Capturing the genetic makeup of the active microbiome in situ

Abstract

More than any other technology, nucleic acid sequencing has enabled microbial ecology studies to be complemented with the data volumes necessary to capture the extent of microbial diversity and dynamics in a wide range of environments. In order to truly understand and predict environmental processes, however, the distinction between active, inactive and dead microbial cells is critical. Also, experimental designs need to be sensitive toward varying population complexity and activity, and temporal as well as spatial scales of process rates. There are a number of approaches, including single-cell techniques, which were designed to study in situ microbial activity and that have been successively coupled to nucleic acid sequencing. The exciting new discoveries regarding in situ microbial activity provide evidence that future microbial ecology studies will indispensably rely on techniques that specifically capture members of the microbiome active in the environment. Herein, we review those currently used activity-based approaches that can be directly linked to shotgun nucleic acid sequencing, evaluate their relevance to ecology studies, and discuss future directions.

Figure 1

Methods that yield activity-labeled samples and are targeting cell processes in an ‘active’ microbial cell that can be coupled with shotgun sequencing. Colors denote resources (green), cell components (blue) and cell processes (orange). Raman: Raman microspectroscopy. For DNA- and RNA-SIP, total nucleic acids are extracted from the samples, and labeled and unlabeled DNA/RNA is separated by density gradient centrifugation. The ‘heavier’ labeled nucleic acid fractions can be used for construction of metagenomic libraries (Neufeld et al., 2007; Whiteley et al., 2007), whereas PLFAs are analyzed on a mass spectrometer and cannot be combined with nucleic acid sequencing (Jehmlich et al., 2010). Many ¹³C-, ¹⁸O-, ¹⁵N-labeled fine chemicals are available (for example, phenol, methanol, ammonia, methane, carbonate, etc.), but the wide-ranging application of SIP is limited by the commercial availability of complex labeled compounds that require expensive custom synthesis. Furthermore, sensitivity of the SIP technique is a function of substrate concentration and the duration of substrate incorporation. Successful SIP is dependent on optimization of substrate concentration to guarantee a significant signal-to-noise ratio and incubation length and avoid enrichment bias (Neufeld et al., 2006) (Table 1). ‘Cross-feeding’, that is, the flow of the isotope label from primary metabolizers to secondary consumers has also been documented (Hutchens et al., 2004; Dumont et al., 2006). RSG is a fluorogenic redox indicator dye available from Molecular Probes, Invitrogen (Carlsbad, CA, USA). RSG yields green fluorescence (488 nm excitation) when modified by bacterial reductases, many of which are parts of electron transport systems. SIP-Raman microspectroscopy has been performed using ¹³C-, ¹⁵N-labeled compounds, as well as with D₂O. The addition of D₂O (up to a certain concentration and for limited time) is expected to have negligible effects on the microbial community composition and activity patterns, for example, compared with nutrient substrates (Lester et al., 1960; Berry et al., 2015; Kopf et al., 2015) that are traditionally used for SIP experiments. Incorporation of D₂O-derived deuterium into the biomass of autotrophic and heterotrophic bacteria and archaea can be unambiguously detected via C-D signature peaks in single-cell Raman spectra (Ashkin, 1970; Berry et al., 2015). However, for comparative studies between active taxa it should be kept in mind that microbes with different physiologies will incorporate different amounts of deuterium at similar growth rates.

Figure 2

Project statistics by method over the last decade. Counts displayed exclusively include projects using high-throughput sequencing. Metagenome (MetaG) and metatranscriptome (MetaT) projects are depicted by lines (primary y axis), whereas BrdU, DNA-SIP, RNA-SIP and PTR projects are displayed as bars (secondary y axis). Project abundances are cumulative. Number of MetaG and MetaT projects include public sequencing projects as recorded in the Genomes OnLine Database (GOLD) (Pagani et al., 2011) retrieved 15 January 2016. Number of all other activity-based projects include published records that feature high-throughput (next-generation) shotgun sequence data.

Figure 3

High-throughput workflows of current and emerging in situ microbial activity approaches linked to sequencing. Metatranscriptomics and stable isotope labeling are the most commonly used techniques coupled with next-generation shotgun sequencing technology. Emerging methods that are currently still subject to development and/or optimization involve the incubation of cells and cell clusters with, for example, fluorescent compounds or D₂O before selective sorting of active cells using FACS or Raman OT.