Hydrogenogenic carbon monoxide (CO)-oxidizing bacteria (hereafter referred to as CO-oxidizing bacteria) conserve energy using an anaerobic CO dehydrogenase containing Ni (Ni-CODH) and hydrogen-producing hydrogenase (ECH). In this study, we promoted the exploration and isolation of undescribed microbial species by using the established enrichment culture method in which the gas phase is replaced by CO to expand the culture strain collection as a genetic resource. Parageobacillus sp. strain G301 was successfully isolated from the sediment of Unagi-ike Lake in Kagoshima Prefecture, Japan, and closely related to Parageobacillus toebii, which lacks CODH/ECH. Furthermore, the genome of strain G301 encoded a gene cluster for aerobic CODH containing Mo (Mo-CODH). Culture experiments demonstrated that the strain enabled both hydrogenic anaerobic and aerobic CO oxidation, which had not been previously reported. We also obtained a CO oxidizing bacterial isolate of Thermolongibacillus altinsuensis which has not been reported to be capable of CO oxidation, from a sediment sample from the bottom of Lake Biwa. A comprehensive search in public metagenomes showed that Ni-CODH was distributed in a wide range of prokaryotes spanning 55 bacterial and 11 archaeal phyla. Although Ni-CODH is a universal enzyme that exists in a wide variety of microbiome, including aquatic environments such as oceans, artificial environments, and mammalian microflora including humans, Ni-CODH responsible for hydrogenogenic CO oxidation was rarely found in the metagenomic data, suggesting the importance of the isolation and cultivation. We also found an acetyl CoA production pathway, in which Ni-CODH is a key enzyme, in heterotrophic, human gut-derived enterobacteria, and expanded the genomic database of potential aerobic marine CO-oxidizing bacteria in the ocean. These results in this study could expand our knowledge on Ni-CODH and CO oxidizers and are expected to provide a basis not only for utilization as a genetic resource, but also for elucidating unpreceded metabolisms of CO-oxidizing bacteria and their ecological functions on the surrounding environment.

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How cells wake up from dormancy remains enigmatic, although it has long been known that Schizosaccharomyces pombe spores break dormancy in response to glucose uptake from their environment. Expression profiling upon dormancy breaking must be a clue for the question. As RNA-seq profiles made from bulk spores may not display sufficient temporal resolution, we established a system to perform single cell RNA-seq (scRNA-seq) to capture the transcriptional landscape of a single spore upon dormancy breaking. scRNA-seq was performed over 64 single spores in dormancy or germination, and each single-cell transcriptome was mutually compared to predict how transcriptomes changed over time upon dormancy breaking. Thus, each transcriptome was aligned along the 'Virtual Timeline' and a histone H3 gene was highlighted as a differentially expressed gene upon dormancy breaking. The data highlighted a possibility that chromatin dramatically change in accordance with progression of germination.

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Olive bacterial wilt was first reported in Kagawa prefecture in Japan in 2017. Ralstonia pseudosolanacearum, the causal agent of bacterial wilt disease in Solanaceae plants, was identified as the causal agent of olive bacterial wilt disease. In this study, we aimed to identify specific virulence factors for R. pseudosolanacearum isolated from olive bacterial wilt and to elucidate the mechanism of infection of olives. To search for specific virulence factors of the pathogen, 26 strains isolated from 6 prefectures in Japan were classified based on phylotype, sequevar, and biovar. The characterization of the strains showed that 20 out of 26 strains were classified as phylotype I, sequevar 14, and biovar 3. These results suggest that R. pseudosolanacearum strains with specific genetic backgrounds infect olives and cause olive bacterial wilt. Whole genome sequence analyses of eight olive bacterial wilt strains were performed by next- generation sequencing (NGS), and comparative genomic analyses were conducted. Especially, the type III secretion effectors (T3Es) repertoires of each strain were compared with bacterial wilt strains isolated from tomato, and eggplant, and identified three effectors, RipBE, RipBK, or RipBQ specific to the genome of olive bacterial wilt strains. To investigate the molecular functions of the effectors from olive bacterial wilt strains, 72 T3Es from the olive bacterial wilt strain, YPPS1660, were systematically expressed in the budding yeast Saccharomyces cerevisiae. Among them, we found that expression of RipBK, which is specifically harbored by some olive bacterial wilt strains, strongly inhibited yeast growth and that this growth inhibition was dependent on the Toll/Interleukin-1 receptor (TIR) domain in the RipBK molecule. The TIR domain has recently been shown to possess NADase activity that degrades NAD⁺. Therefore, we measured the NADase activity of recombinant RipBK protein expressed in Escherichia coli and found that it showed only a very low activity. Interestingly, the addition of yeast lysate to the recombinant RipBK protein increased NADase activity in a concentration-dependent manner. This suggests that NADase activity of RipBK is stimulated by a substance(s) derived from host eukaryotes.

