Kei Kimura

First evidence for the existence of pennate diatom viruses.

Diatoms are considered the most successful and widespread group of photosynthetic eukaryotes. Their contribution to primary production is remarkably significant to the earths ecosystems. Diatoms are composed of two orders: Centrales and Pennales. Thus far, viruses infecting centric diatom species have been isolated and characterized; however, viruses infecting pennates have not been reported. Here, we describe the first isolations and preliminary characterizations of two distinct pennate diatom viruses, AglaRNAV (31 nm in diameter, accumulates in the host cytoplasm) and TnitDNAV (35 nm in diameter, accumulates in the host nuclei) infecting Asterionellopsis glacialis and Thalassionema nitzschioides, respectively. Their genomes contain a single-stranded RNA of approximately 9.5 kb, and a closed, circular single-stranded DNA of approximately 5.5 kb harboring a partially double-stranded region, respectively. Further analysis of these viruses may elucidate many aspects of diatom host–virus relationships.

Discovery of Two Novel Viruses Expands the Diversity of Single-Stranded DNA and Single-Stranded RNA Viruses Infecting a Cosmopolitan Marine Diatom

ABSTRACT Recent studies have suggested that diatom viruses are an important factor affecting diatom population dynamics, which in turn are important in considering marine primary productivity. The marine planktonic diatom Chaetoceros tenuissimus Meunier is a cosmopolitan species and often causes blooms off the western coast of Japan. To date, two viruses, C. tenuissimus DNA virus (CtenDNAV) type I and CtenRNAV type I, have been identified that potentially affect C. tenuissimus population dynamics in the natural environment. In this study, we successfully isolated and characterized two additional novel viruses (CtenDNAV type II and CtenRNAV type II). This paper reports the basic characteristics of these new viruses isolated from surface water or sediment from the Hiroshima Bay, Japan. The physiological and morphological characteristics of the two new viruses were similar to those of the previously isolated viruses. However, the amino acid sequences of the structural proteins of CtenDNAV type II and CtenRNAV type II were clearly distinct from those of both type I viruses, with identity scores of 38.3% and 27.6%, respectively. Our results suggest that at least four genetically distinct viruses sharing the same diatom host are present in western Japan and affect the population dynamics of C. tenuissimus. Moreover, the result that CtenRNAV type II lysed multiple diatom species indicates that RNA viruses may affect various diatom populations in the natural environment.

New single-stranded DNA virus with a unique genomic structure that infects marine diatom Chaetoceros setoensis

Diatoms are among the most abundant organisms in nature; however, their relationships with single-stranded DNA (ssDNA) viruses have not yet been defined in detail. We report the isolation and characterisation of a virus (CsetDNAV) that lytically infects the bloom-forming diatom Chaetoceros setoensis. The virion is 33 nm in diameter and accumulates in the nucleus of its host. CsetDNAV harbours a covalently closed-circular ssDNA genome comprising 5836 nucleotides and eight different short-complementary fragments (67–145 nucleotides), which have not been reported in other diatom viruses. Phylogenetic analysis based on the putative replicase-related protein showed that CsetDNAV was not included in the monophyly of the recently established genus Bacilladnavirus. This discovery of CsetDNAV, which harbours a genome with a structure that is unique among known viruses that infect diatoms, suggests that other such undiscovered viruses possess diverse genomic architectures.

Isolation and characterization of a single-stranded RNA virus that infects the marine planktonic diatom Chaetoceros sp. (SS08-C03)

Diatoms are the major primary producers in the worlds aquatic environment; hence, their dynamics are an important focus in current studies. Viruses, along with other physical, chemical, and biological factors, have recently been recognized as potential factors of diatom mortality. We isolated and characterized a new diatom virus (Csp03RNAV) that causes lysis of the marine planktonic diatom Chaetoceros sp. strain SS08‐C03 isolated from Hiroshima Bay, Japan. Here, we present the physiology, morphology, and genome characteristics of this virus. Csp03RNAV was isolated from surface waters of Yatsushiro Sea, Japan. Virions were icosahedral and 32 nm in diameter, and accumulated in the cytoplasm of the host cells. The latent period was estimated to be <48 h. Csp03RNAV harbors a single‐stranded RNA genome, which has 9417 bases encoding two open reading frames that code for putative replication‐related proteins and putative structural proteins, respectively. The monophyly of Csp03RNAV and the other known diatom‐infecting single‐stranded RNA viruses (genus Bacillarnavirus), Rhizosolenia setigera RNA virus, Chaetoceros socialis f. radians RNA virus, and Chaetoceros tenuissimus RNA virus was strongly supported by phylogenetic analysis based on the amino acid sequence of the RNA‐dependent RNA polymerase domain. On the basis of these results, Csp03RNAV is considered to be a new member of the genus Bacillarnavirus.

