Episode notes
Prokaryotes are ubiquitous and form a fundamental component of the biosphere, contributing substantially to global biomass and playing essential roles in carbon, nitrogen, and other biogeochemical cycles. The microbial world is extraordinarily diverse with only a small fraction of the estimated millions to billions of microbial species have been formally described, and an even smaller fraction has been cultured and experimentally characterized. Microorganisms live in intimate association with plants and animals and can profoundly influence their metabolism, development, immunity, and health. The collective genomes of microorganisms associated with a host are therefore sometimes referred to as its “second genome.”
The composition and abundance of microbial communities vary dramatically with their environment. A teaspoon of soil, for example, can contain an enormous diversity of microorganisms, often including thousands of bacterial and archaeal taxa, with highly uneven abundances. The human gut contains a much smaller but still remarkably diverse community, comprising hundreds to more than a thousand bacterial species depending on the individual and the criteria used to define a species. Plant roots are particularly rich microbial habitats because they interact directly with soil and release nutrients that support specialized microbial communities. Marine environments are similarly diverse with even a milliliter of seawater containing hundreds of thousands to millions of microbial cells representing thousands of different taxa.
Before the advent of next-generation sequencing (NGS), microbiology depended heavily on isolating microorganisms and growing them in culture. Individual organisms could then be studied for their biochemical properties, or their genomes could be sequenced. This approach, however, was fundamentally limited by cultivability. Many microorganisms cannot readily be grown under standard laboratory conditions. The first complete bacterial genome to be sequenced was that of Haemophilus influenzae, published in 1995. For many years thereafter, genome sequencing remained largely an organism-by-organism exercise and was constrained by the cost and labor required for Sanger sequencing.
The enormous microbial diversity present in natural environments far exceeds the diversity that can be cultured in the laboratory. This created a major blind spot in traditional microbiology leading to organisms that could not be isolated could not easily be studied. Genome sequencing of cultured bacteria nevertheless revealed extensive diversity in gene content and provided a foundation for assigning functions to conserved genes. Many genes retain significant homology across bacterial species, particularly at the protein level. However, because of the degeneracy of the genetic code, nucleotide sequences can diverge considerably while encoding similar or even identical proteins. Consequently, DNA-level homology can be insufficient for designing universal PCR primers for a gene family.
One solution is to target genomic regions that are sufficiently conserved across diverse microbial groups. Ribosomal RNA (rRNA) genes are particularly valuable because ribosomes are essential for protein synthesis and are present in all cellular organisms. Several regions of rRNA genes are highly conserved, while other regions evolve sufficiently rapidly allowing to distinguish between related organisms. This combination of conserved and variable regions makes rRNA genes excellent molecular markers for microbial identification and community profiling.