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Escherichia coli produces extracellular vesicles called outer membrane vesicles (OMVs) by releasing part of its outer membrane. We previously reported that the combined deletion of nlpI and mlaE, related to envelope structure and phospholipid accumulation in the outer leaflet of the outer membrane, respectively, resulted in the synergistic increase of OMV production. In this study, the analysis of ΔmlaEΔnlpI cells using quick-freeze, deep- etch electron microscopy (QFDE-EM) revealed that plasmolysis occurred at the tip of the long axis in cells and that OMVs formed from this tip. Furthermore, intracellular vesicles and multilamellar OMV were observed in the ΔmlaEΔnlpI cells. QFDE-EM analysis also revealed that ΔmlaEΔnlpI sacculi contained many holes noticeably larger than the mean radius of the peptidoglycan pores in wild-type E. coli. These results suggest that in ΔmlaEΔnlpI cells, cytoplasmic membrane materials protrude into the periplasmic space through the peptidoglycan holes and are released as outer-inner membrane vesicles (OIMVs). Meanwhile, the secretion of recombinant GFP expressed in the cytosol of the ΔmlaEΔnlpI cells was about 2mg/L which was more than 100 times higher than that of WT, suggesting that OMV phenomenon is able to be applied for the secretory protein expression system of E. coli. Next, secretory production of fatty acid via OMVs was also examined using the multiple gene knockout mutant strain. However, it was failed due to the sever suppression of cell growth and fatty acid production. Expression suppression of mlaE and nlpI using Clustered Regularly Interspaced Palindromic Repeats Interference (CRISPRi) achieved the increase of OMV production while maintaining cell activity. In the future, we will optimize the expression levels of each gene for secrete production of useful compounds via OMVs.

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Among the many d-amino acids which have healthy functions, D-serine reportedly prevents cognitive impairment of the human brain. After lactic acid bacteria capable of producing D-serine were selected, we investigated the bacteria cultivation conditions for production of D-serine. Many microorganisms that have serine racemase produce D-serine from L-serine. For this study, D-serine was assayed using colorimetry, for which peroxide produced by amino acid oxidase reacting with D-serine was colored using methyl benzothiazolinonehydrazone (MBTH) and dimethylaniline (DMA). Lactic acid bacteria (129 strains) were cultivated in media containing L-serine. After cultivation, the bacteria-converted D-serine was assayed. Results demonstrated high D-serine conversion potential for the H74 strain, which was identified as Lactococcus lactis ssp. lactis according to its morphological, physiological, and molecular biological characteristics. The H74 strain converted efficiently L-serine to D-serine in a medium containing 1.0% L-serine. The strain converted efficiently L-serine to D-serine at 15°C, and the conversion rate at 15°C was found to be four times higher than that found at 30°C. The high conversion rate at 15 °C continued for 8 days. Serine racemase activity in the strain was found in the cell membrane and cytoplasm fractions. These findings suggest that serine racemase is a membrane-binding cytoplasmic enzyme. From these findings, we infer that the strain has high potential for the production of D-serine and foods that contain D-serine as supplements supporting health.

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Filamentous fungi, which inhabit diverse environments on earth, are ecologically important as they contribute to material cycles based on biomass decomposition, and they also have a strong influence on human society through their effects on animal, plant, and human diseases and their application in the fermentation industry. In the environment, they interact with microorganisms with different growth mechanisms, such as viruses, bacteria, and fungi, and a detailed and comprehensive understanding of these interactions is important for understanding the true ecology of filamentous fungi. Therefore, our laboratory has focused on 1) viruses in filamentous fungi cells, 2) co-cultures between filamentous fungi, and 3) interactions with microbial community, and has been analyzing these interactions. In particular, we have explored filamentous fungal viruses (mycoviruses) on a large scale, which have not been given much importance, and have contributed to our understanding of the diversity of mycoviruses and the mechanisms by which they express their functions in the host. Co-culture experiments with other fungi and filamentous fungi have also revealed the molecular basis of the mutual response that induces the production of secondary metabolites. We believe that this detailed and bird's-eye understanding of the interactions between organisms based on filamentous fungi will establish a new field of filamentous fungi research in the future.

