David D. Dunigan

The Chlorella variabilis NC64A Genome Reveals Adaptation to Photosymbiosis, Coevolution with Viruses, and Cryptic Sex

This report describes the genome sequence of Chlorella variabilis NC64A. Surprisingly, given that NC64A has been thought to be asexual and nonmotile, this work identifies homologs of genes involved in meiosis, gamete fusion, and flagella. Chlorella variabilis NC64A, a unicellular photosynthetic green alga (Trebouxiophyceae), is an intracellular photobiont of Paramecium bursaria and a model system for studying virus/algal interactions. We sequenced its 46-Mb nuclear genome, revealing an expansion of protein families that could have participated in adaptation to symbiosis. NC64A exhibits variations in GC content across its genome that correlate with global expression level, average intron size, and codon usage bias. Although Chlorella species have been assumed to be asexual and nonmotile, the NC64A genome encodes all the known meiosis-specific proteins and a subset of proteins found in flagella. We hypothesize that Chlorella might have retained a flagella-derived structure that could be involved in sexual reproduction. Furthermore, a survey of phytohormone pathways in chlorophyte algae identified algal orthologs of Arabidopsis thaliana genes involved in hormone biosynthesis and signaling, suggesting that these functions were established prior to the evolution of land plants. We show that the ability of Chlorella to produce chitinous cell walls likely resulted from the capture of metabolic genes by horizontal gene transfer from algal viruses, prokaryotes, or fungi. Analysis of the NC64A genome substantially advances our understanding of the green lineage evolution, including the genomic interplay with viruses and symbiosis between eukaryotes.

The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation

BackgroundLittle is known about the mechanisms of adaptation of life to the extreme environmental conditions encountered in polar regions. Here we present the genome sequence of a unicellular green alga from the division chlorophyta, Coccomyxa subellipsoidea C-169, which we will hereafter refer to as C-169. This is the first eukaryotic microorganism from a polar environment to have its genome sequenced.ResultsThe 48.8 Mb genome contained in 20 chromosomes exhibits significant synteny conservation with the chromosomes of its relatives Chlorella variabilis and Chlamydomonas reinhardtii. The order of the genes is highly reshuffled within synteny blocks, suggesting that intra-chromosomal rearrangements were more prevalent than inter-chromosomal rearrangements. Remarkably, Zepp retrotransposons occur in clusters of nested elements with strictly one cluster per chromosome probably residing at the centromere. Several protein families overrepresented in C. subellipsoidae include proteins involved in lipid metabolism, transporters, cellulose synthases and short alcohol dehydrogenases. Conversely, C-169 lacks proteins that exist in all other sequenced chlorophytes, including components of the glycosyl phosphatidyl inositol anchoring system, pyruvate phosphate dikinase and the photosystem 1 reaction center subunit N (PsaN).ConclusionsWe suggest that some of these gene losses and gains could have contributed to adaptation to low temperatures. Comparison of these genomic features with the adaptive strategies of psychrophilic microbes suggests that prokaryotes and eukaryotes followed comparable evolutionary routes to adapt to cold environments.

DNA Viruses: The Really Big Ones (Giruses)

Viruses with genomes greater than 300 kb and up to 1200 kb are being discovered with increasing frequency. These large viruses (often called giruses) can encode up to 900 proteins and also many tRNAs. Consequently, these viruses have more protein-encoding genes than many bacteria, and the concept of small particle/small genome that once defined viruses is no longer valid. Giruses infect bacteria and animals although most of the recently discovered ones infect protists. Thus, genome gigantism is not restricted to a specific host or phylogenetic clade. To date, most of the giruses are associated with aqueous environments. Many of these large viruses (phycodnaviruses and Mimiviruses) probably have a common evolutionary ancestor with the poxviruses, iridoviruses, asfarviruses, ascoviruses, and a recently discovered Marseillevirus. One issue that is perhaps not appreciated by the microbiology community is that large viruses, even ones classified in the same family, can differ significantly in morphology, lifestyle, and genome structure. This review focuses on some of these differences than on extensive details about individual viruses.

