Probing Identity and Physiology of Uncultured Microorganisms with Isotope Labeling Techniques

Abstract

The global nutrient cycles are mainly driven by microorganisms, which are the ­caretakers of our past, present, and future biosphere. Microorganisms also interact intimately with the geosphere, thereby mediating fluxes of elements from the abiotic to the biotic world. While the functional importance of microorganisms for sustaining life on Earth is generally acknowledged, we are just beginning to understand how physiological activities of specific microbial guilds and taxa contribute to large-scale processes on the ecosystem and the global levels. In the last 2 decades, molecular methods targeting ribosomal RNA (rRNA) or rRNA genes for microbial identification and quantification in environmental samples have been instrumental in deciphering the vast natural diversity of microorganisms. More than 85 new bacterial phyla were discovered with this approach, but for most not a single cultivated representative is available and virtually nothing is known about the metabolic capabilities of their constituents (Achtman and Wagner 2008). It is expected that this gap in knowledge will further increase because the discovery of novel microbial diversity has not yet come to an end (Sogin et al. 2006), fueled by new high-throughput sequencing technologies that generate an unprecedented amount of data such as the GS FLX Titanium sequencer from 454 Life Sciences Corporation. A single run of the GS FLX Titanium sequencer produces about 1,250,000 sequences of 400–450 bp from a complex mixture of bacterial 16S rRNA genes (e.g., recovered by PCR or reverse transcriptase PCR from an environmental DNA or RNA extract, respectively), which is more than have been generated during 20 years of application of the 16S rRNA approach and traditional Sanger sequencing. In addition, metagenomics and genome sequencing of recently isolated microorganisms continually reveal the presence of variants of ­phylogenetic marker genes which are not covered by the “universal” primer sets widely used in phylogenetic studies. In terms of the natural 16S rRNA sequence diversity, one can thus expect a continuous filling of the missing branches in the tree of life. While a comprehensive analysis of microbial richness in environments having high microbial diversity (such as soils, sediments, and the gastrointestinal tracts of humans and animals) now seems within reach, a major conceptual and technical challenge confronting the field of microbiology is to uncover the physiological capabilities and ecological functions of the many microorganisms for whom only the 16S rRNA sequence is presently known. Of great importance for detecting the physiological properties and activity of uncultivated microorganisms are holistic molecular approaches targeting whole natural communities rather than individual community members, such as metagenomics, metatranscriptomics, and environmental proteomics. There are, however, limitations with the aforementioned methods because neither the mere presence of genes coding for specific metabolic functions nor their expression (transcription and translation) in an environmental sample definitively proves the existence of a specific physiological processes (Wagner 2009). Furthermore, the function of many genes and proteins recovered by these methods is still unknown, and therefore the absence of known functional genes involved in a specific metabolic pathway does not prove that a particular metabolic capability is absent, but rather that there may exist another, not yet elucidated, pathway.