PCR-Denaturing Gradient Gel Electrophoresis of Complex Microbial Communities: A Two-Step Approach to Address the Effect of Gel-to-Gel Variation and Allow Valid Comparisons Across a Large Dataset

Denaturing gradient gel electrophoresis (DGGE) is widely used in microbial ecology to profile complex microbial communities over time and in response to different stimuli. However, inherent gel-to-gel variability has always been a barrier toward meaningful interpretation of DGGE profiles obtained from multiple gels. To address this problem, we developed a two-step methodology to align DGGE profiles across a large dataset. The use of appropriate inter-gel standards was of vital importance since they provided the basis for efficient within- and between-gel alignment and a reliable means to evaluate the final outcome of the process. Pretreatment of DGGE profiles by a commercially available image analysis software package (TL120 v2006, Phoretix 1D Advanced) followed by a simple interpolation step in Matlab minimized the effect of gel-to-gel variation, allowing for comparisons between large numbers of samples with a high degree of confidence. At the same time, data were obtained in the form of whole densitometric curves, rather than as band presence/absence or intensity information, and could be readily analyzed by a collection of well-established multivariate methods. This work clearly demonstrates that there is still room for significant improvements as to the way large DGGE datasets are processed and statistically interrogated.     

Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-melting-temperature (T_m(L)) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to T_m(L). The average T_m(L) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.     

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。     

Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene

The genotypic diversity of insoluble macromolecules degraded myxobacteria, provided an opportunity to discover new bacterial resources and find new ecological functions. In this study, we developed a semi-nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to determine the presence and genotypic diversity of myxobacteria in soil. After two rounds of PCR with myxobacteria-specific primers, an 194 bp fragment of mglA, a key gene involved in gliding motility, suitable for DGGE was obtained. A large number of bands were observed in DGGE patterns, indicating diverse myxobacteria inhabiting in soils. Furthermore, sequencing and BLAST revealed that most of the bands belonged to the myxobacteria-group, and only three of the twenty-eight bands belonged to other group, i.e., Deinococcus maricopensis. The results verified that myxobacterial strains with discrepant sequence compositions of gene mglA could be discriminated by DGGE with myxobacteria-specific primers. Collectively, the developed semi-nested-PCR-DGGE strategy is a useful tool for studying the diversity of myxobacteria.     

Development and Application of a Selective PCR-Denaturing Gradient Gel Electrophoresis Approach To Detect a Recently Cultivated Bacillus Group Predominant in Soil

The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance and cultivability of at least some of these microorganisms makes them an appropriate subject for studies on their biogeographical dissemination and diversity. Since cultivability can vary significantly with the physiological state and even between closely related strains, we developed a culture-independent 16S rRNA gene-targeted DGGE fingerprinting protocol for the detection of these bacteria from soil samples. The composition of the B. benzoevorans relatives in the soil samples from The Netherlands, Bulgaria, Russia, Pakistan, and Portugal showed remarkable differences between the different countries. Differences in the DGGE profiles of these communities in archived soil samples from the Dutch Wieringermeer polder were observed over time during which a shift from anaerobic to aerobic and from saline to freshwater conditions occurred. To complement the molecular methods, we additionally cultivated B. benzoevorans-related strains from all of the soil samples. The highest number of B. benzoevorans relatives was found in the soils from the northern part of The Netherlands. The present study contributes to our knowledge of the diversity and abundance of this interesting group of microbes in soils throughout the world.     

Denaturing Gradient Gel Electrophoresis as a Tool To Determine Batch Similarity of Probiotic Cultures of Porcine Cecal Bacteria

A continuous-flow porcine cecal bacterial culture has been used experimentally as treatment against enterotoxigenic Escherichia coli in weanling pigs. Periodically, the cultures must be started from frozen stock. Our results indicate that denaturing gradient gel electrophoresis can be applied as an indirect indication of culture similarity for each new batch generated from frozen stock.     

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。     

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.     

