Biology subjects

Hiraoka, S.

Publications and source records attributed to Hiraoka, S..

9 recordsLinked to original sources

bioRxiv2026

Epigenomes in thermophilic microbial communities and their impact on the interaction across prokaryotes and mobilomes

DNA chemical modifications, including methylation, are widespread in prokaryotes and mobilomes, including viruses, plasmids, and other extrachromosomal DNAs, and play important roles in their ecology and interactions. However, current knowledge of these modification systems and their association with interactions between hosts and mobilomes across communities, including those in extreme environments, is severely limited. Here, using single-molecule real-time sequencing and single-cell genome sequencing technologies, we conducted a culture-independent metaepigenomic analysis of microbial communities in hot spring biofilms. A total of 248, 332, and 465 genomes were constructed from diverse prokaryotes, viruses, and extrachromosomal circular DNAs, respectively, from 10 biofilm samples collected from 3 hot spring sites. In total, 1106 candidate methylated motifs and 3280 genes associated with the restriction-modification (RM) system, including DNA methyltransferases (MTases), were identified. In contrast to the varied methylated motifs, the nucleotide-level modification ratios were consistent with those of a common Escherichia coli genome, and an environment-dependent epigenomic preference attributed to the lack of C5-methylcytosine was observed, as supported by direct measurements of modified bases by liquid chromatography-tandem mass spectrometry. A systematic survey revealed various defense systems in the genome, and almost half of the MTase genes were estimated to be genetically involved in defense mechanisms against extracellular DNA, such as RM systems. The mobilomes and their predicted hosts shared epigenomic patterns within each interactive subnetwork, suggesting that mobilome DNA was modified by host MTase during the current infection or transfection, rather than serving as historical records. Our findings highlight that DNA modification shapes multiple ecological and evolutionary strategies in interactions between prokaryotes and mobilomes, and that epigenomes serve as a potential signature for accurate prediction of current host-phage interactions.

microbiology↗

bioRxiv2025

Direct carbon monoxide fixation via the bacterial and archaeal Wood-Ljungdahl pathways

The Wood-Ljungdahl (WL) pathway, which is widely distributed in both archaea and bacteria, is an ancient carbon fixation pathway from CO2. CO2 fixation proceeds via two branches of pathway: progressive reduction to the methyl group in the methyl branch and one-step reduction to CO in the carbonyl branch. In the final step of the pathway, the methyl group, CO, and CoA are combined into a carbon monoxide dehydrogenase (CODH)/acetyl-CoA synthase (ACS) complex to form acetyl-CoA. Here, we show direct CO fixation to the carbonyl group of acetyl-CoA in both archaeal and bacterial WL pathways under hydrogenogenic growth conditions using 13C tracer-based metabolomics. A combination of metabolomics and proteomics suggested that the hydrogenotrophically grown Thermodesulfatator indicus and Archaeoglobus sp. strain MCR cells, directly fixed CO using free-form ACS in the carbonyl branch with relatively low CO availability. In contrast, carboxydotrophically grown Archaeoglobus cells utilize the CODH/ACS complex for CO2 fixation rather than CO fixation. Direct CO fixation by free-form ACS is more advantageous for conserving reduced ferredoxin compared with the thermodynamically challenged CO2 reduction by CODH. These findings provide further insight into the origin and evolution of the most ancient inorganic carbon fixation pathway and geochemical cycles on early Earth.

microbiology↗

bioRxiv2024

Host-encoded DNA methyltransferases modify the epigenome and host tropism of invading phages

Restriction modification (RM) systems are ubiquitous bacterial defense systems; however, some phages evade RM system and adapt to their bacterial hosts. In such cases, phages are thought to stochastically acquire DNA methylation from host-encoded DNA methyltransferases (MTases), facilitating host adaptation. However, no studies have directly compared the methylomes of host bacteria and their infecting phages. Here, we demonstrate the epigenetic landscape of adapted phages with diverse infection histories, focusing on the broad host-range phage KHP30T as its adapts to three Helicobacter pylori strains. Using a multistage infection system, we observed that the adapted phages displayed significantly high titers against the last infected H. pylori strain, suggesting an attendant change in host tropism. Single-molecule real-time sequencing revealed that methylated motifs were predominantly shared between the adapted phages and their most recent host. Our findings enhance our understanding of epigenetic phage-host interactions, which have significant implications for microbial ecology.

