Metagenomics: application of genomics to uncultured microorganisms

Abstract

Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na(+)(Li(+))/H(+) antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.

FIG. 1.

Phylogenetic tree of Bacteria showing established phyla (italicized Latin names) and candidate phyla described previously (70, 74, 114) with the November 2003 ARB database (http://arb-home.de) (90) with 16,964 sequences that are >1,000 bp. The vertex angle of each wedge indicates the relative abundance of sequences in each phylum; the length of each side of the wedge indicates the range of branching depth found in that phylum; the redness of each wedge corresponds to the proportion of sequences in that phylum obtained from cultured representatives. Candidate phyla do not contain any cultured members.

FIG. 2.

Phylogenetic stains. Fluorescent in situ hybridization biofilm samples from Iron Mountain Mine, Calif. Nucleic acid probes were labeled with indodicarbocyanine, and DNA was stained nonspecifically with 4′,6′-diamidino-2-phenylindole. The nucleic acid probes are specific for (top left) Sulfobacillus spp., (top right) Archaea, (bottom left) Archaea on fungal filaments, and (bottom right) Eukarya. Reproduced from reference with permission of the publisher.

FIG. 3.

Construction and screening of metagenomic libraries. Schematic representation of construction of libraries from environmental samples. The images at the top from left to right show bacterial mats at Yellowstone, soil from a boreal forest in Alaska, cabbage white butterfly larvae, and a tube worm.

FIG. 4.

Antibiotics discovered in metagenomic libraries.

FIG. 5.

Intracellular screen for quorum-sensing inducers. The biosensor, which detects molecules that induce luxR-regulated genes, resides inside the same cell as the metagenomic clone. An active clone fluoresces due to accumulation of green fluorescent protein and can be detected by fluorescence microscopy or captured by fluorescence-activated cell sorting.

FIG. 6.

Metagenomics-based model of biogeochemical cycles mediated by prokaryotes in acid mine drainage. Cell metabolic cartoons constructed from the annotation of 2,180 open reading frames identified in the Leptospirillum group II genome and 1,931 open reading frames in the Ferroplasma type II genome. The cells are shown within a biofilm that is attached to the surface of an acid mine drainage stream. Reproduced from reference with permission of the publisher.