Continuous year-round isolation of giant viruses from brackish shoreline soils.

Giant viruses, categorized under Nucleocytoviricota, are believed to exist ubiquitously in natural environments. However, comprehensive reports on isolated giant viruses remain scarce, with limited information available on unrecoverable strains, viral proliferation sites, and natural hosts. Previously, the author highlighted Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. styx, isolated from brackish water soil, as potential hotspots for giant virus multiplication. This study presents findings from nearly a year of monthly sampling within the same brackish water region after isolating the three aforementioned strains. This report details the recurrent isolation of a wide range of giant viruses. Each month, four soil samples were randomly collected from an approximately 5 × 10 m plot, comprising three soil samples and one water sample containing sediment from the riverbed. Acanthamoeba castellanii was used as a host for virus isolation. These efforts consistently yielded at least one viral species per month, culminating in a total of 55 giant virus isolates. The most frequently isolated species was Mimiviridae (24 isolates), followed by Marseilleviridae (23 isolates), Pandoravirus (6 isolates), and singular isolates of Pithovirus and Cedratvirus. Notably, viruses were not consistently isolated from any of the four samples every month, with certain sites yielding no viruses. Cluster analysis based on isolate numbers revealed that soil samples from May and water and sediment samples from January produced the highest number of viral strains. These findings underscore brackish coastal soil as a significant site for isolating numerous giant viruses, highlighting the non-uniform distribution along coastlines.

2-Dimensional genetic algorithm exhibited an essentiality of gene interaction for evolution.

Organisms consist of several genetic factors differing between species. However, the evolutionary effects of gene interactions on the evolutionary rate, adaptation, and divergence of organisms remain unknown. In a previous study, the 2-dimensional genetic algorithm (2DGA) program, including a gene interaction parameter, could simulate punctuated equilibrium under the disparity mode. Following this, we verified the effect of the number of gene interactions (gene cluster size) on evolution speed, adaptation, and divergence using the advanced 2DGA program. In this program, the population was replicated, mutated, and selected for 200,000 generations, and the fitness score, divergence, number of population, and genotype were output and plotted. The genotype data were used for evaluating the phylogenetic relations among the population. The gene cluster size 1) affected the disparity and parity mutagenesis modes differently, 2) determined the growth/exclusion rate and error threshold, and 3) accelerated or decelerated the population's speed of evolutionary advancement. In particular, when the gene cluster size expanded, the rate of increase in fitness scores decreased independently of the mutation rate and mode of mutation (disparity mode/parity mode). The mutation rate at the error threshold was also decreased by expanding the gene cluster size. Dendrograms traced the genotypes of the simulated population, indicating that the disparity mode caused the evolutionary process to enter 1) a stun mode, 2) an evolution mode, or 3) a divergence mode based on the mutation rate and gene cluster size, while the parity mode did not cause the population to enter a stun mode. Based on the above findings, we compared the predictions of the present study with evolution observed in the laboratory or the natural world and the processes of ongoing virus evolution, suggesting that our findings possibly explained the real evolution.

Co-Isolation and Characterization of Two Pandoraviruses and a Mimivirus from a Riverbank in Japan.

Giant viruses, like pandoraviruses and mimiviruses, have been discovered from diverse environments, and their broad global distribution has been established. Here, we report two new isolates of Pandoravirus spp. and one Mimivirus sp., named Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. isolate styx, co-isolated from riverbank soil in Japan. We obtained nearly complete sequences of the family B DNA polymerase gene (polB) of P. hades and P. persephone; the former carried two known intein regions, while the latter had only one. Phylogenetic analysis revealed that the two new pandoravirus isolates are closely related to Pandoravirus dulcis. Furthermore, random amplified polymorphic DNA analysis revealed that P. hades and P. persephone might harbor different genome structures. Based on phylogenetic analysis of the partial polB sequence, Mimivirus sp. isolate styx belongs to mimivirus lineage A. DNA staining suggested that the Pandoravirus spp. asynchronously replicates in amoeba cells while Mimivirus sp. replicates synchronously. We also observed that P. persephone- or Mimivirus sp. isolate styx-infected amoeba cytoplasm is extruded by the cells. To the best of our knowledge, we are the first to report the isolation of pandoraviruses in Asia. In addition, our results emphasize the importance of virus isolation from soil to reveal the ecology of giant viruses.

