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

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.

FIG. 1

Representation of insert size range in the soil metagenome libraries. Clones within a range of 10 kb are grouped together. For SL1, n = 81; for SL2, n = 132.

FIG. 2

Phylogenetic placement of 16S rRNA sequences from SL1. Neighbor-joining analysis on 75 sequences from the represented phyla were used to construct the tree.

FIG. 3

Schematic diagram of sequence analysis of clone SL1-36C7. Putative ORFs are indicated by the arrows; below is a list of the ORFs with the protein to which each is most similar. The potential operon includes ORFs 15 to 21. The antibacterial ORF is number 27.

FIG. 4

(A) Predicted amino acid sequence of the antibacterial ORF. The predicted protein has 543 residues. Highly conserved sequences within the repeats are in bold, underlined, or italics to highlight the repeats. (B) Kyte-Doolittle hydrophobicity profile of the antibacterial ORF.

FIG. 5

(A) Restriction digest analysis of amylase and lipase clones from SL1 digested with HindIII. The arrow indicates the BAC vector. Lanes 1 to 8, amylase clones; lanes 9 and 10, lipase clones. (B) Restriction digests of hemolytic clones from SL2, digested with NotI. The arrow indicates the BAC vector. Lane 1, size markers.