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In recent years, virus surveillance in filamentous fungi have revealed the existence of numerous novel viruses, which revolutionized our understanding of viral diversity on earth. In this study, we applied a novel virus discovery method to filamentous fungi, which has enhanced sensitivity and various advantages compared to conventional approaches. This enabled us to identify a large number of viruses that had been overlooked until now. In the present study, we discovered a divided RNA-dependent RNA polymerase (RdRp), which is an essential RNA viral replication enzyme and empirically considered to be encoded by a single gene. Due to such diversity of viral structure and untapped fungal resources, filamentous fungi currently are a promising model host organism for the research of RNA viruses.

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Several mycoviruses that have a significant impact on fungal pathogenicity have been extensively studied over the years. However, researchers have also noticed that a number of mycoviruses that do not exhibit any noticeable effect on host phenotypes exist. This empirical fact suggests that the previously analyzed effects of mycoviruses represent exceptional cases and only provide a partial view of their function. Therefore, it is necessary to study the comprehensive effects of mycoviruses regardless of visible changes in the host phenotype. Firstly, to facilitate the study of a wide range of mycoviruses, we developed an efficient virus elimination system. In this regard, we conducted comparative analyses of virus elimination rates using various nucleoside analogs and observed that the efficacy of elimination depends on the specific combination of the analog and the viral genome type. Secondly, we also obtained virus-free strains from multiple virus-harboring fungal strains/species. Based on the comparative analyses, it was revealed that most mycoviruses induced host-phenotype change but it was limited to the specific conditions. Our data suggested that mycoviruses are not destructive but mild parasites of filamentous fungi, possibly to expand fungal phenotypic diversity without genetic change.

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Fungi produce various secondary metabolites, and thus are exploited as a source of useful compounds. Studies of fungal genomes have revealed that fungi have the potential to produce more secondary metabolites than expected, as they have a large number of genes for secondary metabolism. To date, some knowledge on control of fungal secondary metabolite production by mycoviruses has accumulated. In this study, we evaluated the influence of mycoviruses on fungal secondary metabolism using a rice blast fungus Magnaporthe oryzae that is infected with three types of mycoviruses: a totivirus, a chrysovirus, and a partitivirus. As a result, it was revealed that a mycotoxin tenuazonic acid was produced in a manner dependent on the totivirus infection. Transcriptome analysis revealed the regulator y mechanism under viral induction of tenuazonic acid: the totivirus activates the transcription factor gene TAS2 that upregulates the tenuazonic acid biosynthetic gene TAS1. To the best of our knowledge, this is the first report that confirmed mycovirus-associated regulation of secondary metabolism at a transcriptional level. Our finding highlights the potential of mycoviruses as a epigenomic factor activating fungal secondary metabolism.

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Fungi have the potential to produce various biologically active secondary metabolites. Fungal genomes contain far more genes for secondary metabolites than the number of metabolites that have been identified. In several studies, novel fungal metabolites were discovered on co-culturing fungus with another microorganism. Therefore, the coculturing method has attracted much attention in this field. However, study of the molecular mechanisms underlying this effect is in its infancy. In this research, we used strains of the fungal genus Aspergillus in co-culture to enhance secondary metabolites production. Aspergillus nidulans showed enhanced production of antibacterial compounds called diphenyl ethers when it was co-cultured with Aspergillus fumigatus. We identified a gene cluster (the ors cluster) that are involved in production of these metabolites. Notably, this cluster was previously demonstrated to be responsible for production of a different compound, orsellinic acid, when Aspergillus nidulans was co-cultured with the bacterium Streptomyces rapamycinicus. To gain insight into the differences in metabolite biosynthesis during fungal–bacterial and fungal–fungal interaction, the biosynthetic pathway of co-culture-induced metabolites were analyzed and reconstructed in Aspergillus oryzae. Our model co-culture system serves as a foundation for understanding fungal secondary metabolic response to fungal–fungal interaction.

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