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Applied and Environmental Microbiology, June 2000, p. 2541-2547, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms

Michelle R. Rondon,1,|| Paul R. August,2 Alan D. Bettermann,1 Sean F. Brady,3 Trudy H. Grossman,2,dagger Mark R. Liles,1 Kara A. Loiacono,2 Berkley A. Lynch,2,Dagger Ian A. MacNeil,2 Charles Minor,2 Choi Lai Tiong,2 Michael Gilman,2,§ Marcia S. Osburne,2 Jon Clardy,3 Jo Handelsman,1,* and Robert M. Goodman1

Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin1; ARIAD Pharmaceuticals, Inc., Cambridge, Massachusetts2; and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York3

Received 29 November 1999/Accepted 23 March 2000

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-G+C, 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.

* Corresponding author. Mailing address: Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden Drive, Madison, WI 53706. Phone: (608) 263-8783. Fax: (608) 262-8643. E-mail: joh{at}plantpath.wisc.edu.

dagger Present address: Vertex Pharmaceuticals, Inc., Cambridge, MA 02139.

Dagger Present address: UCB Research Inc., Cambridge, MA 02139.

§ Present address: Biogen, Inc., Cambridge, MA 02142.

|| Present address: The Ohio State University, Columbus, OH 43210.

Applied and Environmental Microbiology, June 2000, p. 2541-2547, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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