Sizing up metatranscriptomics

Abstract

A typical marine bacterial cell in coastal seawater contains only ∼200 molecules of mRNA, each of which lasts only a few minutes before being degraded. Such a surprisingly small and dynamic cellular mRNA reservoir has important implications for understanding the bacterium's responses to environmental signals, as well as for our ability to measure those responses. In this perspective, we review the available data on transcript dynamics in environmental bacteria, and then consider the consequences of a small and transient mRNA inventory for functional metagenomic studies of microbial communities.

Figure 1

The use of internal standards (artificial mRNAs produced by in vitro transcription of vector templates) in metatranscriptomic studies allows calculation of average per-cell mRNA inventories. (a) A known number of internal standards are spiked into a microbial sample. In this example, 917 and 971 nt standards were added to a filter in an extraction tube containing lysis buffer just before initiating RNA extraction (see Gifford et al., 2011 for complete protocol). The ratio of standards added:standards recovered in the high-throughput sequence library allows estimation of the numbers of natural mRNAs in the sampled community. (b) Recovery ratio of internal standards in Illumina libraries from free-living (FL) and particle-associated (PA) metatranscriptomes from two locations in the Amazon River plume in May 2010. Standards were produced by reverse transcription from the T7 promoter (green arrowhead) of two linearized commercial cloning vectors (Promega, Fitchburg, WI, USA; New England Biolabs, Ipswich, MA, USA). (c) Based on internal standard recovery in the mRNA library, the average number of transcripts per SYBR green-stained bacterial cell was calculated for the free-living (0.2 to <2 μm size range; purple cells) and particle-associated (>2 μm size range; orange cells) size fractions at two stations in the Amazon River plume. The total abundance of prokaryotic transcripts was 2.3 × 10¹¹ l⁻¹ at Station 27 and 8.5 × 10¹¹ l⁻¹ at Station 10. The background color is modified from a MODIS Aqua image of chlorophyll a concentrations.

Figure 2

Bacterioplankton macromolecule inventories in a milliliter of typical coastal seawater. Bacterial mRNAs are an order of magnitude less abundant than genes, and almost four orders of magnitude less abundant than proteins.

Figure 3

Simulation model of levels of mRNA (green lines) and protein (blue lines) of the same gene in a single cell (a, c) or averaged for a population of 100 cells (b, d) during a 24-h period. In the steady-state version of the model (a, b), which could represent either constitutive gene expression or an unchanging extrinsic regulatory signal, each cell experiences up to 10 randomly timed transcription events per day and produces a single mRNA molecule at each event (Supplementary Materials). In the dynamic version (c, d), extrinsic signaling upregulates gene transcription for a 4-h period (500–740 min; blue shading) through an increase in transcriptional burst size to three mRNA molecules per transcription event. Both model types were initialized with 900 protein molecules per cell, and both assume 7 proteins are translated from each mRNA template, that the half-life of mRNA is 1.5 min, and that the half-life of protein is 12 h. Varying the parameter values (for example, frequency of transcription events, mRNA burst size, proteins translated per message, macromolecule half-lives) changes the size of the final mRNA and protein pools, but they remain poorly synchronized under dynamic extrinsic conditions.