Department of Global Agricultural Sciences, Graduate School of Agricultural and Life Sciences1, and Institute of Molecular and Cellular Biosciences2, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113, Japan
Microbial Resources and Chemotaxonomy Research Group, National Institute of Bioscience and Human-technology, Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan3
Author for correspondence: Hongik Kim (National Institute of Bioscience and Human-technology). Tel: +81 298 61 6590. Fax: +81 298 61 6587. e-mail: hongik{at}nibh.go.jp
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ABSTRACT |
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Keywords: novel soil clones, unculturable micro-organisms, paddy soil, novel phylotype
Abbreviations: ML, maximum-likelihood; MP, maximum-parsimony; NJ, neighbour-joining
The GenBank/EMBL/DDBJ accession numbers for the sequences of the novel soil clones and their aligned data set are D88480D88489 and ds36901, respectively.
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INTRODUCTION |
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Organisms in many disparate environments still remain uncultured because of the difficulty of cultivation based on conventional techniques. In recent years, the phylogenetic positions of uncultured bacteria have been revealed by 16S rDNA sequence analyses (Ward et al., 1990 , 1992
; Weller & Ward, 1991
; Fuhrman et al., 1992
). Uncultured archaeal organisms have been detected in different biotopes, including marine environments (DeLong, 1992
; Fuhrman et al., 1992
), hot springs (Barns et al., 1994
, 1996
), abyssal environments (McInerney et al., 1995
), freshwater (Schleper et al., 1997
) and soils (Bintrim et al., 1997
; Großkopf et al., 1998
). Crenarchaeotal lineages, which are branched very deeply from cultivated Crenarchaeota, were revealed to be predominant in marine and abyssal environments (Fuhrman et al., 1992
; McInerney et al., 1995
). In addition to this bunch of deeply branched crenarchaeotal lineages, a number of phylotypes within the Crenarchaeota have also been detected in non-extreme environments, such as marine, freshwater and terrestrial soils (DeLong, 1998
). These findings indicate that members of this kingdom of the Archaea are globally distributed and they are playing an ecologically important role in their habitats. Euryarchaeotal clones have also been detected in marine environments (DeLong, 1992
), hot springs (Barns et al., 1994
) and paddy fields (Großkopf et al., 1998
). Discovering unknown organisms in environmental microbial communities is perhaps not as difficult as was once thought. Recently, Barns et al. (1996)
found an ancient lineage of the Archaea in a hot spring and proposed a new kingdom, Korarchaeota.
In the course of diversity analysis of paddy soil archaea, we have found a number of unusual 16S rDNA clones (Kudo et al., 1996 , 1997
). Small-subunit rRNA genes were partially amplified by PCR from DNA extracted directly from paddy soil, by using primer sets consisting of reverse primers designed to amplify the 16S rRNA encoding genes of a novel lineage selectively and archaeal forward primer. In this report, we describe a novel phylotype with no known close relatives deduced from molecular phylogenetic analysis.
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METHODS |
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Phylogenetic analysis.
Soil clone sequences were compared to sequences in the available databases using the BLASTN algorithm (Altschul et al., 1997 ) to search for close evolutionary relatives (the last survey was performed in March 21, 2000). As no sequences in the DNA database showed similarities higher than 80%, representative organisms were selected from the Archaea, Eukarya and Bacteria. Partial 16S rDNA sequences of these organisms were then compiled for phylogenetic analyses with those of soil clones. The data set was aligned manually using 16S rRNA secondary structure to remove some unalignable regions on the domain level and sequences of identified homologous regions were realigned using the program CLUSTAL W version 1.7 (Thompson et al., 1994
). The compared sequences were positions 632820, 880996, 10411132 and 11421229. A maximum-parsimony (MP) tree reconstruction was performed using the PAUP software version 3.1.1 (Swofford, 1991
). The applied ratio of transition and transversion was 2:1. A heuristic search was used with a random stepwise addition sequence of 100 replicates, tree-bisection-reconnection branch swapping and the MULPARS option. A further analysis was run with 100 bootstrap replicates, each consisting of 10 additional random replicates. A maximum-likelihood (ML) analysis was carried out using a program package, MOLPHY version 2.3b3 (Adachi & Hasegawa, 1996
). A ML distance matrix was calculated using NucML, and a neighbour-joining (NJ) topology as the starting tree for the ML method was reconstructed by NJdist in MOLPHY. A ML tree was obtained using NucML with R (local rearrangement search) option based on the HKY model (Hasegawa et al., 1985
). Local bootstrap probabilities were estimated by the RELL (resampling of estimated log-likelihood) method (Kishino et al., 1990
; Hasegawa & Kishino, 1994
). NJ analysis was performed using the PHYLIP software version 3.572 (Felsenstein, 1995
). DNADIST from this program package was used to create a distance matrix based on the two-parameter method of Kimura (1980)
. This distance matrix was used to construct a NJ tree using the NJ algorithm from NEIGHBOR (PHYLIP version 3.572), and the bootstrap analyses utilized 1000 replicate data sets.
