Of the vast array of prokaryotes on earth, only few (less than 1%) can be isolated on artificial laboratory media. This has greatly hampered our knowledge about such prokaryotes. These uncultivable species are important because they may yield many novel enzymes and catalysts with industrial applications such as antibiotics. The inability to grow and isolate these microbes has led to the emergence of a new technique viz. Metagenomics i.e. the culture-independent analysis of a mixture of microbial genomes. It differs from conventional methods as it deals with analysis of microbial DNA extracted directly from environmental samples. Due to the fact that 99% of prokaryotes are uncultivable, the possibility of discovery of novel enzymes is enormous and invaluable contributions to evolutionary biology, medicine and agricultural science can be expected from this newly developed technique in forthcoming years.
The total number of prokaryotes on earth has been estimated at 4–6×1030 (Whitman et al. 1998). The first step in investigating any bacterial species comprises of obtaining its pure culture. However, recent estimates indicate that less than 1% of the prokaryotes in most environments can be isolated (Amann et al. 1990). Until recently, this limitation meant that the genomes of most microbial life could not be dissected because these contain no cultured representatives. It is valuable to find the species that cannot grow in culture because they depend on other organisms for critical processes. The need to develop a method to analyze genomes under various conditions outside of the laboratory setting has led to the emergence of a new field—metagenomics. Soil microbial communities contain the highest level of prokaryotic diversity of any environment, and metagenomic approaches involving the extraction of DNA from soil can improve our access to these communities. Most analyses of soil biodiversity and function assume that the DNA extracted represents the microbial community in the soil, but subsequent interpretations are limited by the DNA recovered from the soil. Unfortunately, extraction methods do not provide a uniform and unbiased subsample of metagenomic DNA, and as a consequence, accurate species distributions cannot be determined.
What is Metagenomics?
Metagenomics is the culture-independent analysis of a mixture of microbial genomes (termed the metagenome). The term is derived from the statistical concept of meta-analysis (the process of statistically combining separate analyses) and genomics (the comprehensive analysis of an organism’s genetic material) (Rondon et al. 2000). To be precise, metagenomics is the sequencing and analysis of DNA of microorganisms recovered from an environment, without the need for culturing them. The term “metagenomics” was first used by Jo Handelsman, Jon Clardy, Robert M. Goodman, and others, and first appeared in publication in 1998 (Handelsman et al. 1998). It has been commonly referred to as “community genomics” or “environmental genomics” and is different from conventional techniques in two respects. First, metagenomics is based on the genomic analysis of microbial DNA extracted directly from environmental samples, and not samples cultured in the laboratory. Second, unlike traditional methods of genetic manipulation and genome analysis, metagenomics focuses on the use of DNA sequencing to predict features of organisms and to understand the genetic basis of certain traits (Handelsman, 2005).
Torsvik’s report in 1980 was the first report on extraction and digestion of genomic DNA from bacterial samples prepared from soil (Torsvik and Goksoyr 1980) and provided proof for the concept of generating gene libraries directly from environmental DNA (Pace et al. 1986). Only in the next decade the first such metagenome libraries were reported.
Metagenomics has revealed several interesting discoveries. The most extensive metagenomic sequencing effort to sequence the prokaryotic genomes in the water of the Sargasso Sea, a well characterized region of the Atlantic near Bermuda that has unusually low nutrient levels has been made only recently (Venter et al. 2004). The study uncovered a new class of rhodopsin genes in alpha-proteobacteria. Rhodopsins are proteins that respond to light and serve a range of purposes in a wide variety of organisms, including the detection of light in the retinal cells of humans and other animals. Further studies of the newly discovered bacterial rhodopsins found that the light response of the proteins was tuned to match that of the light that was reaching the alpha-proteobacteria at different ocean depths.
Approaches to Metagenomic Analysis
Traditional genomics focuses on the sequencing and analysis of the genomes of individual organisms. When applied to microbes, it typically involves culturing the organism of interest followed by sequencing. Metagenomics is a new area of microbial genomics that aims to sequence the full or partial genomes of all members of a microbial community (also called a consortium). Since only a very small minority of single-cell organisms has been successfully cultured in the laboratory, metagenomics becomes a very powerful technique for sequencing genes from organisms that cannot be cultured.
The first step in metagenomic analysis usually involves the isolation of DNA from an environmental sample. Different types of samples often require specialized extraction techniques; however, once the DNA is isolated, it is cloned, entered into some vector (bacterial artificial chromosome, plasmid, cosmid, etc.), and then inserted into an appropriate bacterial host. This is followed by extraction of information from metagenome libraries. Two approaches, the function-driven analysis and the sequence-driven analysis, have emerged to extract biological information from metagenomic libraries.
The function-driven analysis is initiated by identification of clones that express a desired trait, followed by characterization of the active clones by sequence and biochemical analysis. This approach quickly identifies clones that have potential applications in medicine, agriculture or industry by focusing on natural products or proteins that have useful activities. The limitations of the approach are that it requires expression of the function of interest in the host cell and clustering of all of the genes required for the function.
Sequence-driven analysis relies on the use of conserved DNA sequences to design hybridization probes or PCR primers to screen metagenomic libraries for clones that contain sequences of interest. Significant discoveries have resulted from random sequencing of metagenomic clones. Sequencing of clones carrying phylogenetic anchors, such as the 16S rRNA gene and the Archaeal DNA repair gene radA (Beja et al. 2000, Suzuki et al. 2001), has led to functional information about the organisms from which these clones were derived.
Future applications and Limitations
Most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches promise the accessibility of the genetic resources and their potential applications. Genetic resources from terrestrial environments can be accessed by exploring the soil metagenome. Soil metagenomic analyses are usually initiated by the isolation of environmental DNAs. Several methods have been described for the direct isolation of environmental DNAs from soil and sediments. Application of metagenomics largely depends on the construction of genomic DNA libraries and subsequent high-throughput sequencing or library screening. Thus, obtaining large quantities of pure cloneable DNA from the environment is a prerequisite. The practical applications of metagenomics are vast. The screening of genes from thousands of microbial species will undoubtedly yield many novel enzymes and catalysts with industrial applications e.g. cellulases, chitinases, lipases, antibiotics, other natural products. The possibility of finding novel enzymes in metagenomics screens is high when one considers that the samples of ocean water from the Sargasso Sea yielded over one million new open reading frames. Moreover, the diversity patterns of microorganisms obtained by metagenomics can be used for monitoring and predicting environmental conditions and their change. Metagenomics will also aid industry by circumventing the need to culture microbial organisms. In addition, the technique of metagenomics can reveal new insights about the metabolic pathways of organisms and this information can be utilized for designing culture media for the growth of previously uncultured microbes.
Despite the great strides made by metagenomics researchers, significant technical hurdles remain. The complexity of sample from environments often makes purification of DNA challenging. Sample contamination can also be a problem. Problems can also arise because sequence similarities in distinct species can lead to errors in assembly. Perhaps the greatest challenge of metagenomics is attempting to sequence the genomes of underrepresented species (i.e. comprising less than 1% of the microbial community). Such cases require the sequencing of gigabases of DNA for adequate coverage. Presently, this is outside the reach of sequencing technology.
Metagenomics is a young and exciting technique that has broad application in biology and biotechnology. Although many advances in gene expression, library construction, vector design, and screening will improve it, the current technology is sufficiently powerful to yield products like new antibiotics and enzymes. In the next few years, invaluable contributions to evolutionary biology, medicine and agricultural science can be expected from this newly emerging field.
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