A New Approach To Utilize PCR–Single-Strand-Conformation Polymorphism for 16S rRNA Gene-Based Microbial Community Analysis

Single-strand-conformation polymorphism (SSCP) of DNA, a method widely used in mutation analysis, was adapted to the analysis and differentiation of cultivated pure-culture soil microorganisms and noncultivated rhizosphere microbial communities. A fragment (approximately 400 bp) of the bacterial 16S rRNA gene (V-4 and V-5 regions) was amplified by PCR with universal primers, with one primer phosphorylated at the 5′ end. The phosphorylated strands of the PCR products were selectively digested with lambda exonuclease, and the remaining strands were separated by electrophoresis with an MDE polyacrylamide gel, a matrix specifically optimized for SSCP purposes. By this means, reannealing and heteroduplex formation of DNA strands during electrophoresis could be excluded, and the number of bands per organism was reduced. PCR products from 10 of 11 different bacterial type strains tested could be differentiated from each other. With template mixtures consisting of pure-culture DNAs from 5 and 10 bacterial strains, most of the single strains could be detected from such model communities after PCR and SSCP analyses. Purified bands amplified from pure cultures and model communities extracted from gels could be reamplified by PCR, but by this process, additional products were also generated, as detected by further SSCP analysis. Profiles generated with DNAs of rhizosphere bacterial communities, directly extracted from two different plant species grown in the same field site, could be clearly distinguished. This study demonstrates the potential of the selected PCR–single-stranded DNA approach for microbial community analysis.     

Emission of Climate-Relevant Trace Gases and Succession of Microbial Communities during Open-Windrow Composting

Determination of different indicators of microbial biomass, community structure, and bioactivity by the fumigation extraction method, as well as determination of phospholipid fatty acids (PLFAs) and their subfractions and the measurement of trace gases, respectively, provides valuable information about microbial succession in composting processes. The emission rates of carbon dioxide (CO(inf2)), methane (CH(inf4)), and nitrous oxide (N(inf2)O) increased successively during compost maturation: initially in the presence of easily degradable nutrients, during high temperature, and after the temperature had cooled down, respectively. The emission rate patterns of these trace gases corresponded to the concentrations of PLFAs and their particular subfractions. (i) Similar to the CO(inf2) emission rates, microbial biomass estimations by fumigation extraction and by determination of the amount of total PLFAs showed a discontinuous decrease during the composting process, with a slight increase at the end of the observation period. (ii) An increase in ether lipids, indicating the enhanced presence of archaean methanogens, and an elevated CH(inf4) emission were observed at the same time. (iii) The period of enhanced N(inf2)O emission corresponded to the increase in beta and omega hydroxy fatty acids derived from the outer membrane. Additionally, the continuous increase in branched-chain fatty acids suggested an increase in gram-positive bacteria and actinomycetes, and the decrease in polyunsaturated fatty acids indicated a decrease in eukaryotic cells during the composting.     

Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5′-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5′-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5′-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry.     

Microbial Diversity and Succession in the Chinese Luzhou-Flavor Liquor Fermenting Cover Lees as Evaluated by SSU rRNA Profiles

The microbial diversity and the community succession in the fermenting cover lees of Chinese Luzhou-flavor liquor were investigated by small-subunit rRNA (SSU rRNA) culture independent method. All sequences retrieved from the 1, 7 and 60 days fermented cover lees were respectively assigned into the genera of Streptococcus, Acetobacter, Arthrobacter, Bacillus, Staphylococcus, Serratia, Nocardia, Methanoculleus, Clostridium, Aneurinibacillus, Corynebacterium, Lactobacillus, Microbacterium, Trichosporon, Saccharomycopsis, Sagenomella, Talaromyces, Eurotium, Issatchenkia, Zygosaccharomyces, Saccharomyces and TM7 phylum. The fungal Issatchenkia, Saccharomycopsis and Talaromyces and the bacteria Staphylococcus and Lactobacillus were most abundant in the 1 day fermented cover lees, the fungal Issatchenkia, Saccharomyces and Talaromyces and the bacteria Bacillus and Streptococcus were dominant in the 7 days cover lees, the archaea Methanoculleus and the fungal Eurotium and Talaromyces were prevalent in the 60 days cover lees. When the microbial community profiles in three samples were compared at species level, the prokaryotic community similarity coefficient was from 0.4042 to 0.5703 and descended to 0.2222, and that of eukaryotic community was from 0.3000 to 0.6000 and followed to 0.5215. These results suggested that microbial diversity variability and community succession have happened in the cover lees associated with fermentation proceeding and such variability and succession respond for the appearance of some unique flavor of Luzhou-flavor liquor.     

