Dead or alive: molecular assessment of microbial viability

Abstract

Nucleic acid-based analytical methods, ranging from species-targeted PCRs to metagenomics, have greatly expanded our understanding of microbiological diversity in natural samples. However, these methods provide only limited information on the activities and physiological states of microorganisms in samples. Even the most fundamental physiological state, viability, cannot be assessed cross-sectionally by standard DNA-targeted methods such as PCR. New PCR-based strategies, collectively called molecular viability analyses, have been developed that differentiate nucleic acids associated with viable cells from those associated with inactivated cells. In order to maximize the utility of these methods and to correctly interpret results, it is necessary to consider the physiological diversity of life and death in the microbial world. This article reviews molecular viability analysis in that context and discusses future opportunities for these strategies in genetic, metagenomic, and single-cell microbiology.

FIG 1

rRNA synthesis and maturation in bacteria. RNA polymerase (RNAP), reading from right to left in this diagram, produces a long transcript (30S rRNA) containing 3 rRNA subunits interspersed with external transcribed spacers (ETS) and internal transcribed spacers (ITS). This transcript is rapidly converted by RNase activity and other endonucleolytic activities to pre-RNA subunits with leader and tail sequences. The leaders and tails are trimmed in exonucleolytic processes closely tied to ribosome assembly and the initiation of protein synthesis. RT-qPCRs can be designed to target the pre-rRNA exclusively, or to straddle a pre-rRNA-mature rRNA junction as shown here, such that intact pre-rRNA is needed for successful amplification. The 5′ leader region (ETS1) is especially useful for MVT because of its species specificity and relative abundance when transcription is active; however, other pre-rRNA sequences (e.g., ITS1) can also be targeted by RT-qPCR primers.