Targeted metagenomic recovery of four divergent viruses reveals shared and distinctive characteristics of giant viruses of marine eukaryotes.

Giant viruses have remarkable genomic repertoires-blurring the line with cellular life-and act as top-down controls of eukaryotic plankton. However, to date only six cultured giant virus genomes are available from the pelagic ocean. We used at-sea flow cytometry with staining and sorting designed to target wild predatory eukaryotes, followed by DNA sequencing and assembly, to recover novel giant viruses from the Pacific Ocean. We retrieved four 'PacV' partial genomes that range from 421 to 1605 Kb, with 13 contigs on average, including the largest marine viral genomic assembly reported to date. Phylogenetic analyses indicate that three of the new viruses span a clade with deep-branching members of giant Mimiviridae, incorporating the Cafeteria roenbergensis virus, the uncultivated terrestrial Faunusvirus, one PacV from a choanoflagellate and two PacV with unclear hosts. The fourth virus, oPacV-421, is phylogenetically related to viruses that infect haptophyte algae. About half the predicted proteins in each PacV have no matches in NCBI nr (e-value < 10-5), totalling 1735 previously unknown proteins; the closest affiliations of the other proteins were evenly distributed across eukaryotes, prokaryotes and viruses of eukaryotes. The PacVs encode many translational proteins and two encode eukaryotic-like proteins from the Rh family of the ammonium transporter superfamily, likely influencing the uptake of nitrogen during infection. cPacV-1605 encodes a microbial viral rhodopsin (VirR) and the biosynthesis pathway for the required chromophore, the second finding of a choanoflagellate-associated virus that encodes these genes. In co-collected metatranscriptomes, 85% of cPacV-1605 genes were expressed, with capsids, heat shock proteins and proteases among the most highly expressed. Based on orthologue presence-absence patterns across the PacVs and other eukaryotic viruses, we posit the observed viral groupings are connected to host lifestyles as heterotrophs or phototrophs. This article is part of a discussion meeting issue 'Single cell ecology'.

Ecophysiology of uncultivated marine euryarchaea is linked to particulate organic matter.

Particles in aquatic environments host distinct communities of microbes, yet the evolution of particle-specialized taxa and the extent to which specialized microbial metabolism is associated with particles is largely unexplored. Here, we investigate the hypothesis that a widely distributed and uncultivated microbial group--the marine group II euryarchaea (MGII)--interacts with living and detrital particulate organic matter (POM) in the euphotic zone of the central California Current System. Using fluorescent in situ hybridization, we verified the association of euryarchaea with POM. We further quantified the abundance and distribution of MGII 16 S ribosomal RNA genes in size-fractionated seawater samples and compared MGII functional capacity in metagenomes from the same fractions. The abundance of MGII in free-living and >3 µm fractions decreased with increasing distance from the coast, whereas MGII abundance in the 0.8-3 µm fraction remained constant. At several offshore sites, MGII abundance was highest in particle fractions, indicating that particle-attached MGII can outnumber free-living MGII under oligotrophic conditions. Compared with free-living MGII, the genome content of MGII in particle-associated fractions exhibits an increased capacity for surface adhesion, transcriptional regulation and catabolism of high molecular weight substrates. Moreover, MGII populations in POM fractions are phylogenetically distinct from and more diverse than free-living MGII. Eukaryotic phytoplankton additions stimulated MGII growth in bottle incubations, providing the first MGII net growth rate measurements. These ranged from 0.47 to 0.54 d(-1). However, MGII were not recovered in whole-genome amplifications of flow-sorted picoeukaryotic phytoplankton and heterotrophic nanoflagellates, suggesting that MGII in particle fractions are not physically attached to living POM. Collectively, our results support a linkage between MGII ecophysiology and POM, implying that marine archaea have a role in elemental cycling through interactions with particles.

Microbubble sizing and shell characterization using flow cytometry.

Experiments were performed to size, count, and obtain shell parameters for individual ultrasound contrast microbubbles using a modified flow cytometer. Light scattering was modeled using Mie theory, and applied to calibration beads to calibrate the system. The size distribution and population were measured directly from the flow cytometer. The shell parameters (shear modulus and shear viscosity) were quantified at different acoustic pressures (from 95 to 333 kPa) by fitting microbubble response data to a bubble dynamics model. The size distribution of the contrast agent microbubbles is consistent with manufacturer specifications. The shell shear viscosity increases with increasing equilibrium microbubble size, and decreases with increasing shear rate. The observed trends are independent of driving pressure amplitude. The shell elasticity does not vary with microbubble size. The results suggest that a modified flow cytometer can be an effective tool to characterize the physical properties of microbubbles, including size distribution, population, and shell parameters.

