Shellie R. Bench

Globally Distributed Uncultivated Oceanic N2-Fixing Cyanobacteria Lack Oxygenic Photosystem II

Biological nitrogen (N2) fixation is important in controlling biological productivity and carbon flux in the oceans. Unicellular N2-fixing cyanobacteria have only recently been discovered and are widely distributed in tropical and subtropical seas. Metagenomic analysis of flow cytometry–sorted cells shows that unicellular N2-fixing cyanobacteria in “group A” (UCYN-A) lack genes for the oxygen-evolving photosystem II and for carbon fixation, which has implications for oceanic carbon and nitrogen cycling and raises questions regarding the evolution of photosynthesis and N2 fixation on Earth.

Metabolic streamlining in an open-ocean nitrogen-fixing cyanobacterium

Nitrogen (N2)-fixing marine cyanobacteria are an important source of fixed inorganic nitrogen that supports oceanic primary productivity and carbon dioxide removal from the atmosphere. A globally distributed, periodically abundant N2-fixing marine cyanobacterium, UCYN-A, was recently found to lack the oxygen-producing photosystem II complex of the photosynthetic apparatus, indicating a novel metabolism, but remains uncultivated. Here we show, from metabolic reconstructions inferred from the assembly of the complete UCYN-A genome using massively parallel pyrosequencing of paired-end reads, that UCYN-A has a photofermentative metabolism and is dependent on other organisms for essential compounds. We found that UCYN-A lacks a number of major metabolic pathways including the tricarboxylic acid cycle, but retains sufficient electron transport capacity to generate energy and reducing power from light. Unexpectedly, UCYN-A has a reduced genome (1.44 megabases) that is structurally similar to many chloroplasts and some bacteria, in that it contains inverted repeats of ribosomal RNA operons. The lack of biosynthetic pathways for several amino acids and purines suggests that this organism depends on other organisms, either in close association or in symbiosis, for critical nutrients. However, size fractionation experiments using natural populations have so far not provided evidence of a symbiotic association with another microorganism. The UCYN-A cyanobacterium is a paradox in evolution and adaptation to the marine environment, and is an example of the tight metabolic coupling between microorganisms in oligotrophic oceanic microbial communities.

Metagenomic Characterization of Chesapeake Bay Virioplankton

ABSTRACT Viruses are ubiquitous and abundant throughout the biosphere. In marine systems, virus-mediated processes can have significant impacts on microbial diversity and on global biogeocehmical cycling. However, viral genetic diversity remains poorly characterized. To address this shortcoming, a metagenomic library was constructed from Chesapeake Bay virioplankton. The resulting sequences constitute the largest collection of long-read double-stranded DNA (dsDNA) viral metagenome data reported to date. BLAST homology comparisons showed that Chesapeake Bay virioplankton contained a high proportion of unknown (homologous only to environmental sequences) and novel (no significant homolog) sequences. This analysis suggests that dsDNA viruses are likely one of the largest reservoirs of unknown genetic diversity in the biosphere. The taxonomic origin of BLAST homologs to viral library sequences agreed well with reported abundances of cooccurring bacterial subphyla within the estuary and indicated that cyanophages were abundant. However, the low proportion of Siphophage homologs contradicts a previous assertion that this family comprises most bacteriophage diversity. Identification and analyses of cyanobacterial homologs of the psbA gene illustrated the value of metagenomic studies of virioplankton. The phylogeny of inferred PsbA protein sequences suggested that Chesapeake Bay cyanophage strains are endemic in that environment. The ratio of psbA homologous sequences to total cyanophage sequences in the metagenome indicated that the psbA gene may be nearly universal in Chesapeake Bay cyanophage genomes. Furthermore, the low frequency of psbD homologs in the library supports the prediction that Chesapeake Bay cyanophage populations are dominated by Podoviridae.