Monitoring the Bacterial Population Dynamics in Sourdough Fermentation Processes by Using PCR-Denaturing Gradient Gel Electrophoresis

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.     

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。     

Detection of Helicobacter Colonization of the Murine Lower Bowel by Genus-Specific PCR-Denaturing Gradient Gel Electrophoresis

Helicobacter genus-specific PCR and denaturing gradient gel electrophoresis can detect and speciate the helicobacters that colonize the lower bowel of laboratory mice. The method's sensitivity is comparable to that of species-specific PCR and may detect unnamed Helicobacter species. This approach should prove useful for commercial and research murine facilities.     

Identification and Population Dynamics of Yeasts in Sourdough Fermentation Processes by PCR-Denaturing Gradient Gel Electrophoresis

Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40°C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C.     

Combination of Multiplex PCR and PCR-Denaturing Gradient Gel Electrophoresis for Monitoring Common Sourdough-Associated Lactobacillus Species

A combination of denaturing gradient gel electrophoresis (DGGE) and a previously described multiplex PCR approach was employed to detect sourdough lactobacilli. Primers specific for certain groups of Lactobacillus spp. were used to amplify fragments, which were analyzed by DGGE. DGGE profiles obtained from Lactobacillus type strains acted as standards to analyze lactobacilli from four regional Abruzzo (central Italy) sourdoughs.     

Characterization of Variation in Rumen Methanogenic Communities under Different Dietary and Host Feed Efficiency Conditions,as Determined by PCR-Denaturing Gradient Gel Electrophoresis Analysis

Understanding ruminal methanogens is essential for greenhouse gas mitigation, as well as for improving animal performance in the livestock industry. It has been speculated that ruminal methanogenic diversity affects host feed efficiency and results in differences in methane production. This study examined methanogenic profiles in the rumen using culture-independent PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis for 56 beef cattle which differed in feed efficiency, as well as diet (the cattle were fed a low-energy diet or a high-energy diet). The methanogenic PCR-DGGE profiles detected were greatly affected by diet, and the major pattern changed from a community containing predominantly Methanobrevibacter ruminantium NT7 with the low-energy diet to a community containing predominantly Methanobrevibacter smithii, Methanobrevibacter sp. AbM4, and/or M. ruminantium NT7 with the high-energy diet. For each diet, the methanogenic PCR-DGGE pattern was strongly associated with the feed efficiency of the host. Diet-associated bands for Methanobrevibacter sp. AbM4 and M. smithii SM9 and a feed efficiency-related band for M. smithii PS were identified. The abundance of total methanogens was estimated by determining the numbers of copies of the 16S rRNA genes of methanogens. However, the size of the methanogen population did not correlate with differences in feed efficiency, diet, or metabolic measurements. Thus, the structure of the methanogenic community at the species or strain level may be more important for determining host feed efficiency under different dietary conditions.Ruminal methanogens use methanogenesis pathways to maintain low hydrogen partial pressure and to facilitate fiber digestion in the rumen by converting hydrogen into methane gas (24, 37). However, although it is necessary, this process also has adverse effects because the released methane represents a significant loss of dietary energy for the host animal (14) and it constitutes a large proportion of the agricultural greenhouse gas emitted (4, 10). Many studies to obtain a better understanding of rumen methanogens have been conducted in order to improve the efficiency of ruminal function and to mitigate methane release. Assessments by both cultivation-dependent and cultivation-independent methods have found that members of the genus Methanobrevibacter account for the majority of the methanogens in the rumens of sheep and cattle (11, 18, 21-23, 28, 31, 33, 34). In addition, Methanosphaera stadtmanae, Methanobacterium species, and Methanosarcina barkeri have also been found in some studies (13, 32). Although the phylogenetic positions of the methanogens in the rumen are diverse, these organisms utilize only three major pathways for methanogenesis: the CO₂ reduction pathway, the C₁ compound (e.g., methanol and methylamine) conversion pathway, and the acetate fermentation pathway. Each methanogen species has a substrate preference, and most methanogens can use only one or two substrates (37).Previous studies of rumen methanogens focused primarily on determining the methanogen species composition in different samples and developing strategies to reduce the methane yield from ruminants. Recently, there has been a strong desire to understand the impact of methanogens on host biology. Two primary studies found that feedlot beef cattle with higher feed efficiency (designated “efficient” animals) produced about 20% less methane gas than animals with lower feed efficiency (designated “inefficient” animals) (8, 19). The methanogenic communities of efficient and inefficient animals fed a low-energy diet have been compared, and divergence between the two communities has been reported (36). However, it is not clear how the methanogens in the rumen of cattle change when the animals are fed a different diet.The aims of this study were to describe the methanogenic communities in 56 steers with different feed efficiencies that were fed two distinct diets (a low-energy diet and a high-energy diet) and to understand how methanogenic communities change in response to diet modification using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and sequence analysis. Multivariate analysis was used to analyze the association of PCR-DGGE bands with the daily dry matter intake (DMI), average daily gain (ADG), feed conversion ratio (FCR), and residual feed intake (RFI). Methanogens that were associated with diet and with host feed efficiency were identified. In addition, the methanogen population of each rumen sample was examined by quantitative real-time PCR (qRT-PCR), and the results for different RFI groups and both diets were compared.     

Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, ~99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.     

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.     

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.     

Correlation of Particular Bacterial PCR-Denaturing Gradient Gel Electrophoresis Patterns with Bovine Ruminal Fermentation Parameters and Feed Efficiency Traits

The influence of rumen microbial structure and functions on host physiology remains poorly understood. This study aimed to investigate the interaction between the ruminal microflora and the host by correlating bacterial diversity with fermentation measurements and feed efficiency traits, including dry matter intake, feed conversion ratio, average daily gain, and residual feed intake, using culture-independent methods. Universal bacterial partial 16S rRNA gene products were amplified from ruminal fluid collected from 58 steers raised under a low-energy diet and were subjected to PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Multivariate statistical analysis was used to relate specific PCR-DGGE bands to various feed efficiency traits and metabolites. Analysis of volatile fatty acid profiles showed that butyrate was positively correlated with daily dry matter intake (P < 0.05) and tended to have higher concentration in inefficient animals (P = 0.10), while isovalerate was associated with residual feed intake (P < 0.05). Our results suggest that particular bacteria and their metabolism in the rumen may contribute to differences in host feed efficiency under a low-energy diet. This is the first study correlating PCR-DGGE bands representing specific bacteria to metabolites in the bovine rumen and to host feed efficiency traits.A fundamental understanding of microbial ecology and relationships to ruminant physiology is essential for successful manipulation of ruminal microflora and subsequent improvement in animal production since rumen microflora play important roles in the nutrient and energy uptake of the host (25). Hence, principles such as niche occupancy, selective pressure, adaptation, and interactions among populations (42) as well as the kinetics of substrate utilization (18) have to be taken into account when evaluating the ruminal microflora and host interactions. Bacterial density in the rumen is high, with direct counts as high as 10 billion cells per gram of ruminal contents (19, 33). Due to the limited understanding of the complex nature of the microbial component and activities in the rumen, the mechanisms of host-microbe and microbe-microbe interactions and whether such interactions impact host biology have not been well established.Many recent studies have employed molecularly based culture-independent techniques to investigate bacterial profiles (11, 22, 24, 39). PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis has been applied to assess ruminal microbial diversity based upon PCR-amplified 16S rRNA fragments to study community interactions (34), monitor populations shifts (23), and screen clone libraries (10). The PCR-DGGE banding patterns are considered to be representative of the dominant bacterial groups (26) and can be applied to screen changes of dominant species in the microflora for large numbers of environmental samples. A new terminology of “microbiome” has been applied to the study of the rumen microbial community, and such studies have further confirmed the complexity of this environment (7). However, many questions remain unanswered. For example, how does the microbiome change in large numbers of animals in response to host, diet, environment, health, and other factors? Which is more important to the host, the whole microbiome or the core microbiome? What is the function of a particular microbiome? Therefore, defining the ruminal microbiome to study its functions and interactions with the host has been an immense challenge. The selection of the rumen microbiome with particular functions after screening by culture-independent methods such as PCR-DGGE, therefore, is essential for high-throughput sequence analysis.Feed efficiency is one of the most critical factors that impact feed utilization by cattle. We hypothesized that particular bacterial populations in the rumen are associated with fermentation metabolites, which can also influence host feed efficiency. A recent study suggested that the bacterial structure may be associated with cattle''s residual feed intake (14); however, the small number of animals used in this study did not provide a direct linkage between a particular microbial population and host feed efficiency traits. The rumen microbial community changes in response to the feeding time (20). Since previous studies have shown that the concentration of volatile fatty acids (VFA) at prefeeding had less variation by diet (31) or by feeding cycles (43) and because of limited access to rumen fluid sampling from the examined commercial population in this study, we centered on the characterization of prefeeding dynamics in the ruminal bacteria and in the fermentation metabolites in 58 steers to test our hypothesis. Therefore, we focused on investigating the associations between rumen bacteria and host feed efficiency traits using PCR-DGGE analysis, aiming to identify the functional rumen microflora. The traits evaluated were daily dry matter intake (DMI), average daily gain (ADG), feed conversion ratio (FCR) (feed/gain), and residual feed intake (RFI) to measure the feed efficiency of cattle (1, 2, 28). Furthermore, we developed a multivariate statistical analysis to correlate bacterial PCR-DGGE profiles with fermentation measurements such as VFA and ammonia-nitrogen (NH₃-N) in the rumen and with feed efficiency traits, including, DMI, FCR, ADG, and RFI.     

