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

Abstract

The biosphere is dominated by microorganisms (32), yet most microbes in nature have not been studied. Traditional methods for culturing microorganisms limit analysis to those that grow under laboratory conditions (14,25). The recent surge of research in molecular microbial ecology provides compelling evidence for the existence of many novel types of microorganisms in the environment in numbers and varieties that dwarf those of the comparatively few microorganisms amenable to laboratory cultivation (7,13,31). Corroboration comes from estimates of DNA complexity and the discovery of many unique 16S rRNA gene sequences from numerous environmental sources (8,10,28). Collectively, the genomes of the total microbiota found in nature, which we termed the metagenome (11), contain vastly more genetic information than is contained in the culturable subset. Given the profound utility and importance of microorganisms to all biological systems, methods are needed to access the wealth of information within the metagenome.Cloning large fragments of DNA isolated directly from microbes in natural environments provides a method to access soil metagenomic DNA. Previously, we investigated the use of the bacterial artificial chromosome (BAC) vector to express Bacillus cereus genomic DNA (20). The advantage of BAC vectors is that they maintain very large DNA inserts (greater than 100 kb) stably in Escherichia coli (23), facilitating the cloning of large fragments of DNA. Our results demonstrated that expression of heterologous DNA from B. cereus in an E. coli BAC system was detectable at a reasonable frequency (20), validating the idea that the low-copy BAC vector (one to two per cell) (23) could be used to express foreign DNA from foreign promoters in E. coli.Here we describe the construction and initial screening of two BAC libraries made with DNA isolated directly from soil. We found detectable levels of several biochemical activities from BAC library clones. Sequence analysis of selected BAC plasmids encoding such activities and of 16S rRNA genes in one of the libraries confirms the novelty of the genomic information cloned in our libraries. The results show that DNA extracted directly from soil is a valuable source of new genetic information and is accessible by using BAC libraries. Our results demonstrate that both traditional and functional genomics of uncultured microorganisms can be carried out by this approach and that screening of metagenome libraries for activities or gene sequences can provide a basis for conducting genomic analyses of uncultured microorganisms. MATERIALS AND METHODSBacterial strains and plasmids. E. coli strain DH10B and the BAC vector pBeloBAC11 were provided by H. Shizuya (15). Bacillus subtilis strain BR151(pPL608) is strain 1E32 (lys-3 metB10 trpC2) from the Bacillus Genetic Stock Center, Ohio State University. -TnphoA was used as described before (20).

Fundamental issues in systems biology

“…Analyses of metagenomes, or composite genomes of microbial assemblages in particular habitats, (72,73) provide a new way at looking at in situ microbial communities and the relationships between genomes, organisms, populations and environments. (74,75,76,77,78,79,80) Although metagenomics is still at an early stage of constructing massive sequence inventories or gathering functional information, (81,82,83,84) the field could potentially lead to radical reappraisals of the nature of boundaries between biological entities and the organization of life itself. At the very least, it challenges highly individualistic assumptions of biological and molecular interaction.…”

The search for the ideal biocatalyst

“…Modern bioprospecting methods such as multiplex or metagenome cloning [43][44][45][46] and similar methods used by companies such as Diversa Corporation (La Jolla, CA) and TerraGen Discovery (Vancouver, BC, Canada) directly access environmental genomes (whether culturable or unculturable). These methods access sequence space in a virtually random manner and have been successfully employed to isolate novel nahR (ref.…”

“…These methods access sequence space in a virtually random manner and have been successfully employed to isolate novel nahR (ref. 45) and amylase, lipase, and hemolytic enzyme genes 46 .…”

Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules

“…Culture-independent analysis of the genetic and biochemical diversity present in naturally occurring bacterial communities, which began in the 1990s (see refs. [14][15][16][17][18][19]) is now a rapidly growing area of research and has been given the name metagenomics 20 .…”