Metabolic versatility of the Riftia pachyptila endosymbiont revealed through metagenomics

The facultative symbiont of Riftia pachyptila, named here Candidatus Endoriftia persephone, has evaded culture to date, but much has been learned regarding this symbiosis over the past three decades since its discovery. The symbiont population metagenome was sequenced in order to gain insight into its physiology. The population genome indicates that the symbionts use a partial Calvin-Benson Cycle for carbon fixation and the reverse TCA cycle (an alternative pathway for carbon fixation) that contains an unusual ATP citrate lyase. The presence of all genes necessary for heterotrophic metabolism, a phosphotransferase system, and dicarboxylate and ABC transporters indicate that the symbiont can live mixotrophically. The metagenome has a large suite of signal transduction, defence (both biological and environmental) and chemotaxis mechanisms. The physiology of Candidatus Endoriftia persephone is explored with respect to functionality while associated with a eukaryotic host, versus free-living in the hydrothermal environment.

Lysogenic virus-host interactions predominate at deep-sea diffuse-flow hydrothermal vents.

The consequences of viral infection within microbial communities are dependent on the nature of the viral life cycle. Among the possible outcomes is the substantial influence of temperate viruses on the phenotypes of lysogenic prokaryotes through various forms of genetic exchange. To date, no marine microbial ecosystem has consistently shown a predisposition for containing significant numbers of inducible temperate viruses. Here, we show that deep-sea diffuse-flow hydrothermal vent waters display a consistently high incidence of lysogenic hosts and harbor substantial populations of temperate viruses. Genetic fingerprinting and initial metagenomic analyses indicate that temperate viruses in vent waters appear to be a less diverse subset of the larger virioplankton community and that these viral populations contain an extraordinarily high frequency of novel genes. Thus, it appears likely that temperate viruses are key players in the ecology of prokaryotes within the extreme geothermal ecosystems of the deep sea.

Low genomic diversity in tropical oceanic N2-fixing cyanobacteria

High levels of genomic and allelic microvariation have been found in major marine planktonic microbial species, including the ubiquitous open ocean cyanobacterium, Prochlorococcus marinus. Crocosphaera watsonii is a unicellular cyanobacterium that has recently been shown to be important in oceanic N2 fixation and has been reported from the Atlantic and Pacific oceans in both hemispheres, and the Arabian Sea. In direct contrast to the current observations of genomic variability in marine non-N2-fixing planktonic cyanobacteria, which can range up to >15% nucleotide sequence divergence, we discovered that the marine planktonic nitrogen-fixing cyanobacterial genus Crocosphaera has remarkably low genomic diversity, with <1% nucleotide sequence divergence in several genes among widely distributed populations and strains. The cultivated C. watsonii WH8501 genome sequence was virtually identical to DNA sequences of large metagenomic fragments cloned from the subtropical North Pacific Ocean with <1% sequence divergence even in intergenic regions. Thus, there appears to be multiple strategies for evolution, adaptation, and diversification in oceanic microbial populations. The C. watsonii genome contains multiple copies of several families of transposases that may be involved in maintaining genetic diversity through genome rearrangements. Although genomic diversity seems to be the rule in many, if not most, marine microbial lineages, different forces may control the evolution and diversification in low abundance microorganisms, such as the nitrogen-fixing cyanobacteria.

In situ transcriptomic analysis of the globally important keystone N 2 -fixing taxon Crocosphaera watsonii

The diazotrophic cyanobacterium Crocosphaera watsonii supplies fixed nitrogen (N) to N-depleted surface waters of the tropical oceans, but the factors that determine its distribution and contribution to global N2 fixation are not well constrained for natural populations. Despite the heterogeneity of the marine environment, the genome of C. watsonii is highly conserved in nucleotide sequence in contrast to sympatric planktonic cyanobacteria. We applied a whole assemblage shotgun transcript sequencing approach to samples collected from a bloom of C. watsonii observed in the South Pacific to understand the genomic mechanisms that may lead to high population densities. We obtained 999 C. watsonii transcript reads from two metatranscriptomes prepared from mixed assemblage RNA collected in the day and at night. The C. watsonii population had unexpectedly high transcription of hypothetical protein genes (31% of protein-encoding genes) and transposases (12%). Furthermore, genes were expressed that are necessary for living in the oligotrophic ocean, including the nitrogenase cluster and the iron-stress-induced protein A (isiA) that functions to protect photosystem I from high-light-induced damage. C. watsonii transcripts retrieved from metatranscriptomes at other locations in the southwest Pacific Ocean, station ALOHA and the equatorial Atlantic Ocean were similar in composition to those recovered in the enriched population. Quantitative PCR and quantitative reverse transcriptase PCR were used to confirm the high expression of these genes within the bloom, but transcription patterns varied at shallower and deeper horizons. These data represent the first transcript study of a rare individual microorganism in situ and provide insight into the mechanisms of genome diversification and the ecophysiology of natural populations of keystone organisms that are important in global nitrogen cycling.