Significant Association between Sulfate-Reducing Bacteria and Uranium-Reducing Microbial Communities as Revealed by a Combined Massively Parallel Sequencing-Indicator Species Approach

Massively parallel sequencing has provided a more affordable and high-throughput method to study microbial communities, although it has mostly been used in an exploratory fashion. We combined pyrosequencing with a strict indicator species statistical analysis to test if bacteria specifically responded to ethanol injection that successfully promoted dissimilatory uranium(VI) reduction in the subsurface of a uranium contamination plume at the Oak Ridge Field Research Center in Tennessee. Remediation was achieved with a hydraulic flow control consisting of an inner loop, where ethanol was injected, and an outer loop for flow-field protection. This strategy reduced uranium concentrations in groundwater to levels below 0.126 μM and created geochemical gradients in electron donors from the inner-loop injection well toward the outer loop and downgradient flow path. Our analysis with 15 sediment samples from the entire test area found significant indicator species that showed a high degree of adaptation to the three different hydrochemical-created conditions. Castellaniella and Rhodanobacter characterized areas with low pH, heavy metals, and low bioactivity, while sulfate-, Fe(III)-, and U(VI)-reducing bacteria (Desulfovibrio, Anaeromyxobacter, and Desulfosporosinus) were indicators of areas where U(VI) reduction occurred. The abundance of these bacteria, as well as the Fe(III) and U(VI) reducer Geobacter, correlated with the hydraulic connectivity to the substrate injection site, suggesting that the selected populations were a direct response to electron donor addition by the groundwater flow path. A false-discovery-rate approach was implemented to discard false-positive results by chance, given the large amount of data compared.Massively parallel sequencing has increased our ability to study microbial communities to a greater depth and at decreased sequencing costs to an extent that replication and gradient interrogation are now reasonably attainable. However, this massive throughput has mostly been used in exploratory studies, given the challenges to analysis of the big data sets generated and the relative novelty of the technique. To date, no report of a study that has used this method to describe the microbial community over a large area influenced by complicated hydrogeochemical factors during bioremediation has been published. Here, we used pyrosequencing technology complemented with a hypothesis-based approach to identify bacteria associated with biostimulation of U(VI) reduction at Area 3 of the U.S. Department of Energy''s (DOE''s) Oak Ridge Field Research Center (FRC) at Oak Ridge, TN.The Oak Ridge FRC is one of the most-studied sites for uranium bioremediation (2, 8, 19-22, 27, 37, 45-48). Previously used as a uranium enrichment plant, the site remains contaminated with depleted uranium, nitrate, and acidity. To deal with uranium contamination, dissimilatory metal reduction has been studied as an alternative that reduces risk by converting toxic soluble metals and radionuclides to insoluble, less toxic forms (2, 3, 16, 21, 26, 45). For example, some microbes can use metals such as Cr(VI), Se(VI), and the radionuclides U(VI) and Tc(VII) as final electron acceptors, producing a reduced insoluble species, thus blocking dispersal and reducing bioavailability.The ability to reduce U(VI) to U(IV) has been found in several unrelated