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  1. 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 ofProchlorococcus, 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 grazeProchlorococcus. 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 haptophyteChrysochromulinawas most abundant among the isolated mixotrophs at Station ALOHA, with 76–250 cells mL−1across 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.

  2. 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.

  3. 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.

  4. Viruses are the most abundant and diverse biological entities on the planet and constitute a significant proportion of Earth’s genetic diversity. Most of this diversity is not represented by isolated viral-host systems and has only been observed through sequencing of viral metagenomes (viromes) from environmental samples. Viromes provide snapshots of viral genetic potential, and a wealth of information on viral community ecology. These data also provide opportunities for exploring the biochemistry of novel viral enzymes. The in vitro biochemical characteristics of novel viral DNA polymerases were explored, testing hypothesized differences in polymerase biochemistry according to protein sequence phylogeny. Forty-eight viral DNA Polymerase I (PolA) proteins from estuarine viromes, hot spring metagenomes, and reference viruses, encompassing a broad representation of currently known diversity, were synthesized, expressed, and purified. Novel functionality was shown in multiple PolAs. Intriguingly, some of the estuarine viral polymerases demonstrated moderate to strong innate DNA strand displacement activity at high enzyme concentration. Strand-displacing polymerases have important technological applications where isothermal reactions are desirable. Bioinformatic investigation of genes neighboring these strand displacing polymerases found associations with SNF2 helicase-associated proteins. The specific function of SNF2 family enzymes is unknown for prokaryotes and viruses. In eukaryotes, SNF2 enzymes have chromatin remodelingmore »functions but do not separate nucleic acid strands. This suggests the strand separation function may be fulfilled by the DNA polymerase for viruses carrying SNF2 helicase-associated proteins. Biochemical data elucidated from this study expands understanding of the biology and ecological behavior of unknown viruses. Moreover, given the numerous biotechnological applications of viral DNA polymerases, novel viral polymerases discovered within viromes may be a rich source of biological material for further in vitro DNA amplification advancements.« less
    Free, publicly-accessible full text available April 21, 2023
  5. It has become increasingly apparent that the lipid composition of cell membranes affects the function of transmembrane proteins such as ion channels. Here, we leverage the structural and functional diversity of small viral K+ channels to systematically examine the impact of bilayer composition on the pore module of single K+ channels. In vitro–synthesized channels were reconstituted into phosphatidylcholine bilayers ± cholesterol or anionic phospholipids (aPLs). Single-channel recordings revealed that a saturating concentration of 30% cholesterol had only minor and protein-specific effects on unitary conductance and gating. This indicates that channels have effective strategies for avoiding structural impacts of hydrophobic mismatches between proteins and the surrounding bilayer. In all seven channels tested, aPLs augmented the unitary conductance, suggesting that this is a general effect of negatively charged phospholipids on channel function. For one channel, we determined an effective half-maximal concentration of 15% phosphatidylserine, a value within the physiological range of aPL concentrations. The different sensitivity of two channel proteins to aPLs could be explained by the presence/absence of cationic amino acids at the interface between the lipid headgroups and the transmembrane domains. aPLs also affected gating in some channels, indicating that conductance and gating are uncoupled phenomena and that the impactmore »of aPLs on gating is protein specific. In two channels, the latter can be explained by the altered orientation of the pore-lining transmembrane helix that prevents flipping of a phenylalanine side chain into the ion permeation pathway for long channel closings. Experiments with asymmetrical bilayers showed that this effect is leaflet specific and most effective in the inner leaflet, in which aPLs are normally present in plasma membranes. The data underscore a general positive effect of aPLs on the conductance of K+ channels and a potential interaction of their negative headgroup with cationic amino acids in their vicinity.« less