Distribution of SNSs in Mimivirus Genomes and the Classification of Mimiviruses Isolated from Japan.

Mimiviruses have been detected in various habitats. Analyses of single nucleotide substitutions (SNSs) have revealed that SNSs are mainly localized on both ends of the mimivirus genome, and mimivirus lineage A has been split into three genotype groups; therefore, mimiviruses may be classified into lineages and genotype groups based on SNSs. We isolated 9 mimiviruses from Japan and analyzed SNSs. These isolates were classified as lineage A genotype group type 2, suggesting that the local diversity of members of the family Mimiviridae isolated from Acanthamoeba spp. is lower than that of giant viruses from other families isolated in Japan.

Fifteen Marseilleviruses Newly Isolated From Three Water Samples in Japan Reveal Local Diversity of Marseilleviridae.

The family Marseilleviridae, defined as a group of icosahedral double-stranded DNA viruses with particle size of approximately 250 nm and genome size of 350-380 kbp, belongs to the nucleo-cytoplasmic family of large DNA viruses. The family Marseilleviridae is currently classified into lineages A-E. In this study, we isolated 12 or 15 new members of the family Marseilleviridae from three sampling locations in Japan. Molecular phylogenetic analysis of the MCP genes showed that the new viruses could be further classified into three groups, hokutoviruses, kashiwazakiviruses, and kyotoviruses. Hokutoviruses were closely related to lineage B, kyotoviruses were related to lineage A, and kashiwazakiviruses were also classified into lineage B but a new putative subgroup of lineage B, revealing the diversity of this lineage. Interestingly, more than two viruses with slightly different MCP genes were isolated from a single water sample from a single location, i.e., two hokutoviruses and one kashiwazakivirus were isolated from a small reservoir, five kashiwazakiviruses from the mouth of a river, and five kyotoviruses from fresh water of a river, suggesting that several milliliters of water samples contain several types of giant viruses. Amoeba cells infected with hokutoviruses or kashiwazakiviruses exhibited a "bunch" formation consisting of normal and infected cells similarly to a tupanvirus, whereas cells infected with kyotoviruses or tokyovirus did not. These results suggest the previously unrecognized local diversity of the family Marseilleviridae in aquatic environments.

Visualization of giant virus particles and development of "VIRAMOS" for high school and university biology course.

Several educational trials on handling viruses and or virology have been reported. However, given their small size, direct visualization of these viruses under a microscope has been rarely performed. The so-called "giant viruses" are larger than other viruses with a particle size greater than 200-300 nm. This enables their direct visualization under a light microscope more easily than other viruses. In this study, we developed two new types of teaching material for learning about viruses and cellular organisms using mimivirus, one of the well-known giant viruses. One teaching material involves using glass slides with enclosed mimivirus particles, and another is a paper-based teaching material, named VIRAMOS (http://tlab-edusys.azurewebsites.net/content/viramos_en.pdf). Using these, students can investigate and learn about viruses and cellular organisms. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):426-431, 2019.

Gram-Positive Bacteria-Like DNA Binding Machineries Involved in Replication Initiation and Termination Mechanisms of Mimivirus.

