Adam R. Rivers

Sizing up metatranscriptomics

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.

Transcriptional response of bathypelagic marine bacterioplankton to the Deepwater Horizon oil spill.

The Deepwater Horizon blowout released a massive amount of oil and gas into the deep ocean between April and July 2010, stimulating microbial blooms of petroleum-degrading bacteria. To understand the metabolic response of marine microorganisms, we sequenced ∼66 million community transcripts that revealed the identity of metabolically active microbes and their roles in petroleum consumption. Reads were assigned to reference genes from ∼2700 bacterial and archaeal taxa, but most assignments (39%) were to just six genomes representing predominantly methane- and petroleum-degrading Gammaproteobacteria. Specific pathways for the degradation of alkanes, aromatic compounds and methane emerged from the metatranscriptomes, with some transcripts assigned to methane monooxygenases representing highly divergent homologs that may degrade either methane or short alkanes. The microbial community in the plume was less taxonomically and functionally diverse than the unexposed community below the plume; this was due primarily to decreased species evenness resulting from Gammaproteobacteria blooms. Surprisingly, a number of taxa (related to SAR11, Nitrosopumilus and Bacteroides, among others) contributed equal numbers of transcripts per liter in both the unexposed and plume samples, suggesting that some groups were unaffected by the petroleum inputs and blooms of degrader taxa, and may be important for re-establishing the pre-spill microbial community structure.

Minimum information about a single amplified genome (MISAG) and a metagenome-assembled genome (MIMAG) of bacteria and archaea

We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.

Iron stress genes in marine Synechococcus and the development of a flow cytometric iron stress assay

Marine Synechococcus are frequently found in environments where iron (Fe) is a limiting nutrient. To understand their capacity to respond to Fe stress, we screened picoplankton genomes and the Global Ocean Survey metagenome for known Fe stress genes. Many open ocean strains of Synechococcus lack most known genes for Fe stress, while coastal and upwelling strains contain many, suggesting that maintaining multiple Fe limitation compensation strategies is not a selective advantage in the open ocean. All genomes contained iron deficiency-induced protein A (IdiA) and its complementary Fe(3+) transport proteins. The ubiquity of IdiA was exploited to develop an in situ Fe stress bioassay based on immunolabelling and flow cytometry. As a test of field applicability, we used the assay on natural Synechococcus populations from one station in the Costa Rica Upwelling Dome where total Fe ranged from <0.08 to 0.14 nM in the upper water column. The bioassay found Fe stress in 5-54% of the population. Based on our findings, we believe that when reactive strains are present this assay can reveal environmental and clade-specific differences in the response of Synechococcus to Fe stress.

IMG/VR: A database of cultured and uncultured DNA viruses and retroviruses

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from >6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs are grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparing with external sequences, thus serving as an essential resource in the viral genomics community.

The transcriptional response of prokaryotes to phytoplankton-derived dissolved organic matter in seawater.

To better understand the functional responses in prokaryotes to dissolved organic matter (DOM), we compared the transcriptional pattern of natural prokaryotic communities grown in continuous cultures on seawater amended with phytoplankton-derived DOM. Metatranscriptomic reads were classified taxonomically (by genomic binning) and functionally (using Kyoto Encyclopedia of Genes and Genomes), and the relative gene expression of individual taxa (genome bins) was compared with the total community response. In the first experiment comparing seawater and seawater amended with diatom-derived DOM, metatranscriptomes revealed pronounced differences in pathways involved in carbohydrate and lipid metabolism. In the second experiment comparing seawater amended with cyanobacteria- and diatom-derived DOM, metatranscriptomes had similar functional profiles, likely reflecting more similar DOM regimes in this experimental setup. Among the five most abundant taxa investigated in more detail, two featured pronounced differences in transcript abundance between treatments suggesting that they were specialized in the use of only one of the two DOM regimes. However, these two taxa were less involved in carbohydrate metabolism than others and had few genes that were significantly regulated in response to the DOM source. Our results indicate that both substrate composition and the competitive interplay of community members were decisive for the functional response of a microbial system.