Genetic Diversity of Benzoyl Coenzyme A Reductase Genes Detected in Denitrifying Isolates and Estuarine Sediment Communities

Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to α-, β-, or γ-Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems.     

Investigation of the Microbial Ecology of Intertidal Hot Springs by Using Diversity Analysis of 16S rRNA and Chitinase Genes

The microbial diversity of intertidal hot springs on the seashore of northwest Iceland was examined by combining directed in situ enrichments, artificial support colonization, and mat sampling. Analysis of 16S rRNA genes revealed the presence of clones related to both marine and terrestrial, thermophilic, mesophilic, and psychrophilic microorganisms scattered among 11 bacterial divisions. No archaea were found. The species composition of the enrichments was affected by the length of the hot periods experienced at low tide and was very different from those found in the biomass. A total of 36 chitinase genes were detected by molecular screening of the samples with degenerate primers for glycoside hydrolase family 18. The chitinase gene diversity was at least twofold higher in the enrichment samples than in the controls, indicating that a much higher diversity of hydrolytic genes can be accessed with this approach.     

Phylogenetic Analysis of Bacterial Communities Associated with Leaves of the Seagrass Halophila stipulacea by a Culture-Independent Small-Subunit rRNA Gene Approach

Abstract The phylogenetic diversity of the bacterial community associated with leaves of the marine plant Halophila stipulacea in the northern Gulf of Elat was examined by 16S rRNA gene (rDNA) sequence analyses of a clone library. For 59 clones corresponding to 51 ARDRA (amplified rDNA restriction analysis) groups, the sequence of ∼1 kb was determined, and the fraction of the corresponding ARDRA groups of the leaf library was calculated. The class Proteobacteria was represented by 62.6% of the clone sequences. Most sequences originated from members of the γ-subclass (27.3%), affiliated with members of the genera Pseudomonas, Vibrio, Marinomonas, Oceanospirillum, and other marine groups. Affiliation to the α-subclass was determined for 24.2% of the sequences. They were related to the genera Hyphomonas, Roseobacter, Ruegeria, and Rhizobiaceae. Several α-proteobacterial sequences were distantly related to known sequences. Only 4% of the clone sequences were related to β-Proteobacteria. Additionally, 7.1% of the sequences possibly belonged to the class Proteobacteria, but branched deeply from known subclasses. Several sequences were affiliated to members of the orders Verrucomicrobiales and Planctomycetales, the Holophaga/Acidobacterium phylum, and chloroplasts of marine diatoms. Received: 20 March 1999; Accepted: 13 August 1999; Online Publication: 2 March 2000     

Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.     

Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes

A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4-V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8-V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of gamma-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.     

Bacterial Primary Colonization and Early Succession on Surfaces in Marine Waters as Determined by Amplified rRNA Gene Restriction Analysis and Sequence Analysis of 16S rRNA Genes