Repeating patterns of virioplankton production within an estuarine ecosystem

The Chesapeake Bay, a seasonally variable temperate estuary, provides a natural laboratory for examining the fluctuations and impacts of viral lysis on aquatic microorganisms. Viral abundance (VA) and viral production (VP) were monitored in the Chesapeake Bay over 4 1/2 annual cycles, producing a unique, long-term, interannual study of virioplankton production. High and dynamic VP rates, averaging 7.9 × 106 viruses per mL per h, indicate that viral lysis impacts a significant fraction of microorganisms in the Chesapeake. Viral-mediated bacterial mortality, VA, VP, and organic carbon release all displayed similar interannual and seasonal trends with higher values in 2003 and 2006 than in 2004 and 2005 and peaks in early spring and summer. Surprisingly, higher rates of viral lysis occurred in winter, resulting in a magnified effect of viral lysis on bacterioplankton during times of reduced productivity. Viral lysis directly impacted the organic carbon pool, contributing on average 76 μg of C per L per d, an amount capable of sustaining ∼55% of Chesapeake Bay bacterial production. The observed repeating interannual patterns of VP and lysis are likely interlinked with seasonal cycles of host abundance and diversity, which are in turn driven by annual cycles in environmental conditions, emphasizing the complex interplay of seasonality and microbial ecology in the Chesapeake Bay.

Methods for the isolation of viruses from environmental samples.

Viruses are omnipresent and extraordinarily abundant in the microbial ecosystems of water, soil, and sediment. In nearly every reported case for aquatic and porous media environments (soils and sediments) viral abundance exceeds that of co-occurring host populations by 10-100-fold. If current estimates based on metagenome DNA sequence data are correct, then viruses represent the largest reservoir of unknown genetic diversity on Earth. Microscopy and molecular genetic tools have been critical in demonstrating that viruses are a dynamic component of microbial ecosystems capable of significantly influencing the productivity and population biology of their host communities. Moreover, these approaches have begun to describe and constrain the immense genetic diversity of viral communities. A critical first step in the application of many cultivation-independent approaches to virus ecology is obtaining a concentrate of viruses from an environmental sample. Culture-dependent methods also rely on viruses being present at a high enough abundance to detect. Here, methodological details for the isolation and concentration of viruses from water, soil, and aquatic sediment samples are covered in detail.

Two Strains of Crocosphaera watsonii with Highly Conserved Genomes are Distinguished by Strain-Specific Features

Unicellular nitrogen-fixing cyanobacteria are important components of marine phytoplankton. Although non-nitrogen-fixing marine phytoplankton generally exhibit high gene sequence and genomic diversity, gene sequences of natural populations and isolated strains of Crocosphaera watsonii, one of the two most abundant open ocean unicellular cyanobacteria groups, have been shown to be 98–100% identical. The low sequence diversity in Crocosphaera is a dramatic contrast to sympatric species of Prochlorococcus and Synechococcus, and raises the question of how genome differences can explain observed phenotypic diversity among Crocosphaera strains. Here we show, through whole genome comparisons of two phenotypically different strains, that there are strain-specific sequences in each genome, and numerous genome rearrangements, despite exceptionally low sequence diversity in shared genomic regions. Some of the strain-specific sequences encode functions that explain observed phenotypic differences, such as exopolysaccharide biosynthesis. The pattern of strain-specific sequences distributed throughout the genomes, along with rearrangements in shared sequences is evidence of significant genetic mobility that may be attributed to the hundreds of transposase genes found in both strains. Furthermore, such genetic mobility appears to be the main mechanism of strain divergence in Crocosphaera which do not accumulate DNA microheterogeneity over the vast majority of their genomes. The strain-specific sequences found in this study provide tools for future physiological studies, as well as genetic markers to help determine the relative abundance of phenotypes in natural populations.