Chloroviruses: Not Your Everyday Plant Virus

Viruses infecting higher plants are among the smallest viruses known and typically have four to ten protein-encoding genes. By contrast, many viruses that infect algae (classified in the virus family Phycodnaviridae) are among the largest viruses found to date and have up to 600 protein-encoding genes. This brief review focuses on one group of plaque-forming phycodnaviruses that infect unicellular chlorella-like green algae. The prototype chlorovirus PBCV-1 has more than 400 protein-encoding genes and 11 tRNA genes. About 40% of the PBCV-1 encoded proteins resemble proteins of known function including many that are completely unexpected for a virus. In many respects, chlorovirus infection resembles bacterial infection by tailed bacteriophages.

Bcl-2 Family Members Inhibit Oxidative Stress-Induced Programmed Cell Death in Saccharomyces cerevisiae

Selected antiapoptotic genes were expressed in bakers yeast (Saccharomyces cerevisiae) to evaluate cytoprotective effects during oxidative stress. When exposed to treatments resulting in the generation of reactive oxygen species (ROS), including H(2)O(2), menadione, or heat shock, wild-type yeast died and exhibited apoptotic-like characteristics, consistent with previous studies. Yeast strains were generated expressing nematode ced-9, human bcl-2, or chicken bcl-xl genes. These transformants tolerated a range of oxidative stresses, did not display features associated with apoptosis, and remained viable under conditions that were lethal to wild-type yeast. Yeast strains expressing a mutant antiapoptotic gene (bcl-2 deltaalpha 5-6), known to be nonfunctional in mammalian cells, were unable to tolerate any of the ROS-generating insults. These data are the first report showing CED-9 has cytoprotective effects against oxidative stress, and add CED-9 to the list of Bcl-2 protein family members that modulate ROS-mediated programmed cell death. In addition, these data indicate that Bcl-2 family members protect wild-type yeast from physiological stresses. Taken together, these data support the concept of the broad evolutionary conservation and functional similarity of the apoptotic processes in eukaryotic organisms.

Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice

Significance Human mucosal surfaces contain a wide range of microorganisms. The biological effects of these organisms are largely unknown. Large-scale metagenomic sequencing is emerging as a method to identify novel microbes. Unexpectedly, we identified DNA sequences homologous to virus ATCV-1, an algal virus not previously known to infect humans, in oropharyngeal samples obtained from healthy adults. The presence of ATCV-1 was associated with a modest but measurable decrease in cognitive functioning. A relationship between ATCV-1 and cognitive functioning was confirmed in a mouse model, which also indicated that exposure to ATCV-1 resulted in changes in gene expression within the brain. Our study indicates that viruses in the environment not thought to infect humans can have biological effects. Chloroviruses (family Phycodnaviridae) are large DNA viruses known to infect certain eukaryotic green algae and have not been previously shown to infect humans or to be part of the human virome. We unexpectedly found sequences homologous to the chlorovirus Acanthocystis turfacea chlorella virus 1 (ATCV-1) in a metagenomic analysis of DNA extracted from human oropharyngeal samples. These samples were obtained by throat swabs of adults without a psychiatric disorder or serious physical illness who were participating in a study that included measures of cognitive functioning. The presence of ATCV-1 DNA was confirmed by quantitative PCR with ATCV-1 DNA being documented in oropharyngeal samples obtained from 40 (43.5%) of 92 individuals. The presence of ATCV-1 DNA was not associated with demographic variables but was associated with a modest but statistically significant decrease in the performance on cognitive assessments of visual processing and visual motor speed. We further explored the effects of ATCV-1 in a mouse model. The inoculation of ATCV-1 into the intestinal tract of 9–11-wk-old mice resulted in a subsequent decrease in performance in several cognitive domains, including ones involving recognition memory and sensory-motor gating. ATCV-1 exposure in mice also resulted in the altered expression of genes within the hippocampus. These genes comprised pathways related to synaptic plasticity, learning, memory formation, and the immune response to viral exposure.

Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the release of the internal viral membrane that was linked to the capsid by many copies of a viral membrane protein in the mature infectous virus. Simultaneously, part of the trisymmetrons around the unique vertex disassemble, probably in part because two minor capsid proteins are absent, causing Paramecium bursaria chlorella virus and the cellular contents to merge, possibly as a result of enzyme(s) within the spike assembly. This may be one of only a few recordings of successive stages of a virus while infecting a eukaryotic host in pseudoatomic detail in three dimensions.

Virion-Associated Restriction Endonucleases of Chloroviruses

ABSTRACT Chloroviruses are large, double-stranded-DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae. The prototype of the genus is Paramecium bursaria chlorella virus 1 (PBCV-1). Chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded DNA methyltransferases (MTases); about 25% of the MTases are associated with companion DNA site-specific (restriction) endonucleases (REases). These enzymes constitute virally encoded restriction-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the host DNA early in infection. To test this hypothesis, host chromosomal DNA from PBCV-1-infected cells was examined by pulsed-field gel electrophoresis. Initiation of host chromosomal DNA degradation occurred within 5 min postinfection (p.i.). The DNA degradation was insensitive to protein synthesis inhibitors or UV inactivation of virus particles, consistent with the agent being a small protein associated with the virion. Nuclease activities, including those of the two predicted REases and an uncharacterized general nuclease(s), were detected in disrupted PBCV-1 particles. The general nuclease(s) degraded both host and viral DNAs in vitro, although the viral DNA was not degraded in vivo, suggesting differential intracellular trafficking of the virion-associated nucleases. Infection with chloroviruses lacking an R/M system(s) resulted in either delayed host chromosomal DNA degradation or no detectable host chromatin changes. These immediate-early events associated with chlorovirus infections may facilitate rapid switching of the host transcriptional apparatus to viral transcription, which begins within 5 to 10 min p.i.

Towards defining the chloroviruses: a genomic journey through a genus of large DNA viruses

BackgroundGiant viruses in the genus Chlorovirus (family Phycodnaviridae) infect eukaryotic green microalgae. The prototype member of the genus, Paramecium bursaria chlorella virus 1, was sequenced more than 15 years ago, and to date there are only 6 fully sequenced chloroviruses in public databases. Presented here are the draft genome sequences of 35 additional chloroviruses (287 – 348 Kb/319 – 381 predicted protein encoding genes) collected across the globe; they infect one of three different green algal species. These new data allowed us to analyze the genomic landscape of 41 chloroviruses, which revealed some remarkable features about these viruses.ResultsGenome colinearity, nucleotide conservation and phylogenetic affinity were limited to chloroviruses infecting the same host, confirming the validity of the three previously known subgenera. Clues for the existence of a fourth new subgenus indicate that the boundaries of chlorovirus diversity are not completely determined. Comparison of the chlorovirus phylogeny with that of the algal hosts indicates that chloroviruses have changed hosts in their evolutionary history. Reconstruction of the ancestral genome suggests that the last common chlorovirus ancestor had a slightly more diverse protein repertoire than modern chloroviruses. However, more than half of the defined chlorovirus gene families have a potential recent origin (after Chlorovirus divergence), among which a portion shows compositional evidence for horizontal gene transfer. Only a few of the putative acquired proteins had close homologs in databases raising the question of the true donor organism(s). Phylogenomic analysis identified only seven proteins whose genes were potentially exchanged between the algal host and the chloroviruses.ConclusionThe present evaluation of the genomic evolution pattern suggests that chloroviruses differ from that described in the related Poxviridae and Mimiviridae. Our study shows that the fixation of algal host genes has been anecdotal in the evolutionary history of chloroviruses. We finally discuss the incongruence between compositional evidence of horizontal gene transfer and lack of close relative sequences in the databases, which suggests that the recently acquired genes originate from a still largely un-sequenced reservoir of genomes, possibly other unknown viruses that infect the same hosts.

Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus

ABSTRACT The 331-kbp chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its host Chlorella variabilis NC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.