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

TLDR

Traditional culturing methods recover only a small fraction of viable microorganisms, leaving much microbial diversity unrepresented, while metagenomic libraries provide access to the genetic information of uncultured soil microorganisms. The authors aim to develop BAC‑based metagenomic libraries to investigate the full extent of soil microbial diversity and to enable genomic studies linking phylogeny and function in uncultured communities. They constructed BAC metagenomic libraries from soil genomic DNA and screened them in *Escherichia coli*, demonstrating that the vector can maintain, express, and analyze environmental DNA. The two >1 Gbp BAC libraries contain DNA from diverse microbial phyla, as shown by 16S rRNA phylogeny, and clones express functional phenotypes including antibacterial, lipase, amylase, nuclease, and hemolytic activities.

Abstract

ABSTRACT 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.