Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5 end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computersimulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems. Species richness (the number of species within a community) and species evenness (the sizes of species populations within a community) are two essential parameters for defining community structure and diversity. Quantitation of these two parameters is severely limited by conventional culture-dependent methods by virtue of the fact that large fractions (Ͼ85 to 99.999%) of the organisms existing in nature appear to be refractory to cultivation Several molecular approaches now provide powerful adjuncts to the culture-dependent techniques. One approach in particular that couples PCR and rRNA-based phylogeny has been effective in the exploration of microbial environments and the identification of uncultured organisms. The stepwise strategy of this approach is to isolate total community DNA and use this DNA as a template for PCR amplification of 16S rRNA genes with universal or domain-specific primers. This is usually followed by construction of a clone library for genes encoding rRNA (rDNAs) and rapid screening of the library based on sequence differences (4) or by determining restriction fragment length polymorphisms (RFLPs) of the rDNAs (19). The richness and evenness of a community are qualitatively estimated based on the number of unique clones and the relative frequencies of the various "ribotypes" detected. While this approach obviates the requirement for cultivation, construction and screening of clone libraries are laborious. More recently, techniques dependent on DNA melting behavior (20, 21) or single-strand DNA conformation (14) have been developed as a means of circumventing library construction. These methods have been employed to assess community structure and to provide relatively crude estimates of species diversity, but are limited by the comparatively insensitive staining technologies employed and do not provide information concerning the specific phylogenetic groups that comprise a microbial community. Here we report on a quantitative molecular method for rapid analysis of complex microbial communities in which an existing technology was extended. RFLP analysis of 16S rDNA, otherwise known as amplified rDNA restriction analysis (ARDRA), has been used for several years as a method for rapid comparison of rDNAs

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Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected

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A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal

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We used a method that combines microautoradiography with hybridization of fluorescent rRNA-targeted oligonucleotide probes to whole cells (MICRO-FISH) to test the hypothesis that the relative contributions of various phylogenetic groups to the utilization of dissolved organic matter (DOM) depend solely on their relative abundance in the bacterial community. We found that utilization of even simple low-molecular-weight DOM components by bacteria differed across the major phylogenetic groups and often did not correlate with the relative abundance of these bacterial groups in estuarine and coastal environments. The Cytophaga-Flavobacter cluster was overrepresented in the portion of the assemblage consuming chitin, N-acetylglucosamine, and protein but was generally underrepresented in the assemblage consuming amino acids. The amino acid-consuming assemblage was usually dominated by the � subclass of the class Proteobacteria, although the representation of �-proteobacteria in the protein-consuming assemblages was about that expected from their relative abundance in the entire bacterial community. In our experiments, no phylogenetic group dominated the consumption of all DOM, suggesting that the participation of a diverse assemblage of bacteria is essential for the complete degradation of complex DOM in the oceans. These results also suggest that the role of aerobic heterotrophic bacteria in carbon cycling would be more accurately described by using three groups instead of the single bacterial compartment currently used in biogeochemical models.

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G1C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.