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  1. Abstract

    Giant viruses are a large group of viruses that infect many eukaryotes. Although components that do not obey the overall icosahedral symmetry of their capsids have been observed and found to play critical roles in the viral life cycles, identities and high-resolution structures of these components remain unknown. Here, by determining a near-atomic-resolution, five-fold averaged structure of Paramecium bursaria chlorella virus 1, we unexpectedly found the viral capsid possesses up to five major capsid protein variants and a penton protein variant. These variants create varied capsid microenvironments for the associations of fibers, a vesicle, and previously unresolved minor capsid proteins. Our structure reveals the identities and atomic models of the capsid components that do not obey the overall icosahedral symmetry and leads to a model for how these components are assembled and initiate capsid assembly, and this model might be applicable to many other giant viruses.

     
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  2. Abstract

    In sunlit waters, significant predation is performed by unicellular, phagotrophic mixotrophs, that is, predators that also possess plastids. The success of a mixotrophic lifestyle will depend in part on how well mixotrophs acquire prey relative to specialized heterotrophs. Likewise, consequences of mixotrophy for productivity and element cycling will depend on the rate and efficiency at which mixotrophs consume prey biomass relative to heterotrophs. However, trait differences between mixotrophs and heterotrophs are not well characterized. In addition, cell size of mixotrophs varies widely, and constitutive mixotrophs include small flagellates deriving from diverse taxa, while larger species are primarily dinoflagellates. To determine whether similar constraints apply to phagotrophs across this broad range of size and taxa, we compiled 83 measurements of flagellate functional responses and compared maximum clearance rates (Cmax) and maximum ingestion rates (Imax) between trophic modes. We found that the average mixotroph has a 3.7‐fold lowerCmaxand 7.8‐fold lowerImaxthan the average heterotroph, after controlling for cell size. The smaller penalty forCmaxsuggests that relative fitness of mixotrophs will be enhanced under dilute prey concentrations that are common in pelagic ecosystems. We also find that growth efficiency is greater for mixotrophs and for flagellates with lowerCmax, indicating a spectrum of trophic strategies that may be driven by phototrophy vs. phagotrophy allocation as well as fast vs. slow metabolic variation. Allometric scaling shows thatImaxis constrained by a common relationship among dinoflagellates and other taxa, but dinoflagellates achieve a greater volume‐specificCmax. These results should aid in interpreting protistan communities and modeling mixotrophy.

     
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  3. Abstract

    Small eukaryotic phytoplankton are major contributors to global primary production and marine biogeochemical cycles. Many taxa are thought to be mixotrophic, but quantitative studies of phagotrophy exist for very few. In addition, little is known about consumers of Prochlorococcus, the abundant cyanobacterium at the base of oligotrophic ocean food webs. Here we describe thirty-nine new phytoplankton isolates from the North Pacific Subtropical Gyre (Station ALOHA), all flagellates ~2–5 µm diameter, and we quantify their ability to graze Prochlorococcus. The mixotrophs are from diverse classes (dictyochophytes, haptophytes, chrysophytes, bolidophytes, a dinoflagellate, and a chlorarachniophyte), many from previously uncultured clades. Grazing ability varied substantially, with specific clearance rate (volume cleared per body volume) varying over ten-fold across isolates and six-fold across genera. Slower grazers tended to create more biovolume per prey biovolume consumed. Using qPCR we found that the haptophyte Chrysochromulina was most abundant among the isolated mixotrophs at Station ALOHA, with 76–250 cells mL−1 across depths in the upper euphotic zone (5–100 m). Our results show that within a single ecosystem the phototrophs that ingest bacteria come from many branches of the eukaryotic tree, and are functionally diverse, indicating a broad range of strategies along the spectrum from phototrophy to phagotrophy.

     
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  4. Abstract

    Persistent nitrogen depletion in sunlit open ocean waters provides a favorable ecological niche for nitrogen-fixing (diazotrophic) cyanobacteria, some of which associate symbiotically with eukaryotic algae. All known marine examples of these symbioses have involved either centric diatom or haptophyte hosts. We report here the discovery and characterization of two distinct marine pennate diatom-diazotroph symbioses, which until now had only been observed in freshwater environments. Rhopalodiaceae diatomsEpithemia pelagicasp. nov. andEpithemia catenatasp. nov. were isolated repeatedly from the subtropical North Pacific Ocean, and analysis of sequence libraries reveals a global distribution. These symbioses likely escaped attention because the endosymbionts lack fluorescent photopigments, havenifHgene sequences similar to those of free-living unicellular cyanobacteria, and are lost in nitrogen-replete medium. Marine Rhopalodiaceae-diazotroph symbioses are a previously overlooked but widespread source of bioavailable nitrogen in marine habitats and provide new, easily cultured model organisms for the study of organelle evolution.

     
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  5. Abstract

    Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the nonstoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses.

     
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  6. Abstract

    Mixotrophic nanoflagellates can account for more than half of the bacterivory in the sunlit ocean, yet very little is known about their ecophysiology. Here, we characterize the grazing ecology of an open‐ocean mixotroph in the genusFlorenciella(class Dictyochophyceae). Members of this class were indirectly implicated as major consumers ofProchlorococcusandSynechococcusin the oligotrophic North Pacific Subtropical Gyre, but their phagotrophic capabilities have never been investigated. Our studies showed thatFlorenciellareadily consumedProchlorococcus,Synechococcus, and heterotrophic bacteria, and that the ingested prey relieved nutrient limitations on growth.Florenciellagrew faster (3 d−1) in nitrogen‐deplete medium given sufficient liveSynechococcus, than in nitrogen‐replete K medium (2 d−1), but it did not grow in continuous darkness. Grazing rates were substantially higher under nutrient limitation and showed a hint of diel variability, with rates tending to be highest near the end of the light period. An apparent trade‐off between the maximum clearance rate (5 nLFlorenciella−1h−1) and the maximum ingestion rate (up to ∼ 10 prey cellsFlorenciella−1h−1) across experiments suggests that grazing behavior may also vary in response to prey concentration. If the observed grazing rates are representative of other open‐ocean mixotrophs, their collective activity could account for a significant fraction of the daily cyanobacterial mortality. This study provides essential parameters for understanding the grazing ecology of a common marine mixotroph and the first characterization of mixotrophic nanoflagellate functional responses when feeding on unicellular cyanobacteria, the dominant marine primary producers in the oligotrophic ocean.