To assess the phylogenetic placement of the novel soil clones, nucleotide signature analysis was done by using the ARB program package (O. Strunk & W. Ludwig; http://www.mikro.biologie.tu-muenchen.de/pub/ARB/; Technische Universität München, Germany) with data from the Ribosomal Database Project (RDP) (Maidak et al., 1999 ).
Chimera check and prediction of rRNA secondary structure.
Each sequence was submitted to the CHECK-CHIMERA program of the RDP (Maidak et al., 1999 ) to detect the presence of possible chimeric artefacts, and the putative partial secondary structure of 16S rRNA was predicted from the primary structure using the program RNA structure version 2.52 (Mathews & Burkard, 1997
). The Zuker algorithm (1989)
was used for RNA structure prediction based on the free energy minimization, and the putative secondary structure was constructed through comparison with that of published small-subunit rRNA (Neefs et al., 1993
).
Distribution of novel soil organisms in the environment.
To examine the distribution of the novel organisms in the environment, we designed an oligonucleotide primer, 970F (5'-AAT YYA ACT CAA CGC RGA G-3'), with a sequence identical to positions 959976 of 16S rRNA of the novel clones. The presence of the novel soil organisms was tested with a primer set of 970F and SC13 for paddy soils, upland soils and forest soils collected in Japan. For each PCR reaction, approximately 10 ng purified soil DNA was used, and amplification was performed using AmpliTaq Gold (Perkin Elmer) in a thermal cycler (Hybaid) programmed for 9 min at 94 °C, followed by 55 cycles of 30 s at 94 °C, 20 s at 57 °C, and 25 s at 70 °C, and an additional incubation at 70 °C for 10 min. After amplification, 7 µl product was electrophoresed in a 1·5% agarose gel and visualized by ethidium bromide staining.
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RESULTS AND DISCUSSION |
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To amplify nearly complete 16S rDNA fragments of the novel soil organisms, PCR amplification was performed with two primer sets, AS11F-SC13 and AS11F-SW42. However, these primer sets did not give any amplification products. AS343F as a forward primer was subjected to the PCR amplification instead of AS11F, but these primer sets also failed to amplify the target molecules. Partial 16S rDNA was obtained with primer sets AS564F-SC13 and AS564F-SW42, and these products were cloned. Sequences of 10 cloned DNA samples were determined for further analyses, and the sequences of each clone were 8298% similar to each other. The length of sequenced DNA ranged from 644 to 647 nucleotides. Each sequence was folded into a putative secondary structure of 16S rRNA from positions 585 to 1228 (E. coli numbering system; Fig. 1), and they were found to be well-conserved in agreement with previously published models (Neefs et al., 1993
). The correct secondary structure seemed to show some evidence that the individual sequences are a natural occurrence, although this evidence does not give complete proof that the sequences are not chimeric. CHECK-CHIMERA also did not detect chimeras among the novel soil sequences.
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Inference of novel soil micro-organisms
We know nothing about these organisms other than their 16S rDNA sequences, so it might be difficult to predict their phenotypic characteristics. However, it is expected that sequences derived from the paddy fields are from mesophilic organisms. Table 2 compares the G+C contents of the sequenced regions of the 16S rDNA of Archaea and the novel soil clones. The high G+C content of the Crenarchaeota is correlated with their thermophilic characteristic; thermophiles usually have rDNA G+C contents of >60 mol% whereas mesophiles generally have rDNA G+C contents of 55 mol% or less (Dalgaard & Garrett, 1993
; Woese et al., 1991
). Judging from the sampling environments and the G+C contents of the novel soil clones, the sequences probably come from mesophilic organisms.
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ACKNOWLEDGEMENTS |
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Received 2 February 2000;
revised 13 April 2000;
accepted 4 May 2000.