The nearly universal colonization of surfaces in marine waters by bacteria and the formation of biofilms and biofouling communities have important implications for ecological function and industrial processes. However, the dynamics of surface attachment and colonization in situ, particularly during the early stages of biofilm establishment, are not well understood. Experimental surfaces that differed in their degrees of hydrophilicity or hydrophobicity were incubated in a salt marsh estuary tidal creek for 24 or 72 h. The organisms colonizing these surfaces were examined by using a cultivation-independent approach, amplified ribosomal DNA restriction analysis. The goals of this study were to assess the diversity of bacterial colonists involved in early succession on a variety of surfaces and to determine the phylogenetic affiliations of the most common early colonists. Substantial differences in the representation of different cloned ribosomal DNA sequences were found when the 24- and 72-h incubations were compared, indicating that some new organisms were recruited and some other organisms were lost. Phylogenetic analyses of the most common sequences recovered showed that the colonists were related to organisms known to inhabit surfaces or particles in marine systems. A total of 22 of the 26 clones sequenced were affiliated with the Roseobacter subgroup of the α subdivision of the division Proteobacteria (α-Proteobacteria), and most of these clones were recovered at a high frequency from all surfaces after 24 or 72 h of incubation. Two clones were affiliated with the Alteromonas group of the γ-Proteobacteria and appeared to be involved only in the very early stages of colonization (within the first 24 h). A comparison of the colonization patterns on the test surfaces indicated that the early bacterial community succession rate and/or direction may be influenced by surface physicochemical properties. However, organisms belonging to the Roseobacter subgroup are ubiquitous and rapid colonizers of surfaces in coastal environments.     

Genetic Diversity of Pyrenophora teres Isolates as Detected by AFLP Analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to analyse mainly 83 Czech isolates of Pyrenophora teres, P. graminea, P. tritici‐repentis and Helminthosporium sativum. Each species had distinct AFLP profiles. Using 19 primer combinations 948 polymorphic bands were detected. All main clusters in dendrogram correspond to the studied species. Even the two forms of P. teres–P. teres f. teres (PTT) and P. teres f. maculata (PTM) – formed different clusters. Genetic diversity, with regard to the locality and the year of the sample's collection, was analysed separately within the AFLP‐based dendrogram cluster of PTT and PTM. Unweighted pair‐group method (UPGMA) analysis of the 37 isolates of PTT and 30 isolates of PTM, using 469 polymorphic bands, showed that the variability seemed to have been influenced more by the year of sampling than by the geographic origin of the isolate. The presence of intermediate haplotypes with a relatively high number of shared markers between the two groups indicated that hybridization between the forms of P. teres could happen, but it is probably often overlapped by selection pressure or genetic drift.     

Microbial Succession during a Field Evaluation of Phenol and Toluene as the Primary Substrates for Trichloroethene Cometabolism

Microbial community composition and succession were studied in an aquifer that was amended with phenol, toluene, and chlorinated aliphatic hydrocarbons to evaluate the effectiveness of these aromatic substrates for stimulating trichloroethene (TCE) bioremediation. Samples were taken after the previous year's field studies, which used phenol as the primary substrate, and after three successive monthly treatments of phenol plus 1,1-dichloroethene (1,1-DCE) plus TCE, phenol plus TCE, and toluene plus TCE. Dominant eubacteria in the community were assessed after each of the four treatments by characterizing isolates from the most dilute most-probable-number tubes and by extracting DNA from aquifer samples. The succession of dominant phenol- and toluene-degrading strains was evaluated by genomic fingerprinting, cellular fatty acid methyl ester (FAME) analysis, and amplified ribosomal DNA restriction analysis (ARDRA). 1,1-DCE was found to drastically reduce microbial growth and species richness, which corresponded to the reduction in bioremediation effectiveness noted previously for this treatment (G. D. Hopkins and P. L. McCarty, Environ. Sci. Technol. 29:1628-1637, 1995). Only a few gram-positive isolates could be obtained after treatment with 1,1-DCE, and these were not seen after any other treatments. Microbial densities returned to their original levels following the subsequent phenol-TCE treatment, but the original species richness was not restored until after the subsequent toluene-TCE treatment. Genomic fingerprinting and FAME analysis indicated that six of the seven originally dominant microbial groups were still dominant after the last treatment, indicating that the community is quite resilient to toxic disturbance by 1,1-DCE. FAME analysis indicated that six microbial taxa were dominant: three members of the (beta) subclass of the class Proteobacteria (Comamonas-Variovorax, Azoarcus, and Burkholderia) and three gram-positive groups (Bacillus, Nocardia, and an unidentified group). ARDRA revealed that the dominant community members were stable during the three nontoxic treatments and that virtually all of the bands could be accounted for by isolates from five of the dominant taxa, indicating that the isolation protocol used likely recovered most of the dominant members of this community.     