  6. The structures of the four N-linked glycans from the prototype chlorovirus PBCV-1 major capsid protein do not resemble any other glycans in the three domains of life. All known chloroviruses and antigenic variants (or mutants) share a unique conserved central glycan core consisting of five sugars, except for antigenic mutant virus P1L6, which has four of the five sugars. A combination of genetic and structural analyses indicates that the protein coded by PBCV-1 gene a111/114r, conserved in all chloroviruses, is a glycosyltransferase with three putative domains of approximately 300 amino acids each. Here, in addition to in silico sequence analysis and protein modeling, we measured the hydrolytic activity of protein A111/114R. The results suggest that domain 1 is a galactosyltransferase, domain 2 is a xylosyltransferase and domain 3 is a fucosyltransferase. Thus, A111/114R is the protein likely responsible for the attachment of three of the five conserved residues of the core region of this complex glycan, and, if biochemically corroborated, it would be the second three-domain protein coded by PBCV-1 that is involved in glycan synthesis. Importantly, these findings provide additional support that the chloroviruses do not use the canonical host endoplasmic reticulum–Golgi glycosylation pathway to glycosylate their glycoproteins; instead,more »they perform glycosylation independent of cellular organelles using virus-encoded enzymes.« less
  7. Paramecium bursaria chlorella virus-1 (PBCV-1) is a large double-stranded DNA (dsDNA) virus that infects the unicellular green alga Chlorella variabilis NC64A. Unlike many other viruses, PBCV-1 encodes most, if not all, of the enzymes involved in the synthesis of the glycans attached to its major capsid protein. Importantly, these glycans differ from those reported from the three domains of life in terms of structure and asparagine location in the sequon of the protein. Previous data collected from 20 PBCV-1 spontaneous mutants (or antigenic variants) suggested that the a064r gene encodes a glycosyltransferase (GT) with three domains, each with a different function. Here, we demonstrate that: domain 1 is a β- l -rhamnosyltransferase; domain 2 is an α- l -rhamnosyltransferase resembling only bacterial proteins of unknown function, and domain 3 is a methyltransferase that methylates the C-2 hydroxyl group of the terminal α- l -rhamnose (Rha) unit. We also establish that methylation of the C-3 hydroxyl group of the terminal α- l -Rha is achieved by another virus-encoded protein A061L, which requires an O-2 methylated substrate. This study, thus, identifies two of the glycosyltransferase activities involved in the synthesis of the N -glycan of the viral major capsid protein in PBCV-1more »and establishes that a single protein A064R possesses the three activities needed to synthetize the 2-OMe-α- l -Rha-(1→2)-β- l -Rha fragment. Remarkably, this fragment can be attached to any xylose unit.« less
  8. Viruses rely on their host’s translation machinery for the synthesis of their own proteins. Problems belie viral translation when the host has a codon usage bias (CUB) that is different from an infecting virus due to differences in the GC content between the host and virus genomes. Here, we examine the hypothesis that chloroviruses adapted to host CUB by acquisition and selection of tRNAs that at least partially favor their own CUB. The genomes of 41 chloroviruses comprising three clades, each infecting a different algal host, have been sequenced, assembled and annotated. All 41 viruses not only encode tRNAs, but their tRNA genes are located in clusters. While differences were observed between clades and even within clades, seven tRNA genes were common to all three clades of chloroviruses, including the tRNAArg gene, which was found in all 41 chloroviruses. By comparing the codon usage of one chlorovirus algal host, in which the genome has been sequenced and annotated (67% GC content), to that of two of its viruses (40% GC content), we found that the viruses were able to at least partially overcome the host’s CUB by encoding tRNAs that recognize AU-rich codons. Evidence presented herein supports the hypothesis thatmore »a chlorovirus tRNA cluster was present in the most recent common ancestor (MRCA) prior to divergence into three clades. In addition, the MRCA encoded a putative isoleucine lysidine synthase (TilS) that remains in 39/41 chloroviruses examined herein, suggesting a strong evolutionary pressure to retain the gene. TilS alters the anticodon of tRNAMet that normally recognizes AUG to then recognize AUA, a codon for isoleucine. This is advantageous to the chloroviruses because the AUA codon is 12–13 times more common in the chloroviruses than their host, further helping the chloroviruses to overcome CUB. Among large DNA viruses infecting eukaryotes, the presence of tRNA genes and tRNA clusters appear to be most common in the Phycodnaviridae and, to a lesser extent, in the Mimiviridae.« less