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.     

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.     

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.     

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。     

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.     

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.     

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.     

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.     

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.     

变性梯度凝胶电泳在堆肥微生物研究中的应用

对当前堆肥中微生物种群分布及其对有机物分解作用的研究进行分析,论述了分子生物技术中的变性梯度凝胶电泳(DGGE)的特点。将DGCE与PCR扩增技术相结合,可用于研究自然菌种堆肥和人工培养驯化菌种堆肥过程中微生物的演替规律,为研究和筛选堆肥中的微生物提供更加有效、快速的信息,促进堆肥技术的发展。     

Denaturing Gradient Gel Electrophoresis Analysis of 16S Ribosomal DNA Amplicons To Monitor Changes in Fecal Bacterial Populations of Weaning Pigs after Introduction of Lactobacillus reuteri Strain MM53

The diversity and stability of the fecal bacterial microbiota in weaning pigs was studied after introduction of an exogenous Lactobacillus reuteri strain, MM53, using a combination of cultivation and techniques based on genes encoding 16S rRNA (16S rDNA). Piglets (n = 9) were assigned to three treatment groups (control, daily dosed, and 4th-day dosed), and fresh fecal samples were collected daily. Dosed animals received 2.5 × 10¹⁰ CFU of antibiotic-resistant L. reuteri MM53 daily or every 4th day. Mean Lactobacillus counts for the three groups ranged from 1 × 10⁹ to 4 × 10⁹ CFU/g of feces. Enumeration of strain L. reuteri MM53 on MRS agar (Difco) plates containing streptomycin and rifampin showed that the introduced strain fluctuated between 8 × 10³ and 5 × 10⁶ CFU/g of feces in the two dosed groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments, with primers specific for variable regions 1 and 3 (V1 and V3), was used to profile complexity of fecal bacterial populations. Analysis of DGGE banding profiles indicated that each individual maintained a unique fecal bacterial population that was stable over time, suggesting a strong host influence. In addition, individual DGGE patterns could be separated into distinct time-dependent clusters. Primers designed specifically to restrict DGGE analysis to a select group of lactobacilli allowed examination of interspecies relationships and abundance. Based on relative band migration distance and sequence determination, L. reuteri was distinguishable within the V1 region 16S rDNA gene patterns. Daily fluctuations in specific bands within these profiles were observed, which revealed an antagonistic relationship between L. reuteri MM53 (band V1-3) and another indigenous Lactobacillus assemblage (band V1-6).     

Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis

Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.     

Bulk and Rhizosphere Soil Bacterial Communities Studied by Denaturing Gradient Gel Electrophoresis: Plant-Dependent Enrichment and Seasonal Shifts Revealed

The bacterial rhizosphere communities of three host plants of the pathogenic fungus Verticillium dahliae, field-grown strawberry (Fragaria ananassa Duch.), oilseed rape (Brassica napus L.), and potato (Solanum tuberosum L.), were analyzed. We aimed to determine the degree to which the rhizosphere effect is plant dependent and whether this effect would be increased by growing the same crops in two consecutive years. Rhizosphere or soil samples were taken five times over the vegetation periods. To allow a cultivation-independent analysis, total community DNA was extracted from the microbial pellet recovered from root or soil samples. 16S rDNA fragments amplified by PCR from soil or rhizosphere bacterium DNA were analyzed by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints showed plant-dependent shifts in the relative abundance of bacterial populations in the rhizosphere which became more pronounced in the second year. DGGE patterns of oilseed rape and potato rhizosphere communities were more similar to each other than to the strawberry patterns. In both years seasonal shifts in the abundance and composition of the bacterial rhizosphere populations were observed. Independent of the plant species, the patterns of the first sampling times for both years were characterized by the absence of some of the bands which became dominant at the following sampling times. Bacillus megaterium and Arthrobacter sp. were found as predominant populations in bulk soils. Sequencing of dominant bands excised from the rhizosphere patterns revealed that 6 out of 10 bands resembled gram-positive bacteria. Nocardia populations were identified as strawberry-specific bands.     

Analysis of the Dynamics of Bacterial Communities in the Rhizosphere of the Chrysanthemum via Denaturing Gradient Gel Electrophoresis and Substrate Utilization Patterns

In order to gain a better understanding of the spatial and temporal dynamics of bacterial communities of the rhizosphere of the chrysanthemum, two complementary methods were used: a molecular bacterial community profiling method, i.e., 16S rRNA gene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and an agar plate method in which 11 sole-carbon-source utilization tests were used. The DGGE patterns showed that the bacterial communities as determined from direct rhizosphere DNA extracts were largely stable along developing roots of the chrysanthemum, with very little change over time or between root parts of different ages. The patterns were also similar to those produced with DNA extracts obtained from bulk soil samples. The DGGE patterns obtained by using microbial colonies from dilution plates as the source of target DNA were different from those found with the direct DNA extracts. Moreover, these patterns showed differences among plant replicates but also among replicate plates. Results obtained with the sole-carbon-source utilization tests indicated that the metabolic profile of the bacterial communities in the rhizosphere of the root tip did not change substantially during plant growth. This suggests selective development of specific bacterial populations by the presence of a root tip. On the other hand, the metabolic profile of bacterial communities in the rhizosphere of the root base changed during plant growth. With eight sole-carbon-source utilization tests, a significant effect of the development stage of the plant on the number of bacteria which were able to grow on these carbon sources was observed.