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Applied and Environmental Microbiology, October 2008, p. 5975-5985, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.01275-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Amplification of Uncultured Single-Stranded DNA Viruses from Rice Paddy Soil{triangledown} ,{dagger}

Kyoung-Ho Kim,1 Ho-Won Chang,1 Young-Do Nam,1 Seong Woon Roh,1 Min-Soo Kim,1 Youlboong Sung,2 Che Ok Jeon,3 Hee-Mock Oh,1 and Jin-Woo Bae1,4*

Biological Resource Center, KRIBB, Daejeon 305-806, Korea,1 Environmental Research Department, Research Institute of Industrial Science and Technology, Gwangyang 545-090, Korea,2 Department of Life Science, Chung-Ang University, Seoul 156-756, Korea,3 Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea4

Received 9 June 2008/ Accepted 8 August 2008

Viruses are known to be the most numerous biological entities in soil; however, little is known about their diversity in this environment. In order to explore the genetic diversity of soil viruses, we isolated viruses by centrifugation and sequential filtration before performing a metagenomic investigation. We adopted multiple-displacement amplification (MDA), an isothermal whole-genome amplification method with {phi}29 polymerase and random hexamers, to amplify viral DNA and construct clone libraries for metagenome sequencing. By the MDA method, the diversity of both single-stranded DNA (ssDNA) viruses and double-stranded DNA viruses could be investigated at the same time. On the contrary, by eliminating the denaturing step in the MDA reaction, only ssDNA viral diversity could be explored selectively. Irrespective of the denaturing step, more than 60% of the soil metagenome sequences did not show significant hits (E-value criterion, 0.001) with previously reported viral sequences. Those hits that were considered to be significant were also distantly related to known ssDNA viruses (average amino acid similarity, approximately 34%). Phylogenetic analysis showed that replication-related proteins (which were the most frequently detected proteins) related to those of ssDNA viruses obtained from the metagenomic sequences were diverse and novel. Putative circular genome components of ssDNA viruses that are unrelated to known viruses were assembled from the metagenomic sequences. In conclusion, ssDNA viral diversity in soil is more complex than previously thought. Soil is therefore a rich pool of previously unknown ssDNA viruses.

* Corresponding author. Mailing address: Biological Resource Center, KRIBB, Daejeon 305-806, Korea. Phone: 82 42 860 4628. Fax: 82 42 860 4677. E-mail: baejw{at}kribb.re.kr

{triangledown} Published ahead of print on 15 August 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, October 2008, p. 5975-5985, Vol. 74, No. 19
0099-2240/08/$08.00+0     doi:10.1128/AEM.01275-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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