The detailed mechanisms of replication initiation, termination and segregation events were not yet known in Acanthamoeba polyphaga mimivirus (APMV). Here, we show detailed bioinformatics-based analyses of chromosomal replication in APMV from initiation to termination mediated by proteins bound to specific DNA sequences. Using GC/AT skew and coding sequence skew analysis, we estimated that the replication origin is located at 382 kb in the APMV genome. We performed homology-modeling analysis of the gamma domain of APMV-FtsK (DNA translocase coordinating chromosome segregation) related to FtsK-orienting polar sequences (KOPS) binding, suggesting that there was an insertion in the gamma domain which maintains the structure of the DNA binding motif. Furthermore, UvrD/Rep-like helicase in APMV was homologous to Bacillus subtilis AddA, while the chi-like quartet sequence 5'-CCGC-3' was frequently found in the estimated ori region, suggesting that chromosomal replication of APMV is initiated via chi-like sequence recognition by UvrD/Rep-like helicase. Therefore, the replication initiation, termination and segregation of APMV are presumably mediated by DNA repair machineries derived from gram-positive bacteria. Moreover, the other frequently observed quartet sequence 5'-CGGC-3' in the ori region was homologous to the mitochondrial signal sequence of replication initiation, while the comparison of quartet sequence composition in APMV/Rickettsia-genome showed significantly similar values, suggesting that APMV also conserves the mitochondrial replication system acquired from an ancestral genome of mitochondria during eukaryogenesis.

Transposition of insertion sequence IS256Bsu1 in Bacillus subtilis 168 is strictly dependent on recA.

We developed an insertion sequence transposition detection system called the "jumping cat assay" and applied it to the Bacillus subtilis chromosome using IS256Bsu1 derived from B. subtilis natto. The high frequency of transposition enabled us to explore host factors; combining the assay and genetic analyses revealed that recA is essential for the transposition of IS256Bsu1. Detailed analyses using various domain mutants of recA demonstrated that this essentiality is not related to the function of recA in homologous recombination. Instead, the ATP binding and hydrolysis function seemed to be crucial for IS transposition. To elucidate the role of recA, we focused on the muB gene of the enterobacteriophage Mu. Based on information from the NCBI Conserved Domain Database, both MuB and RecA belong to the P-loop dNTPase superfamily. Further experiments revealed that muB complements the transposition-defective phenotype of a recA deletant, although it could not rescue UV sensitivity. These results suggest that recA shares a common function with muB that helps the transposition of IS256Bsu1 in B. subtilis.

Distribution of human single-nucleotide polymorphisms is approximated by the power law and represents a fractal structure.

Single-nucleotide polymorphisms (SNPs) are one of the main causes of evolution. The distribution of human SNPs, which were examined in detail genomewide, was analyzed. Three discrete databases of human SNPs were used for this analysis, and similar results were obtained from these databases. It was found that the distribution of the distance between SNPs was approximated by the power law, and the shape of the regions including SNPs had the so-called fractal structure. Although the reason why the distribution of SNPs obeys such a certain law of physics is unclear, a speculation was attempted in connection with the three-dimensional structure of human chromatin which has a fractal structure.

Relevance of GC content to the conservation of DNA polymerase III/mismatch repair system in Gram-positive bacteria.

The mechanism of DNA replication is one of the driving forces of genome evolution. Bacterial DNA polymerase III, the primary complex of DNA replication, consists of PolC and DnaE. PolC is conserved in Gram-positive bacteria, especially in the Firmicutes with low GC content, whereas DnaE is widely conserved in most Gram-negative and Gram-positive bacteria. PolC contains two domains, the 3'-5'exonuclease domain and the polymerase domain, while DnaE only possesses the polymerase domain. Accordingly, DnaE does not have the proofreading function; in Escherichia coli, another enzyme DnaQ performs this function. In most bacteria, the fidelity of DNA replication is maintained by 3'-5' exonuclease and a mismatch repair (MMR) system. However, we found that most Actinobacteria (a group of Gram-positive bacteria with high GC content) appear to have lost the MMR system and chromosomes may be replicated by DnaE-type DNA polymerase III with DnaQ-like 3'-5' exonuclease. We tested the mutation bias of Bacillus subtilis, which belongs to the Firmicutes and found that the wild type strain is AT-biased while the mutS-deletant strain is remarkably GC-biased. If we presume that DnaE tends to make mistakes that increase GC content, these results can be explained by the mutS deletion (i.e., deletion of the MMR system). Thus, we propose that GC content is regulated by DNA polymerase and MMR system, and the absence of polC genes, which participate in the MMR system, may be the reason for the increase of GC content in Gram-positive bacteria such as Actinobacteria.