Isolation and Characterization of a Single-Stranded DNA Virus Infecting the Marine Diatom Chaetoceros sp. Strain SS628-11 Isolated from Western JAPAN

Diatoms are significant organisms for primary production in the earths aquatic environment. Hence, their dynamics are an important focus area in current studies. Viruses are a great concern as potential factors of diatom mortality, along with other physical, chemical, and biological factors. We isolated and characterized a new diatom virus (Csp07DNAV) that lyses the marine planktonic diatom Chaetoceros sp. strain SS628-11. This paper examines the physiological, morphological, and genomic characteristics of Csp07DNAV. The virus was isolated from a surface water sample that was collected at Hiroshima Bay, Japan. It was icosahedral, had a diameter of 34 nm, and accumulated in the nuclei of host cells. Rod-shaped virus particles also coexisted in the host nuclei. The latent period and burst size were estimated to be <12 h and 29 infectious units per host cell, respectively. Csp07DNAV had a closed circular single-stranded DNA genome (5,552 nucleotides), which included a double-stranded region and 3 open reading frames. The monophyly of Csp07DNAV and other Bacilladnavirus group single-stranded DNA viruses was supported by phylogenetic analysis that was based on the amino acid sequence of each virus protein. On the basis of these results, we considered Csp07DNAV to be a new member of the genus Bacilladnavirus.

RNA Sequencing Revealed Numerous Polyketide Synthase Genes in the Harmful Dinoflagellate Karenia mikimotoi

The dinoflagellate Karenia mikimotoi forms blooms in the coastal waters of temperate regions and occasionally causes massive fish and invertebrate mortality. This study aimed to elucidate the toxic effect of K. mikimotoi on marine organisms by using the genomics approach; RNA-sequence libraries were constructed, and data were analyzed to identify toxin-related genes. Next-generation sequencing produced 153,406 transcript contigs from the axenic culture of K. mikimotoi. BLASTX analysis against all assembled contigs revealed that 208 contigs were polyketide synthase (PKS) sequences. Thus, K. mikimotoi was thought to have several genes encoding PKS metabolites and to likely produce toxin-like polyketide molecules. Of all the sequences, approximately 30 encoded eight PKS genes, which were remarkably similar to those of Karenia brevis. Our phylogenetic analyses showed that these genes belonged to a new group of PKS type-I genes. Phylogenetic and active domain analyses showed that the amino acid sequence of four among eight Karenia PKS genes was not similar to any of the reported PKS genes. These PKS genes might possibly be associated with the synthesis of polyketide toxins produced by Karenia species. Further, a homology search revealed 10 contigs that were similar to a toxin gene responsible for the synthesis of saxitoxin (sxtA) in the toxic dinoflagellate Alexandrium fundyense. These contigs encoded A1–A3 domains of sxtA genes. Thus, this study identified some transcripts in K. mikimotoi that might be associated with several putative toxin-related genes. The findings of this study might help understand the mechanism of toxicity of K. mikimotoi and other dinoflagellates.

Bothrosome Formation in Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles) during Zoospore Settlement

Labyrinthulomycetes are characterized by the presence of ectoplasmic nets originating from an organelle known as the bothrosome, whose evolutionary origin is unclear. To address this issue, we investigated the developmental process from a zoospore to a vegetative cell in Schizochytrium aggregatum. After disappearance of the flagellum during zoospore settlement, the bothrosome emerged at the anterior-ventral pole of the cells. A new Golgi body also appeared at this stage, and the bothrosome was positioned close to both the new and the old Golgi bodies. This observation suggested that the Golgi body is related to the formation of the bothrosome. Actin appeared as a spot in the same location as the newly appeared bothrosome, as determined by immunofluorescence labeling. An immunoelectron microscopic analysis revealed that actin was present in the ectoplasmic nets and in the cytoplasm around the bothrosome, indicating that the electron-dense materials of the bothrosome are not the polar center of F-actin. This suggests that actin filaments pull the endoplasmic reticulum to the bothrosome and induce the membrane to become evaginated within ectoplasmic nets.

