Abstract

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