Metatranscriptomic analysis of autonomously collected and preserved marine bacterioplankton

Abstract

Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically undersampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at room temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.

Local genomic adaptation of coral reef-associated microbiomes to gradients of natural variability and anthropogenic stressors

“…The TonB system transports large molecules (e.g., polysaccharides, proteins, and siderophores) in through the outer membrane of Gram-negative bacteria. Its importance in marine environments is evidenced by the presence of these genes in marine bacterial genomes and pelagic metagenomes (29-31), their high levels of expression in metatranscriptome data (32), and the proteomic identification of their products as the predominant membrane proteins in pelagic bacteria (33). In this study, they accounted for nearly 1% of gene function annotations at some high-nutrient sites (Fig.…”

Single-taxon field measurements of bacterial gene regulation controlling DMSP fate

“…Several previous metagenomic and metatranscriptomic studies had indicated that bacterial populations with high genetic relatedness to HTCC2255 (X95% average amino acid identity) were consistently present at this site (DeLong, 2005;Ottesen et al, 2011;Rich et al, 2011). We therefore retrieved DMSP gene sequences from the available Monterey Bay metagenomic data sets (Rich et al, 2011) and designed species-specific PCR assays that could be employed on the ESP to characterize gene dynamics over time.…”

“…In the coastal upwelling system of Monterey Bay, CA, previous metagenomic and metatranscriptomic surveys indicated consistent and abundant populations of a bacterial taxon with high genetic relatedness to Roseobacter strain HTCC2255 (Ottesen et al, 2011), a bacterium capable of both DMSP cleavage and demethylation (Newton et al, 2010). We took advantage of the predictable presence of this bacterium to design HTC2255-specific PCR primers for in situ observations for the first committed step in each of the two DMSP degradation pathways.…”

Trimethylamine N -oxide metabolism by abundant marine heterotrophic bacteria

“…in situ (15,17,(44)(45)(46). For example, analysis of metatranscriptomic data of bacterioplankton from the Monterey Bay of California showed that the TMAO transporter is one of the most highly expressed transporters in the MRC representative, Rhodobacterales sp.…”

“…ATP-binding cassette (ABC) transporters form one of the largest gene superfamilies found within many bacterial genomes (14), and their expression is frequently detected in the marine environment (15)(16)(17). ABC transporters are essential for bacteria because they are responsible for the uptake of a wide range of compounds, such as sugars, amino acids, metals, and vitamins, at the expense of ATP (18).…”