James L. Van Etten

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 structure and evolution of the major capsid protein of a large, lipid-containing DNA virus

Paramecium bursaria Chlorella virus type 1 (PBCV-1) is a very large, icosahedral virus containing an internal membrane enclosed within a glycoprotein coat consisting of pseudohexagonal arrays of trimeric capsomers. Each capsomer is composed of three molecules of the major capsid protein, Vp54, the 2.0-Å resolution structure of which is reported here. Four N-linked and two O-linked glycosylation sites were identified. The N-linked sites are associated with nonstandard amino acid motifs as a result of glycosylation by virus-encoded enzymes. Each monomer of the trimeric structure consists of two eight-stranded, antiparallel β-barrel, “jelly-roll” domains related by a pseudo-sixfold rotation. The fold of the monomer and the pseudo-sixfold symmetry of the capsomer resembles that of the major coat proteins in the double-stranded DNA bacteriophage PRD1 and the double-stranded DNA human adenoviruses, as well as the viral proteins VP2-VP3 of picornaviruses. The structural similarities among these diverse groups of viruses, whose hosts include bacteria, unicellular eukaryotes, plants, and mammals, make it probable that their capsid proteins have evolved from a common ancestor that had already acquired a pseudo-sixfold organization. The trimeric capsid protein structure was used to produce a quasi-atomic model of the 1,900-Å diameter PBCV-1 outer shell, based on fitting of the Vp54 crystal structure into a three-dimensional cryoelectron microscopy image reconstruction of the virus.

Growth cycle of a virus, PBCV-1, that infects Chlorella-like algae.

The growth and purification of milligram quantities of a large double-stranded DNA virus, PBCV-1, which replicates in a Chlorella-like alga is described. The virus had an adsorption rate constant of ca. 5 x 10(-9) ml/min, a latent period of 150 to 180 min, and a burst size of 200 to 350 PFUs when the host was actively growing in the light. The eclipse period was 30 to 50 min shorter than the latent period. PBCV-1 also replicated in dark grown Chlorella but the burst size was reduced ca. 50%. The photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, had no effect on viral replication. Thus viral replication does not require host photosynthesis. Viral infection rapidly inhibited both growth and CO2 fixation of the host Chlorella.

Virus Infection of Culturable Chlorella-Like Algae and Dlevelopment of a Plaque Assay

Four distinct viruses with double-stranded DNA are known to replicate in Chlorella-like algae symbiotic with hydras and paramecia. An attempt was made to infect a number of cultured Chlorella strains derived from invertebrate hosts with these viruses. One of the viruses, PBCV-1, replicated in two of the algal strains. Restriction endonuclease analysis of the viral DNA showed that the infectious progeny virus was identical to the input virus; thus, Kochs postulates were fulfilled. Viral infection of the two Chlorella strains has allowed the large-scale production of a eukaryotic algal virus and the development of a plaque assay for the virus.

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.

Infection of a chlorella-like alga with the virus, PBCV-1: Ultrastructural studies☆

Ultrastructural studies revealed that the virus, PBCV-1, adsorbs to the surface of the Chlorella-like green alga NC64A and enzymatically digests a portion of the host cell wall. The viral DNA is then released into the interior of the cell leaving an empty capsid on the surface. Thus uncoating of the viral genome occurs at the surface of its host. PBCV-1 also adsorbs to and digests the host wall of either heat-killed, methanol-extracted, or purified cell wall fragments.

Isolation and characterization of a virus that infects Emiliania huxleyi (Haptophyta)

The isolation and characterization of a virus (designated EhV) that infects the marine coccolithophorid Emiliania huxleyi (Lohmann) Hay & Mohler are described. Three independent clones of EhV were isolated from Norwegian coastal waters in years 1999 and 2000. EhV is a double‐stranded DNA‐containing virus with a genome size of ∼415 kilo‐base pairs. The viral particle is an icosahedron with a diameter of 160–180 nm. The virus particle contains at least nine proteins ranging from 10 to 140 kDa; the major capsid protein weighs ∼54 kDa. EhV has a latent period of 12–14 h and a burst size of 400–1000 (mean, 620) viral particles per cell. A phylogenetic tree based on DNA polymerase amino acid sequences indicates EhV should be assigned to the Phycodnaviridae virus family and that the virus is most closely related to viruses that infect Micromonas pusilla and certain Chlorella species.

DNA synthesis in a Chlorella-like alga following infection with the virus PBCV-1☆

Infection of the unicellular, eukaryotic Chlorella-like alga NC64A by the large dsDNA virus, PBCV-1, resulted in a threefold increase in total DNA by 4 hr post infection. Viral infection rapidly inhibited host DNA synthesis which was followed by the degradation of the host chloroplast and nuclear DNA. Viral DNA synthesis began 30 to 40 min after infection and was dependent on de novo protein synthesis. Thus, the virus does not carry all of the components required to form a functional viral DNA polymerase into the cell.