Unveiling a phytoplankton hotspot at a narrow boundary between coastal and offshore waters.

In terrestrial ecosystems, transitional areas between different plant communities (ecotones) are formed by steep environmental gradients and are commonly characterized by high species diversity and primary productivity, which in turn influences the foodweb structure of these regions. Whether comparable zones of elevated diversity and productivity characterize ecotones in the oceans remains poorly understood. Here we describe a previously hidden hotspot of phytoplankton diversity and productivity in a narrow but seasonally persistent transition zone at the intersection of iron-poor, nitrate-rich offshore waters and iron-rich, nitrate-poor coastal waters of the Northeast Pacific Ocean. Novel continuous measurements of phytoplankton cell abundance and composition identified a complex succession of blooms of five distinct size classes of phytoplankton populations within a 100-km-wide transition zone. The blooms appear to be fueled by natural iron enrichment of offshore communities as they are transported toward the coast. The observed succession of phytoplankton populations is likely driven by spatial gradients in iron availability or time since iron enrichment. Regardless of the underlying mechanism, the resulting communities have a strong impact on the regional biogeochemistry as evidenced by the low partial pressure of CO(2) and the nearly complete depletion of nutrients. Enhanced phytoplankton productivity and diversity associated with steep environmental gradients are expected wherever water masses with complementary nutrient compositions mix to create a region more favorable for phytoplankton growth. The ability to detect and track these important but poorly characterized marine ecotones is critical for understanding their impact on productivity and ecosystem structure in the oceans.

Genome size differentiates co-occurring populations of the planktonic diatom Ditylum brightwellii (Bacillariophyta).

BACKGROUND: Diatoms are one of the most species-rich groups of eukaryotic microbes known. Diatoms are also the only group of eukaryotic micro-algae with a diplontic life history, suggesting that the ancestral diatom switched to a life history dominated by a duplicated genome. A key mechanism of speciation among diatoms could be a propensity for additional stable genome duplications. Across eukaryotic taxa, genome size is directly correlated to cell size and inversely correlated to physiological rates. Differences in relative genome size, cell size, and acclimated growth rates were analyzed in isolates of the diatom Ditylum brightwellii. Ditylum brightwellii consists of two main populations with identical 18s rDNA sequences; one population is distributed globally at temperate latitudes and the second appears to be localized to the Pacific Northwest coast of the USA. These two populations co-occur within the Puget Sound estuary of WA, USA, although their peak abundances differ depending on local conditions. RESULTS: All isolates from the more regionally-localized population (population 2) possessed 1.94 +/- 0.74 times the amount of DNA, grew more slowly, and were generally larger than isolates from the more globally distributed population (population 1). The ITS1 sequences, cell sizes, and genome sizes of isolates from New Zealand were the same as population 1 isolates from Puget Sound, but their growth rates were within the range of the slower-growing population 2 isolates. Importantly, the observed genome size difference between isolates from the two populations was stable regardless of time in culture or the changes in cell size that accompany the diatom life history. CONCLUSIONS: The observed two-fold difference in genome size between the D. brightwellii populations suggests that whole genome duplication occurred within cells of population 1 ultimately giving rise to population 2 cells. The apparent regional localization of population 2 is consistent with a recent divergence between the populations, which are likely cryptic species. Genome size variation is known to occur in other diatom genera; we hypothesize that genome duplication may be an active and important mechanism of genetic and physiological diversification and speciation in diatoms.

Virtual-core flow cytometry.

Traditional flow cytometers use a sheath fluid to position particles or cells for cytometric measurements, but the need for sheath fluid greatly complicates flow cytometric instrumentation. A cytometric detector that is free of the requirements of sheath fluid can simplify the design of flow cytometers and can extend their use into a number of areas. We designed a flow cytometer that uses a combination of three photodetectors to sense the position of a particle in sample stream. The position-sensitive detectors create a virtual core in the sample stream that eliminates the need for sheath fluid. In this article, we demonstrate the efficacy of a virtual-core flow cytometer (VCFC) using test particles, immunofluorescently labeled thymocytes, and raw seawater. The VCFC performs accurate measurements that can be used for a number of uses including environmental monitoring or simple immunology tests.