Metagenomics: Studying unculturable microbes

Microorganisms or microbes are often depicted in television commercials as invisible things around us that can cause sickness and therefore must be eliminated by anti-microbial products and disinfectants. But the truth is, although many microbes cause disease, many more are beneficial. Microorganisms perform important functions such as aiding the digestion of food, production of medicines (e.g. antibiotics), converting soil nutrients into forms that plants can use, providing oxygen (photosynthetic microbes are responsible for about half of the photosynthesis on earth) and production of food products (e.g. cheese, beer). Microorganisms have millions of species and are present in almost every environment on earth. However, of all the microbes on earth, only about less than one percent can be cultured or “grown” in the laboratory to be studied. It was only until almost a decade ago, through the introduction of metagenomics, that it has been possible to study and analyze the more than 99 percent that remain untapped in the environment.

Transcending the culturable few

All cells that make up organisms contain deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules, which contain the instructions needed for all structures and functions of an organism. The collection of DNA and RNA of an organism is called a genome. Metagenomics is the culture-independent analysis of a mixture of microbial genomes collected directly from environmental samples, using an approach based either on expression or on sequencing of the DNA or RNA. Metagenomics is from the Greek word “meta” meaning transcendent and “genomics” which is the study of the entire DNA composition of an organism. A relatively new and rapidly developing field of study in molecular biology, metagenomics goes beyond genomics of an individual organism to the “meta” level or genomics of a microbial community. The idea of studying genetic material directly from environmental samples was proposed in 1985 by Dr. Norman R. Pace and colleagues at the Department of Biology, Indiana University after studying the diversity of microbial ribosomal DNA sequences. Later in 1991, they published the first report on isolating and cloning bulk DNA from an environmental sample and in 1998, the term “metagenomics” was first used by Dr. Jo Handelsman and colleagues at the Department of Plant Pathology, University of Wisconsin.

Analyzing the unculturable majority

Metagenomics is the systematic investigation, classification and handling of genetic material isolated directly from the environment, and employs four main steps: (1) isolation of genetic material, (2) processing of the isolated genetic material, (3) construction of the metagenomic library, and (4) analysis of genetic material in the constructed genomic library.

The first step is the isolation of DNA from the sample by cell lysis using either physical or chemical methods. Following this is the processing of the isolated DNA. The collected DNA is cut, at specific sites, into shorter fragments using enzymes called restriction endonucleases. The resulting short, linear DNA fragments are then combined with small units of DNA called vectors that are inserted into cells of host organisms such as the bacterium Escherichia coli. This allows the DNA from the microorganisms in the sample to be multiplied, expressed and studied. The collection of host cell samples containing all of the metagenomic DNA vectors is a metagenomic library while the collection of genetic material from an environmental sample is called the metagenome. The final step is the analysis of the DNA from the metagenomic libraries. This is done either by analyzing the sequences of the genes present in the library or by screening the genes for various functions such as antibiotic production.

Thus, without growing the bacteria, this method allows the study of the diversity of bacteria in many environments and determines which bacteria dominate. Scientists use a type of DNA called the 16S rDNA sequences because they are highly conserved in structure and function among members of the same species of bacteria, differ from distantly related genera and exhibit regions with different degrees of relatedness.

Some of the environments whose metagenomes have been studied are deep sea vents, acid mine drainages, various types of soils, glaciers, hot springs, and even the human gut.

Applications of metagenomics

The development of metagenomics offers numerous possible applications extending through various fields of study and disciplines, including human health, agriculture, environmental science, biotechnology, and forensics. It has led to the discovery of some novel antibiotics and natural products such as turbomycins, terragines and indirubin. Furthermore, important genes and enzymes have been identified through metagenomic analyses.

One big project that is currently being conducted is the Human Microbiome Project initiated by the National Institutes of Health in the US in December 2007. This project aims to study the microbial populations or microflora in different parts of the human body such as the gastrointestinal tract, oral cavity, and skin using metagenomics. The importance of the microflora in human gut lies in their ability to help in the digestion process, stimulate the immune system, produce vitamins and short chain fatty acids, and prevent colonization of potential pathogenic bacteria by competing with them. Knowledge of the gut microflora profile can help in elucidating and understanding gut-associated diseases and in designing better treatment for such diseases. The microbial profile obtained through metagenomics is called microbiome.

Research on a common skin disorder in the inner elbow called atopic dermatitis or eczema is also ongoing. Dr. Julie Segre and her colleagues at the US National Human Genome Research Institute are using metagenomics to understand and later develop possible treatments for this skin disorder as well as other skin conditions such as acne. 

In the Philippines, several metagenomics studies are being conducted. Dr. Nacita B. Lantican and Dr. Asuncion K. Raymundo of the University of the Philippines Los Baños (UPLB) have conducted a study of the microbial community of hot spring water samples from the mudsprings of Mt. Makiling, Laguna. Analyses of the DNA samples revealed that most of the microbial community detected belonged to the Solfolubus group of organisms and one clone has been concluded to possibly be from a novel species of Mt. Makiling mudspring. There are also ongoing efforts to study the rumen microbiome of the native carabao at the Animal and Dairy Science Cluster of the College of Agriculture of UPLB funded by the UP Emerging S&T Program and is under the leadership of its director, Prof. Cesar Sevilla. Work on the Filipino gut microbiome is also being initiated by UP Manila faculty members, Asst. Prof. Leslie Dalmacio and Prof. Raul Destura.

Metagenomics represents a powerful tool to access the rich biodiversity of the majority of microorganisms in the environment which cannot be grown in the laboratory. It is a young and exciting field of molecular biology and with improved techniques/technologies, funding, and more scientists to embark on solving problems related to this field, it will develop and continue to deepen our understanding of the microbial population.

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Laarni Grace Corales and Carlo Miguel Sandoval are MS students in Molecular Biology and Biotechnology (MBB) at the University of the Philippines Los Baños. This science article was written for their MBB 290 Special Topics course under Prof. Evelyn Mae Tecson-Mendoza. E-mail at [email protected] or [email protected].