Molecular phylogeny of Archaea from soil (small subunit ribosomal RNAyplanktonicymicrobial communitiesymicrobial diversityymolecular ecology)

Cultivation methods have contributed to our present knowledge about the presence and diversity of microbes in naturally occurring communities. However, it is well established that only a small fraction of prokaryotes have been cultivated by standard methods and, therefore, the prokaryotes that are cultivated may not ref lect the composition and diversity within those communities. Of the two prokaryotic phylogenetic domains, Bacteria and Archaea, members of the former have been shown to be ubiquitous in nature, with ample evidence of vast assemblages of uncultured organisms. There is also now increasingly compelling evidence that the Archaea, which were once thought to occupy a limited number of environments, are also globally widespread. Here we report the use of molecular phylogenetic techniques, which are independent of microbial cultivation, to conduct an assessment of Archaea in a soil microbial community. Small subunit ribosomal RNA genes of Archaea were amplified from soil and cloned. Phylogenetic and nucleotide signature analyses of these cloned small subunit ribosomal RNA gene sequences revealed a cluster of Archaea from a soil microbial community that diverge deeply from the crenarchaeotal line of descent and has the closest affiliation to the lineage of planktonic Archaea. The identification and phylogenetic classification of this archaeal lineage from soil contributes to our understanding of the ecological significance of Archaea as a component of microbial communities in non-extreme environments. Assessment of the microbial diversity in the environment has long challenged microbiologists and microbial ecologists. Few microbes have a sufficiently distinct cellular morphology to be identified by microscopic techniques. Cultivation of microbes as a means to characterize microbial communities has severe limitations, since the majority of microbes observed microscopically in an environmental sample cannot be cultivated by standard methods (for reviews see refs. 1 and 2). For this reason, methods that are independent of culturing are important to characterize the diversity of microorganisms in their environments. One such method is the molecular phylogenetic analysis of small subunit ribosomal RNA (SSU rRNA) gene sequences (3–15). This approach has resulted in the discovery of entirely new phylogenetic lineages, some of which are major constituents of the environmental communities that were not detected by traditional cultivation techniques (1, 16). Many studies have focused on the identification and diversity of prokaryotes of the domain Bacteria (17) in environmental assemblages of microorganisms. Rather less attention has been given to the diversity and ecological significance of prokaryotes of the domain Archaea (17). This may be due to the fact that the majority of Archaea have been isolated from extreme environments or specialized ecological niches. All cultured members of the Crenarchaeota, one of the two kingdoms of Archaea, are thermophiles. The other kingdom of Archaea, the Euryarchaeota, is comprised of extreme halophiles, sulfur reducers, sulfate reducers, thermophilic heterotrophs, and methanogens. However, recent molecular phylogenetic studies (5, 7, 8, 11, 18, 19) have indicated that Archaea may be more diverse and widespread than what is represented by cultured members of the Archaea. Soil is a familiar, yet poorly characterized, microbial environment in which less than 0.1% of the microscopically observed microorganisms present are cultured by standard techniques (20, 21). Recent surveys of soil microbial populations with molecular phylogenetic techniques revealed an enormous diversity of as yet uncharacterized microbes (6, 11, 15). In these studies, total DNA was isolated directly from soil samples, SSU rRNA gene sequences were amplified by PCR, and the amplified SSU rRNA gene sequences were characterized by nucleotide sequence and phylogenetic analyses. The primer sets used for the amplification were designed to selectively amplify SSU rRNA gene sequences from most members of the domain Bacteria (6) or from most members of the three primary phylogenetic domains, Archaea, Bacteria, and Eucarya (11, 15). Using the latter primer set, one group identified and cloned a partial SSU rRNA gene sequence (FIE16) that was most closely related to those of Archaea (11). Our work was designed to assess the diversity of Archaea present in a soil microbial community and to serve as the starting point for a study of the ecology of Archaea in soil. Small subunit rRNA gene sequences of Archaea were selectively amplified from total DNA extracted directly from soil and cloned. Sequence and phylogenetic analyses of these clones identified a group of Archaea in a soil environment that diverge deeply from the crenarchaeal line of descent and has closest affiliation to the lineage of planktonic Archaea (22). MATERIALS AND METHODS Soil Sampling and Analyses. A subsurface (2–10 cm) soil sample was collected in August 1995, from the West Madison Agricultural Research Station (Madison, WI), and stored on ice until processed. The soil type is a Plano silt loam containing 61% sand, 23% silt, and 16% clay, with 1.7% organic matter. The soil pH was 7.0. Soil analyses were performed by the Soil Testing Laboratory of the University of Wisconsin–Madison, as described (23). Extraction and Purification of Total DNA from Soil Sample. Total DNA was isolated from the soil sample by a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y94277-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: SSU rRNA, small subunit ribosomal RNA; RDP, Ribosomal Database Project. Data deposition: The SSU rRNA gene sequences reported in this paper have been deposited in the GenBank data base [accession nos. U62811 (SCA1145), U62812 (SCA1150), U62813 (SCA1151), U62814 (SCA1154), U62815 (SCA1158), U62816 (SCA1166), U62817 (SCA1170), U62818 (SCA1173), U62819 (SCA1175), U62820 (SCA1180). ‡To whom reprint requests should be addressed. e-mail: [email protected].