Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms

Recent progress in molecular microbial ecology has revealed that traditiona l culturing methods fail to represent the scope of microbial diversity in n ature, since only a small proportion of viable microorganisms in a sample a re recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromos ome (BAC) vector to construct libraries of genomic DNA isolated directly fr om soil (termed metagenomic libraries), To date, we have constructed two su ch libraries, which contain more than 1 Gbp of DNA, Phylogenetic analysis o f 16S rRNA gene sequences recovered from one of the libraries indicates tha t the BAC libraries contain DNA from a wide diversity of microbial phyla, i ncluding sequences from diverse taxa such as the low-G+C, gram-positive Aci dobacterium, Cytophagales, and Proteobacteria. Initial screening of the lib raries in Escherichia coli identified several clones that express heterolog ous genes from the inserts, confirming that the BAC vector can be used to m aintain, express, and analyze environmental DNA, The phenotypes expressed b y these clones include antibacterial, lipase, amylase, nuclease, and hemoly tic activities. Metagenomic libraries are a powerful tool for exploring soi l microbial diversity, providing access to the genetic information of uncul tured soil microorganisms. Such libraries will be the basis of new initiati ves to conduct genomic studies that link phylogenetic and functional inform ation about the microbiota of environments dominated by microorganisms that are refractory to cultivation.