Characterization of a Defined 2,3,5,6-Tetrachlorobiphenyl-ortho-Dechlorinating Microbial Community by Comparative Sequence Analysis of Genes Coding for 16S rRNA

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3,5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the δ subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the δ, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.The extensive industrial use of polychlorinated biphenyls (PCBs) during the 20th century has resulted in the release of an estimated several million pounds of PCBs into the environment (2). Due to the hydrophobicity and chemical stability of these compounds, PCBs ultimately accumulate in subsurface anaerobic sediments, where reductive dechlorination by anaerobic microorganisms is proposed to be an essential step in PCB degradation and detoxification (6). Although anaerobic reductive dechlorination has been documented in the environment and in the laboratory, attempts to identify and isolate anaerobic PCB-dechlorinating microbes by classical enrichment and isolation techniques have been unsuccessful (for a review, see reference 2). Isolation of anaerobic PCB-dechlorinating microbes has been hindered in part by the inability to maintain and sequentially transfer dechlorinating consortia in defined medium. May et al. (24) were the first to demonstrate that single colonies could be obtained by plating highly enriched PCB-dechlorinating enrichment cultures on agar-solidified media. Although two of the colonies exhibited para dechlorination activity when transferred back to liquid enrichment medium, the colonies contained a mixed community of microorganisms and dechlorination required the addition of sediment to the medium. More recently, highly enriched PCB-ortho-dechlorinating enrichment cultures were developed from Baltimore Harbor sediments in minimal media that contained sediments and a single congener (3) or Aroclor 1260 (37). These were the first confirmed reports of sustained ortho dechlorination of PCBs throughout sequential transfers in medium with estuarine sediments. Finally, Cutter et al. demonstrated that a consortium of PCB-ortho-dechlorinating anaerobes from Baltimore Harbor could be sequentially transferred and maintained in minimal medium without the addition of sterile sediment (9). With the ability to maintain PCB dechlorination in a completely defined medium, highly enriched PCB-dechlorinating consortia could be developed by sequential transfers in medium that contained the minimal growth requirements for dechlorinating species.The current study identifies putative PCB-dechlorinating anaerobes in ortho-dechlorinating enrichment cultures by a comprehensive approach that combines traditional selective enrichment techniques with molecular monitoring (SEMM). Microbial consortia enriched for PCB ortho dechlorination in minimal medium were analyzed by comparative sequence analysis of genes coding for 16S rRNA (16S rDNA) amplified from total community DNAs. Protocols were developed for chromosomal DNA extraction from sediment, 16S rDNA amplification by PCR, cloning of partial 16S rDNA PCR fragments, screening by restriction fragment length polymorphism (RFLP) analysis, and DNA sequencing for comparative sequence analysis. By utilizing these techniques, shifts in the microbial community were monitored as the cultures were further enriched for PCB-dechlorinating anaerobes by elimination of undefined medium components (i.e., sediment), changes in carbon source, and addition of selective physiological inhibitors. The results presented herein demonstrate the applicability of the SEMM approach for the selection and monitoring of highly defined PCB-dechlorinating microbial consortia.     

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.     

Worldwide Distribution of Nitrosococcus oceani, a Marine Ammonia-Oxidizing γ-Proteobacterium, Detected by PCR and Sequencing of 16S rRNA and amoA Genes

Diversity of cultured ammonia-oxidizing bacteria in the γ-subdivision of the Proteobacteria was investigated by using strains isolated from various parts of the world ocean. All the strains were very similar to each other on the basis of the sequences of both the 16S rRNA and ammonia monooxygenase genes and could be characterized as a single species. Sequences were also cloned directly from environmental DNA from coastal Pacific and Atlantic sites, and these sequences represented the first Nitrosococcus oceani-like sequences obtained directly from the ocean. Most of the environmental sequences clustered tightly with those of the cultivated strains, but some sequences could represent new species of Nitrosococcus. These findings imply that organisms similar to the cultivated N. oceani strains have a worldwide distribution.     

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.