     
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  7. Many chloroviruses replicate in Chlorella variabilis algal strains that are ex-endosymbionts isolated from the protozoan Paramecium bursaria, including the NC64A and Syngen 2-3 strains. We noticed that indigenous water samples produced a higher number of plaque-forming viruses on C. variabilis Syngen 2-3 lawns than on C. variabilis NC64A lawns. These observed differences led to the discovery of viruses that replicate exclusively in Syngen 2-3 cells, named Only Syngen (OSy) viruses. Here, we demonstrate that OSy viruses initiate infection in the restricted host NC64A by synthesizing some early virus gene products and that approximately 20% of the cells produce a small number of empty virus capsids. However, the infected cells did not produce infectious viruses because the cells were unable to replicate the viral genome. This is interesting because all previous attempts to isolate host cells resistant to chlorovirus infection were due to changes in the host receptor for the virus. 
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  8. Buchan, Alison (Ed.)
    ABSTRACT The ability of Bradyrhizobium spp. to nodulate and fix atmospheric nitrogen in soybean root nodules is critical to meeting humanity’s nutritional needs. The intricacies of soybean bradyrhizobia-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention, even though these interactions may significantly impact soybean yield. In batch culture, four soybean bradyrhizobia strains, Bradyrhizobium japonicum S06B (S06B-Bj), B. japonicum S10J (S10J-Bj), Bradyrhizobium diazoefficiens USDA 122 (USDA 122-Bd), and Bradyrhizobium elkanii USDA 76 T (USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced tailed phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ~3-fold after 48 h of incubation. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each soybean bradyrhizobia genome, preventing accurate identification of spontaneously produced prophage (SPP) genomes. A DNA sequencing and mapping approach accurately delineated the boundaries of four SPP genomes within three of the soybean bradyrhizobia chromosomes and suggested that the SPPs were capable of transduction. In addition to the phages, S06B-Bj and USDA 76-Be contained three to four times more insertion sequences (IS) and large, conjugable, broad host range plasmids, both of which are known drivers of horizontal gene transfer (HGT) in soybean bradyrhizobia. These factors indicate that SPP along with IS and plasmids participate in HGT, drive bradyrhizobia evolution, and play an outsized role in bradyrhizobia ecology. IMPORTANCE Previous studies have shown that IS and plasmids mediate HGT of symbiotic nodulation ( nod ) genes in soybean bradyrhizobia; however, these events require close cell-to-cell contact, which could be limited in soil environments. Bacteriophage-assisted gene transduction through spontaneously produced prophages provides a stable means of HGT not limited by the constraints of proximal cell-to-cell contact. These phage-mediated HGT events may shape soybean bradyrhizobia population ecology, with concomitant impacts on soybean agriculture. 
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  9. Viruses face many challenges on their road to successful replication, and they meet those challenges by reprogramming the intracellular environment. Two major issues challenging Paramecium bursaria chlorella virus 1 (PBCV-1, genus Chlorovirus, family Phycodnaviridae) at the level of DNA replication are (i) the host cell has a DNA G+C content of 66%, while the virus is 40%; and (ii) the initial quantity of DNA in the haploid host cell is approximately 50 fg, yet the virus will make approximately 350 fg of DNA within hours of infection to produce approximately 1000 virions per cell. Thus, the quality and quantity of DNA (and RNA) would seem to restrict replication efficiency, with the looming problem of viral DNA synthesis beginning in only 60–90 min. Our analysis includes (i) genomics and functional annotation to determine gene augmentation and complementation of the nucleotide biosynthesis pathway by the virus, (ii) transcriptional profiling of these genes, and (iii) metabolomics of nucleotide intermediates. The studies indicate that PBCV-1 reprograms the pyrimidine biosynthesis pathway to rebalance the intracellular nucleotide pools both qualitatively and quantitatively, prior to viral DNA amplification, and reflects the genomes of the progeny virus, providing a successful road to virus infection. 
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  10. A large fraction of marine primary production is performed by diverse small protists, and many of these phytoplankton are phagotrophic mixotrophs that vary widely in their capacity to consume bacterial prey. Prior analyses suggest that mixotrophic protists as a group vary in importance across ocean environments, but the mechanisms leading to broad functional diversity among mixotrophs, and the biogeochemical consequences of this, are less clear. Here we use isolates from seven major taxa to demonstrate a tradeoff between phototrophic performance (growth in the absence of prey) and phagotrophic performance (clearance rate when consuming Prochlorococcus ). We then show that trophic strategy along the autotrophy-mixotrophy spectrum correlates strongly with global niche differences, across depths and across gradients of stratification and chlorophyll a . A model of competition shows that community shifts can be explained by greater fitness of faster-grazing mixotrophs when nutrients are scarce and light is plentiful. Our results illustrate how basic physiological constraints and principles of resource competition can organize complexity in the surface ocean ecosystem. 
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