identified bands were shared by both habitats but showed different relative abundance and seasonal variability in the two rivers, probably caused by differences in temperature and pollutants. The data indicate new not-yet-cultivated clusters of river biofilm organisms, some of them probably distributed globally. They confirm the importance of certain known freshwater genera in river biofilms. The high phylogenetic resolution obtained by clone library analysis combined with the high temporal resolution obtained by TGGE suggest that the observed microdiversity in the river biofilm clone libraries might be caused by phylogenetically closely related microbial populations which are adapted to ecological parameters.     

Nested PCR-Denaturing Gradient Gel Electrophoresis Approach To Determine the Diversity of Sulfate-Reducing Bacteria in Complex Microbial Communities

Here, we describe a three-step nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to detect sulfate-reducing bacteria (SRB) in complex microbial communities from industrial bioreactors. In the first step, the nearly complete 16S rRNA gene was amplified using bacterial primers. Subsequently, this product was used as a template in a second PCR with group-specific SRB primers. A third round of amplification was conducted to obtain fragments suitable for DGGE. The largest number of bands was observed in DGGE patterns of products obtained with primers specific for the Desulfovibrio-Desulfomicrobium group, indicating a large diversity of these SRBs. In addition, members of other phylogenetic SRB groups, i.e., Desulfotomaculum, Desulfobulbus, and Desulfococcus-Desulfonema-Desulfosarcina, were detected. Bands corresponding to Desulfobacterium and Desulfobacter were not detected in the bioreactor samples. Comparative sequence analysis of excised DGGE bands revealed the identity of the community members. The developed three-step PCR-DGGE strategy is a welcome tool for studying the diversity of sulfate-reducing bacteria.     

Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.     

Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries

Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.     

GC Fractionation Enhances Microbial Community Diversity Assessment and Detection of Minority Populations of Bacteria by Denaturing Gradient Gel Electrophoresis

Effectively and accurately assessing total microbial community diversity is one of the primary challenges in modern microbial ecology. This is particularly true with regard to the detection and characterization of unculturable populations and those present only in low abundance. We report a novel strategy, GC fractionation combined with denaturing gradient gel electrophoresis (GC-DGGE), which combines mechanistically different community analysis approaches to enhance assessment of microbial community diversity and detection of minority populations of microbes. This approach employs GC fractionation as an initial step to reduce the complexity of the community in each fraction. This reduced complexity facilitates subsequent detection of diversity in individual fractions. DGGE analysis of individual fractions revealed bands that were undetected or only poorly represented when total bacterial community DNA was analyzed. Also, directed cloning and sequencing of individual bands from DGGE lanes corresponding to individual G+C fractions allowed detection of numerous phylotypes that were not recovered using a traditional random cloning and sequencing approach.     

Spatial and Temporal Assessment of Diazotroph Assemblage Composition in Vegetated Salt Marsh Sediments Using Denaturing Gradient Gel Electrophoresis Analysis

Abstract Diazotroph assemblage compositions were assessed in rhizosphere sediments from the tall and short form Spartina alterniflora growth zones over an annual cycle. Sediment cores were collected for DNA extraction and nitrogenase (acetylene reduction) activity assays, and porewater samples were analyzed for several chemical parameters in March, June, September, and December 1997. These data were collected to determine if within- or between-zone differences in the diazotroph assemblage composition correlated with differences in key environmental variables or acetylene reduction activity. Acetylene reduction rates differed between zones and within a zone over an annual period. Soluble sulfide concentrations were higher in the short form S. alterniflora zone on all dates except those in June and differed within both zones on different sample dates. nifH sequences were recovered from rhizosphere sediment DNA by PCR amplification using nifH specific primers. These amplimers were analyzed using denaturing gradient gel electrophoresis (DGGE), and the resulting patterns were compared by neural network and linear discriminant analyses. Ten prominent amplimers, four of which were apparent heteroduplexes, were observed. DGGE banding profiles showed minor differences among sampling dates and between sample zones, but the overall banding pattern was remarkably consistent. This reflects overall similarity between the amplifiable diazotroph assemblages in the tall and short S. alterniflora growth zones and substantial seasonal stability in assemblage composition. Received: 2 March 1999; Accepted: 4 May 1999     

变性梯度凝胶电泳在微生物生态学中的应用

变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)是目前在微生物生态学上应用比较广泛的技术之一,具有简便、准确可靠和重复性好等优点。对DGGE的原理、流程、各项技术要点和在微生物生态学上的应用等方面进行了详细地论述,同时归纳和总结了DGGE的优缺点和局限性。