Establishment of a Bacterial Expression System and Immunoassay Platform for the Major Capsid Protein of HcRNAV, a Dinoflagellate-Infecting RNA Virus

HcRNAV is a small icosahedral virus that infects the shellfish-killing marine dinoflagellate Heterocapsa circularisquama, which harbors a dicistronic linear single-stranded RNA (ssRNA) genome ca. 4.4 kb in length. Its major capsid protein (MCP) gene sequence is not expressed by various strains of Escherichia coli, possibly because of a codon usage problem. To solve this problem, a chemically modified (i.e., de novo synthesized) gene was designed and cloned into the pCold-GST expression vector, and transformed into E. coli strain C41 (DE3), in which codon usage was universally optimized to efficiently express the polypeptide having the viral MCP amino acid sequence. The bacterially expressed protein, which was purified after a procedure involving denaturation and refolding, successfully formed virus-like particles that significantly resembled native HcRNAV particles. The purified, denatured protein was used as an antigen to immunize rabbits, and the resulting antiserum was shown to be strongly reactive to not only the bacterially expressed recombinant protein, but also to native HcRNAV MCP by Western blotting and dot immunoassays, respectively. These results indicate that an antiserum recognizing native HcRNAV MCP was successfully obtained using bacterially expressed HcRNAV MCP as the antigen.

Does suspended matter drained from the Isahaya freshwater reservoir cause organic enrichment in the northern Ariake Bay

The inner part of Isahaya Bay was converted to a freshwater reservoir following the closure of the land claim dike in 1997. Turbid water drains into Isahaya Bay when water levels increase. We investigated whether particulate organic matter (POM) from the reservoir in Isahaya Bay has caused bottom organic enrichment in the northern part of Ariake Bay. Using potential end-members from before to after the rainy seasons, during which a frequent discharge from the reservoir was expected, stable isotope analyses were performed on sediments collected from Isahaya Bay and northern Ariake Bay. Each end-member was isotopically differentiated by δ13C and δ15N (riverine POM: −28.5 to −27.2‰ and 3.3–4.6‰; reservoir POM: −25.7 to −25.3‰ and 7.4–8.4‰; marine POM: −21.8 to −19.7‰ and 6.7–7.6‰; microphytobenthos estimated from consumers: −16.1 to −15.9‰ and 5.2–6.1‰, respectively). Sediment isotopic signatures fell within the mixing space defined by the signatures of the end-members. Marine POM contributed greatly to bottom sediments in both seasons in Isahaya Bay and Ariake Bay, ranging from ca. 60–70 and 40–60%, respectively. Reservoir POM contributed around 10% to bottom sediments. This percentage slightly increased in the sediment of Isahaya Bay after the rainy season, but decreased in the sediment of Ariake Bay. Thus, most of the POM discharged from the reservoir would not reach the northern part of Ariake Bay and would not be a major contributor to organic enrichment. This study is the first to quantitatively describe the contribution of drained reservoir POM outside Isahaya Bay.

Occurrence of the planktonic bloom-forming marine diatom Chaetoceros tenuissimus Meunier and its infectious viruses in western Japan

The genus Chaetoceros is among the most species-rich marine planktonic diatoms. Most Chaetoceros are considered important primary producers in various marine environments, but because of their small size, we know little about their ecology and distribution. Therefore, from 2008 to 2012, we examined the occurrence of C. tenuissimus Meunier, one of the smallest members in the genus, and its infectious viruses in western Japanese coastal waters. Using real-time quantitative PCR, we found that C. tenuissimus was widely detected throughout our study sites, with a maximum concentration of 2.4 × 107 cells/l in May 2012. Sediment analysis revealed that C. tenuissimus resting-stage cells were present at potentially high levels, despite its infectious viruses being detected in the same region. The present study suggests that C. tenuissimus remains highly productive even when surrounded by its infectious viruses. This tolerance to viral infection, along with the diatom’s fast growth rate, suggests that C. tenuissimus might play an important role in maintaining the growth of important filter feeders.