In a metagenomic library clones with useful traits can be traced by mainly two methods viz. function-based screening and sequence-based screening. The functional screening depends on the faithful expression of the cloned gene in a heterologous host. Since no pre-sequence analysis of the gene of interest is required, there is very high chance to identify entirely new classes of genes for new or known functions. The expression of the gene may confer some selective advantage to the desired clone making its visual identification possible in a pool of clones. For example, antibiotic resistance genes or enzyme producing genes can be selected on specific plates (Fig. 7.2). However, very few clones in a metagenomic library express any given activity. Many thousand clones need to be screened before getting one with desirable trait. In the recent years highly efficient screening and selection systems have been developed for selecting positive clones. Uchiyama et al. (2005) have developed Substrate-induced gene expression screening (SIGEX) for rapid identification of clones showing catabolic gene expression. The technique is based on the fact that catabolite-gene expression requires certain substrates as inducers which, in many cases, is controlled by regulatable elements situated near to catabolic genes. A plasmid-based operon-trap expression vector (p18GFP) containing gene for green fluorescent protein (GFP) immediately after the insert site, is constructed and used for creating metagenomic library in this technique. The clones are exposed to an inducer which easily diffuses to the cyptoplasm of all transformed cells in the library. In positive clones (in which the entire operon responding to that substrate/inducer is cloned), the inducer binds to the cloned regulatory elements and causes expression of downstream sequences for GFP. The GFP positive cells can then be separated by fluorescence activated cell sorter (FACS). Another similar approach is METREX in which product of metagenomic DNA induces bacterial quorum sensing. The metagenomic library is created in host cells which already contain a reporter plasmid. The reporter plasmid contains a reporter gene such as gfp under the control of a regulatory promoter. The promoter can be upregulated in the presence of the product whose gene is present only in the positive clones in the library. The positive clones thus express gfp and can be identified and separated from other clones in the library.

This strategy is based on the prior information of target gene sequence. The classical approach of hybridization with labeled probe can be used to identify the positive clones in the metagenomic library (Fig. 7.3). Complete sequencing of clones that contains phylogenetic anchors indicates the taxonomic group of the source of DNA. Random sequencing can also be done to identify a gene of interest followed by assessment of flanking DNA to contain any phylogenetic anchor. The powerful approach of sequence analysis guided by the identification of phylogenetic markers produced the first genomic sequence linked to a 16S rRNA gene that affiliated with γ–Proteobacteria (Handelsman 2004). Sequencing random clones is an alternative to phylogenetic marker-based approach. The field of metagenomics has been transformed by the application of a whole genome shotgun sequencing approach during the last 6–7 years. Next-generation ultra high-throughput sequencing techniques have revolutionized the field with heavy price cut allowing sequencing at scale larger then anytime before. Sequencing projects have been carried out to assess the actual microbial diversity and their ecological inference in different environment such as sea and acid mine drainage, etc. In the past few years, techniques of DNA microarray has also been widely used for screening clones in metagenomic library.

The technique of PCR has been applied to the isolation and detection of novel genes from community genome. However, the major limitation with PCR is to rely on the flanking DNA sequence information for isolation of an unknown gene from an uncultured organism. Furthermore, when PCR is directed at conserved sequence motifs, only partial genes are recovered. In an attempt to solve this problem, a novel strategy for recovering complete open reading frame from environmental DNA samples was proposed by Stokes in 2001. His team designed PCR assays targeted toward the 59-base element family of recombination sites that flank integrons associated gene cassettes. This approach has resulted in the amplification of diverse gene cassettes containing complete open reading frame from the environmental DNA.

Genomic information of the ‘unculturable’ bacteria can help in understanding their physiology and also their role in ecosystems. Metagenomic investigation has revealed that bacteriorhodopsins that function as light- driven proton pump for energy generation in halophilic archaea are also present in eubacteria. Presence of rhodopsin in marine proteobacteria suggested the possibility of a new phototrophic pathway that may influence the flux of carbon and energy in the ocean’s photozone worldwide. By utilizing shotgun sequencing approach complete genomes of Leptospirillum group II and Ferroplasma type II were assembled from a natural acidophilic biofilm. Pathways of carbon and nitrogen fixation and energy generation for every organism of the simple biofilm community were established. The almost complete assembly of the genome of an uncultured bacterium, Kuenenia stuttgartiensis, has revealed unique metabolic adaptations associated with anaerobic ammonium oxidation. PCR-DGGE (Denaturing gradient gel electrophoresis) is a sophisticated method that enables us to visualize a complete microbial community as a simple banding pattern and also allows to easily monitor community dynamics within the environment. In addition to usual 16S rRNA, various functional genes have also been targeted to investigate the diversity and community structure in this approach. Sargasso Sea genome project carried out by Venter et al. (2004) has resulted in the identification of genes for transport of phosphonates and utilization of polyphosphates and pyrophosphates. The finding indicates the microbial community’s capability to survive in the extremely phosphate-limited environment of Sargasoo Sea.