Behavior of Variable V3 Region from 16S rDNA of Lactic Acid Bacteria in Denaturing Gradient Gel Electrophoresis

Separation of amplified V3 region from 16S rDNA by denaturing gradient gel electrophoresis (DGGE) was tested as a tool for differentiation of lactic acid bacteria commonly isolated from food. Variable V3 regions of 21 reference strains and 34 wild strains referred to species belonging to the genera Pediococcus, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, Weissella, and Streptococcus were analyzed. DGGE profiles obtained were species-specific for most of the cultures tested. Moreover, it was possible to group the remaining LAB reference strains according to the migration of their 16S V3 region in the denaturing gel. The results are discussed with reference to their potential in the analysis of LAB communities in food, besides shedding light on taxonomic aspects. Received: 2 August 2000 / Accepted: 5 September 2000     

Combined Phospholipid Biomarker-16S rRNA Gene Denaturing Gradient Gel Electrophoresis Analysis of Bacterial Diversity and Physiological Status in an Intertidal Microbial Mat

A combined lipid biomarker-16S rRNA gene denaturing gradient gel electrophoresis analysis was used to monitor changes in the physiological status, biomass, and microbial composition of a microbial mat. In the morning hours, an increase in the biomass of layers containing a high density of phototrophs and a decrease in the growth rate in the deep layers were observed. The combined approach also revealed differences in major groups of microorganisms, including green nonsulfur, gram-positive, and heterotrophic bacteria.     

16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

Analysis of Endophytic Bacterial Communities of Potato by Plating and Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA Based PCR Fragments

The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desirée) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable endophytic bacterial communities detected in potato stem bases as well as in roots were in most cases on the order 10³ to 10⁵ CFU g⁻¹ of fresh plant tissue. Dilution plating revealed that a range of bacterial types dominated these populations. Dominant isolates fell into the α and γ subgroups of the Proteobacteria, as well as in the Flavobacterium/Cytophaga group. Different representatives of the Firmicutes were also found. The most frequently isolated strains (>5% of the total) were characterized as different Pseudomonas spp. (including P. aureofaciens, P. corrugata, and P. putida), Agrobacterium radiobacter, Stenotrophomonas maltophilia, and Flavobacterium resinovorans, using fatty acid methyl ester (FAME) analysis and/or sequencing of their partial 16S ribosomal RNA genes. Other Proteobacteria or Firmicutes were also found, albeit infrequently, and mainly in potato stem tissue. The fate of three putative potato endophytes, Stenotrophomonas maltophilia, Bacillus sp., and Sphingomonas paucimobilis, was monitored following their release into potato plants via injection, via root dipping, or via the soil. Following stem injection, the S. maltophilia and Bacillus inoculants could be tracked over time periods of, respectively, 22 and 1 day(s) by dilution plating as well as via PCR-DGGE. However, only S. maltophilia was able to colonize, and persist in, plant tissue from soil or dipped roots. S. paucimobilis was never recovered from the plant irrespective of the mode of introduction. The diversity of the indigenous bacterial flora associated with potato was then monitored via PCR-DGGE. The patterns obtained revealed the existence of bacterial communities of limited complexity, with communities from potato stems typically differing from those from stem peel and roots. Evidence was obtained for the endophytic occurrence of a range of organisms falling into the α, β, and γ subgroups of the Proteobacteria as well as in the Firmicutes. Several of the sequences found matched those from isolates, suggesting that the molecular evidence reported culturable organisms. However, a number of sequences did not have matching sequences from isolates, suggesting that non-culturable or as-yet-uncultured endophytic organisms were being detected.     

Analysis of Bacterial Communities in Seagrass Bed Sediments by Double-Gradient Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA Genes

Bacterial communities associated with seagrass bed sediments are not well studied. The work presented here investigated several factors and their impact on bacterial community diversity, including the presence or absence of vegetation, depth into sediment, and season. Double-gradient denaturing gradient gel electrophoresis (DG-DGGE) was used to generate banding patterns from the amplification products of 16S rRNA genes in 1-cm sediment depth fractions. Bioinformatics software and other statistical analyses were used to generate similarity scores between sections. Jackknife analyses of these similarity coefficients were used to group banding patterns by depth into sediment, presence or absence of vegetation, and by season. The effects of season and vegetation were strong and consistent, leading to correct grouping of banding patterns. The effects of depth were not consistent enough to correctly group banding patterns using this technique. While it is not argued that bacterial communities in sediment are not influenced by depth in sediment, this study suggests that the differences are too fine and inconsistent to be resolved using 1-cm depth fractions and DG-DGGE. The effects of vegetation and season on bacterial communities in sediment were more consistent than the effects of depth in sediment, suggesting they exert stronger controls on microbial community structure.     

Succession of Bacterial Community Structure along the Changjiang River Determined by Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H′), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from β-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.     

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。     

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.     

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。     

Analysis of Bacterial Community in the Ginseng Soil Using Denaturing Gradient Gel Electrophoresis (DGGE)

The objective of this work was to determine the shifts in the PCR-DGGE profiles of bacterial communities associated with the rhizosphere soil of ginseng at varying age levels. Differences in the dominance of intense DNA bands in the DGGE profile was observed over the age of the plants indicating the fluctuation in the microbial community structure. The bacterial orders of actinomycetales of Actinobacteria and Spingomonadales and Rhizobiales of α-Proteobacteria were predominant in the ginseng soil.     

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.     