An Updated genome annotation for the model marine bacterium Ruegeria pomeroyi DSS-3

When the genome of Ruegeria pomeroyi DSS-3 was published in 2004, it represented the first sequence from a heterotrophic marine bacterium. Over the last ten years, the strain has become a valuable model for understanding the cycling of sulfur and carbon in the ocean. To ensure that this genome remains useful, we have updated 69 genes to incorporate functional annotations based on new experimental data, and improved the identification of 120 protein-coding regions based on proteomic and transcriptomic data. We review the progress made in understanding the biology of R. pomeroyi DSS-3 and list the changes made to the genome.

Experimental Identification of Small Non-Coding RNAs in the Model Marine Bacterium Ruegeria pomeroyi DSS-3

In oligotrophic ocean waters where bacteria are often subjected to chronic nutrient limitation, community transcriptome sequencing has pointed to the presence of highly abundant small RNAs (sRNAs). The role of sRNAs in regulating response to nutrient stress was investigated in a model heterotrophic marine bacterium Ruegeria pomeroyi grown in continuous culture under carbon (C) and nitrogen (N) limitation. RNAseq analysis identified 99 putative sRNAs. Sixty-nine were cis-encoded and located antisense to a presumed target gene. Thirty were trans-encoded and initial target prediction was performed computationally. The most prevalent functional roles of genes anti-sense to the cis-sRNAs were transport, cell-cell interactions, signal transduction, and transcriptional regulation. Most sRNAs were transcribed equally under both C and N limitation, and may be involved in a general stress response. However, 14 were regulated differentially between the C and N treatments and may respond to specific nutrient limitations. A network analysis of the predicted target genes of the R. pomeroyi cis-sRNAs indicated that they average fewer connections than typical protein-encoding genes, and appear to be more important in peripheral or niche-defining functions encoded in the pan genome.

Phenotypic plasticity in heterotrophic marine microbial communities in continuous cultures

Phenotypic plasticity (PP) is the development of alternate phenotypes of a given taxon as an adaptation to environmental conditions. Methodological limitations have restricted the quantification of PP to the measurement of a few traits in single organisms. We used metatranscriptomic libraries to overcome these challenges and estimate PP using the expressed genes of multiple heterotrophic organisms as a proxy for traits in a microbial community. The metatranscriptomes captured the expression response of natural marine bacterial communities grown on differing carbon resource regimes in continuous cultures. We found that taxa with different magnitudes of PP coexisted in the same cultures, and that members of the order Rhodobacterales had the highest levels of PP. In agreement with previous studies, our results suggest that continuous culturing may have specifically selected for taxa featuring a rather high range of PP. On average, PP and abundance changes within a taxon contributed equally to the organism’s change in functional gene abundance, implying that both PP and abundance mediated observed differences in community function. However, not all functional changes due to PP were directly reflected in the bulk community functional response: gene expression changes in individual taxa due to PP were partly masked by counterbalanced expression of the same gene in other taxa. This observation demonstrates that PP had a stabilizing effect on a community’s functional response to environmental change.

Spatial Homogeneity of Bacterial Communities Associated with the Surface Mucus Layer of the Reef-Building Coral Acropora palmata

Coral surface mucus layer (SML) microbiota are critical components of the coral holobiont and play important roles in nutrient cycling and defense against pathogens. We sequenced 16S rRNA amplicons to examine the structure of the SML microbiome within and between colonies of the threatened Caribbean reef-building coral Acropora palmata in the Florida Keys. Samples were taken from three spatially distinct colony regions—uppermost (high irradiance), underside (low irradiance), and the colony base—representing microhabitats that vary in irradiance and water flow. Phylogenetic diversity (PD) values of coral SML bacteria communities were greater than surrounding seawater and lower than adjacent sediment. Bacterial diversity and community composition was consistent among the three microhabitats. Cyanobacteria, Bacteroidetes, Alphaproteobacteria, and Proteobacteria, respectively were the most abundant phyla represented in the samples. This is the first time spatial variability of the surface mucus layer of A. palmata has been studied. Homogeneity in the microbiome of A. palmata contrasts with SML heterogeneity found in other Caribbean corals. These findings suggest that, during non-stressful conditions, host regulation of SML microbiota may override diverse physiochemical influences induced by the topographical complexity of A. palmata. Documenting the spatial distribution of SML microbes is essential to understanding the functional roles these microorganisms play in coral health and adaptability to environmental perturbations.