phylogenetic groups, i.e., Delta-, Beta-, and Gammaproteobacteria, Firmicutes, Deinococci, and Actinobacteria, among others (42). Most previous studies have focused on the Fe(III)-reducing bacteria (FRB), especially Geobacter, and the sulfate-reducing bacteria (SRB), especially Desulfovibrio. Uranium(VI) reduction for bioremediation purposes has been tested and confirmed in laboratory-scale experiments using serum bottles (13, 18, 48), microcosms (23, 32), sediment columns (14, 43), and in situ field studies (3, 21, 41, 45), with the last one demonstrating the feasibility of U(VI) remediation and the correlation of U(VI) reduction with FRB (3, 6, 18, 31, 41) or SRB (40), or both (8, 19, 49).During field studies at Area 3 of the Oak Ridge site, a hydraulic control system together with ethanol injection successfully promoted U(VI) reduction from 5 μM to levels below U.S. Environmental Protection Agency (EPA) maximum contaminant levels (MCLs) for drinking water (0.126 μM) over a 2-year period (46). Reduction of U(VI) to U(IV) was confirmed by X-ray absorption near edge structure (XANES) (22, 46). Previous microbial surveys of sediments and groundwater from Area 3 wells by the use of 16S rRNA gene clone libraries detected genera known to harbor U(VI)-reducing members, such as Geobacter, Desulfovibrio, Anaeromyxobacter, Desulfosporosinus, and Acidovorax, after U(VI) reduction was established (8, 19). In one study, microbial counts from sediments were correlated with the hydraulic path, suggesting differences in organic carbon availability throughout Area 3 (8). The study that tracked the groundwater microbial communities of four locations of Area 3 over a 1.5-year period during ethanol stimulation found that nitrate, uranium, sulfide, and ethanol were correlated with particular bacterial populations and that the engineering control of dissolved oxygen and delivered nutrients was also significant in explaining the microbial community variability (19). However, the analysis of communities has been focused on limited wells and the community of the entire test area has not been characterized.On the basis of the previous results, we further hypothesized that the hydrological control strategy employed for the remediation of the site constrained the geochemistry of the site by controlling the distribution of organic carbon substrates and other nutrients and that this in turn selected a characteristic microbial community that was distinguishable from its surrounding community. We used massively parallel sequencing of 16S rRNA genes from sediments of 15 wells to characterize the microbial communities along hydrological gradients from the microbiologically active and hydraulically protected inner-loop zone to less active and still contaminated areas outside the treatment area and downgradient. Our sediment-sampling strategy allows a more precise spatial characterization than the use of groundwater samples, where filtering large volumes of water is often required, and also because samples of the attached communities can differ from the planktonic ones, as expected in oligotrophic aquifers (15), such as this site. The deeper sequencing allowed a more extensive survey of the communities, higher confidence in the detection of less dominant but significant members, and a more statistically robust indicator species assessment. We were able to detect groups significantly associated with U(VI) reduction and to explain differences in community structure with hydrogeochemical conditions.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.