16S rDNA克隆文库方法分析太岁样品中细菌的多样性

通过构建16S rDNA克隆文库的方法,分析太岁样品中细菌的群落结构及多样性。太岁样品中的细菌归属于4个门9个目,优势类群依次是芽胞杆菌目(Bacillales,33.01%)、柄杆菌目(Caulobacterales,32.04%)和伯克霍尔德氏菌目(Burkholderiales,12.62%);优势属为短波单胞菌属(Brevundimonas,30.10%)、葡萄球菌属(Staphylococcus,29.13%)和食酸菌属(Acidovorax,7.77%)。并且其中的5个目中含有未培养的细菌,红杆菌目(Rhodobacterales)、伯克霍尔德氏菌目和红环菌目(Rhodocyclales)的11个克隆子的细菌16S rDNA序列同源性低于97%。研究表明太岁样品中细菌多样性较丰富,且蕴藏着许多未知的微生物资源。     

变性梯度凝胶电泳法研究断奶仔猪粪样细菌区系变化

利用PCR和DGGE技术分析了12头仔猪在断奶后其粪样细菌区系的变化。粪样细菌16S rDNA的V6～V8可变区经PCR扩增,扩增产物经DGGE电泳后再进行相似性分析。结果表明,仔猪断奶当天粪样 DGGE谱带少,同窝仔猪间图谱相似。断奶后,随着断奶时间的推移,每头仔猪的DGGE图谱带逐渐增多,变得复杂和多样,仔猪个体间DGGE图谱差异逐渐增大。仔猪是否同窝以及所采食日粮类型对DGGE图谱没有明显影响。相似性分析还表明,日粮中添加寡果糖的仔猪在断奶后第1周,其粪样微生物区系变化迅速,而后缓慢。     

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.     

Microbial Diversity in Uranium Deposits from Jaduguda and Bagjata Uranium Mines,India as Revealed by Clone Library and Denaturing Gradient Gel Electrophoresis Analyses

Microbial communities within subsurface uranium (U) deposits were explored to understand the nature of community composition and their potential role in biogeochemical cycle and bioremediation. Geochemical analysis revealed that the U ores were mainly hosted on metamorphosed chlorite-biotite schists, containing varied organic carbon and elevated level of several heavy metals (U, Cu, Cr, Zn, etc.). Microbial diversity as explored by 16S rRNA gene clone library and DGGE analyses revealed predominance of Proteobacteria, Acidobacteria, Bacteroidetes along with Firmicutes and candidate division OP9 within the domain Bacteria and Euryarchaeota within the domain Archaea. Among the physiochemical parameters, level of organic carbon showed considerable impact on influencing community diversity and composition. Samples from Jaduguda with high organic carbon showed abundance of bacteria known for metabolizing different carbon compounds and affiliated to unclassified uncultured members of Chitnophagaceae (Bacteroidetes), Gp4 of Acidobacteria and unclassified β-Proteobacteria and halophilic, nitrate-reducing γ-Proteobacteria. A relatively diverse assemblage of species capable of autotrophic/heterotrophic N₂ fixation, CH₄ utilization, H₂(0)/Fe(II)/Mn(II)/S(0) oxidation, NO₃/Fe(III) reduction, U and other metal precipitation/mineralization and affiliated to families Rhizobiaceae, Bradyrhizobiaceae, Caulobacteraceae, Comamonadaceae and genera Acinetobacter, Marinobacter and Alcanivorax constituted the Bagjata samples. Distribution and interrelations among abundance of various bacterial groups detected in other U mines/radioactive waste sites were compared with our data. Overall, the study reported distinct compositions of indigenous microbial communities among the samples from two mines and provided a better insight in geomicrobiology of U deposits.     

变性梯度凝胶电泳技术在微生物分子生态学研究中的应用

变性梯度凝胶电泳在微生物分子生态学研究中的应用日益广泛,已成为研究微生物多样性和动态变化的有力工具.对变性梯度凝胶电泳技术中存在的问题,如基因片段的选择、共迁移、背景色和异源双链分子的形成等作了综述,以期为进一步更好地利用该技术进行微生态的研究奠定基础.     

Numerical Analysis of Grassland Bacterial Community Structure under Different Land Management Regimens by Using 16S Ribosomal DNA Sequence Data and Denaturing Gradient Gel Electrophoresis Banding Patterns

Bacterial diversity in unimproved and improved grassland soils was assessed by PCR amplification of bacterial 16S ribosomal DNA (rDNA) from directly extracted soil DNA, followed by sequencing of ~45 16S rDNA clones from each of three unimproved and three improved grassland samples (A. E. McCaig, L. A. Glover, and J. I. Prosser, Appl. Environ. Microbiol. 65:1721–1730, 1999) or by denaturing gradient gel electrophoresis (DGGE) of total amplification products. Semi-improved grassland soils were analyzed only by DGGE. No differences between communities were detected by calculation of diversity indices and similarity coefficients for clone data (possibly due to poor coverage). Differences were not observed between the diversities of individual unimproved and improved grassland DGGE profiles, although considerable spatial variation was observed among triplicate samples. Semi-improved grassland samples, however, were less diverse than the other grassland samples and had much lower within-group variation. DGGE banding profiles obtained from triplicate samples pooled prior to analysis indicated that there was less evenness in improved soils, suggesting that selection for specific bacterial groups occurred. Analysis of DGGE profiles by canonical variate analysis but not by principal-coordinate analysis, using unweighted data (considering only the presence and absence of bands) and weighted data (considering the relative intensity of each band), demonstrated that there were clear differences between grasslands, and the results were not affected by weighting of data. This study demonstrated that quantitative analysis of data obtained by community profiling methods, such as DGGE, can reveal differences between complex microbial communities.