Functional metagenomics involves screening metagenomic libraries for the expression of a function, such as the appearance of a pigment, enzymatic activity, or for an antibiotic. Because, Dr. Handelsman says, clones in these screens are selected by phenotype (such as expression of an antibiotic activity) and not by homology to some known sequence, the strategy can identify new genes as well as novel activities of known gene families.
To date, scientists have used these technologies to both identify novel antibiotics derived from the environment, and to discover novel resistance genes in unexpected places.
In 2012, scientists at the Rockefeller University demonstrated that culture-independent antibiotic discovery methods “have the potential to provide access to novel metabolites with modes of action that differ from those of antibiotics currently in clinical use.”
Dimitris Kallifidas, Ph.D., Hahk-Soo Kang, Ph.D., and Sean F. Brady, Ph.D., working in the Rockefeller’s Laboratory of Genetically Encoded Small Molecules, reported last year in the Journal of the American Chemical Society the identification of a novel, soil-derived antibiotic. The investigators noted that propagation of DNA extracted directly from environmental samples in lab-grown bacteria provides a means to study natural products encoded in the genomes of uncultured bacteria, but that gene silencing can hamper the functional characterization of gene clusters captured on such environmental DNA clones.
The scientists showed that overexpression of transcription factors found in sequenced environmental DNA-derived biosynthetic gene clusters, along with traditional culture-broth extract screening, could identify novel bioactive secondary metabolites from otherwise-silent gene clusters.
The studies led to the successful isolation of Tetarimycin A, a tetracyclic methicillin-resistant Staphylococcus aureus (MRSA)-active antibiotic, from the culture-broth extract of Streptomyces albus cultures. The bacteria growing in the cultures had been cotransformed with an environmentally derived type-II polyketide biosynthetic gene cluster and its pathway-specific Streptomyces antibiotic regulatory protein (SARP) cloned under the control of the constitutive ermE* promoter.
And although overuse of antibiotics has been widely blamed for the evolution and acquisition of antibiotic-resistance genes, scientists say that little is known about the diversity, distribution, and origins of resistance genes, especially for the “unculturable majority” of environmental bacteria.
Metagenomic tools and phylogenetic analysis have revealed that the environment comprises a reservoir of antibiotic resistance gene determinants (ARGDs) and that the majority of ARGDs acquired by human pathogens may have an environmental origin.
While at the University of Wisconsin, Dr. Handelsman and her colleagues investigated antibiotic-resistance genes among uncultured bacteria in an undisturbed soil environment. Their functional metagenomic analysis of a remote Alaskan soil uncovered a reservoir for beta-lactamases that function in E. coli, including divergent beta-lactamases and the first bifunctional beta-lactamase. These enzymes confer resistance to most beta-lactam antibiotics including penicillins, cephalosporins, and the monobactam aztreonam.
The authors concluded that their findings suggest, even without the selective pressure imposed by anthropogenic activity, that the soil microbial community in an unpolluted site harbors unique and ancient beta-lactam resistance determinants. Moreover, despite their evolutionary distance from previously known genes, the Alaskan beta-lactamases confer resistance on E. coli without manipulating its gene expression machinery.
Ed DeLong, Ph.D., professor in the biological engineering division and the department of civil and environmental engineering at MIT, notes in a 2007 commentary on metabolomics in MIT’s Technology Review, “Like the human genome sequence, the results of metagenomic analysis represent a type of ‘parts list’ that does not fully capture the functional properties, interrelationships, and dynamics of living microbial communities. They do, however,” he says, “begin to extend our analytical reach beyond the single organism. Population genomics, ‘community metabolism,’ and genomic comparisons of different microbial communities are all now possible.” Dr. DeLong’s laboratory focuses on investigating the structure, function, and ecological significance of natural microbial communities in natural settings.
And apart from their potential to discover new antibiotics to treat life-threatening microbial diseases, metagenomic approaches, Dr. DeLong says, enable direct assessment of community diversity and provide data sets relevant to both measuring and modeling biological process.