microbiology↗

bioRxiv2023

Genetic and functional diversity of beta-N-acetylgalactosamine residue-targeting glycosidases expanded by deep-sea metagenome

{beta}-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. {beta}-N-Acetylgalactosaminidases hydrolyze {beta}-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-{beta}-N-acetylgalactosaminidases that specifically act on {beta}-N-acetylgalactosamine residues, has been documented so far. In this study, we identified three novel glycoside hydrolase families distributed among all three domains of life and characterized eight novel {beta}-N-acetylgalactosaminidases and {beta}-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and diversify their substrate specificities (endo-, dual-endo/exo-, and exo-) through the accumulation of mutations and insertional amino acid sequences. The diverse {beta}-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present novel evolutionary pathways for expanding their substrate specificity.

biochemistry↗

bioRxiv2023

Probe capture enrichment sequencing of amoA genes discloses diverse ammonia-oxidizing archaeal and bacterial populations

The ammonia monooxygenase subunit A (amoA) gene has been used to investigate the phylogenetic diversity, spatial distribution, and activity of ammonia-oxidizing archaeal (AOA) and bacterial (AOB), which contribute significantly to the nitrogen cycle in various ecosystems. Amplicon sequencing of amoA is a widely used method; however, it produces inaccurate results owing to the lack of a universal primer set. Moreover, currently available primer sets suffer from amplification biases, which can lead to severe misinterpretation. Although shotgun metagenomic and metatranscriptomic analyses are alternative approaches without amplification bias, the low concentration of target genes in heterogeneous environmental DNA restricts a comprehensive analysis to a realizable sequencing depth. In this study, we developed a method for amoA enrichment sequencing using a hybridization capture technique. Using metagenomic mock community samples, our approach effectively enriched amoA genes with low compositional changes, outperforming amplification and meta-omics sequencing analyses. Following the analysis of metatranscriptomic marine samples, we predicted 80 operational taxonomic units (OTUs) assigned to either AOA or AOB, of which 30 OTUs were unidentified using simple metatranscriptomic or amoA gene amplicon sequencing. Mapped read ratios to all the detected OTUs were significantly higher for the capture samples (50.4 {+/-} 27.2%) than for non-capture samples (0.05 {+/-} 0.02%), demonstrating the high enrichment efficiency of the method. The analysis also revealed the spatial diversity of AOA ecotypes with high sensitivity and phylogenetic resolution, which are difficult to examine using conventional approaches.

microbiology↗

bioRxiv2021

Diverse DNA modification in marine prokaryotic and viral communities

Chemical modifications of DNA, including methylation, play an important role in prokaryotes and viruses. However, our knowledge of the modification systems in environmental microbial communities, typically dominated by members not yet cultured, is limited. Here, we conducted metaepigenomic analyses by single-molecule real-time sequencing of marine microbial communities. In total, 233 and 163 metagenomic assembly genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTases were not associated with the defense mechanism. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative motifs in the methylation system that are highly conserved in Alphaproteobacteria, illuminating the co-evolutionary history of the methylation system and host genome. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses.

microbiology↗

bioRxiv2019

Microbial community and geochemical analyses of trans-trench sediments for understanding the roles of hadal environments

Hadal trench bottom (>6,000 m below sea level) sediments harbor higher microbial cell abundance compared to adjacent abyssal plain sediments. This is supported by the accumulation of sedimentary organic matter (OM), facilitated by trench topography. However, the distribution of benthic microbes in different trench systems has not been explored yet. Here, we carried out small subunit ribosomal RNA gene tag sequencing for 92 sediment subsamples of seven abyssal and seven hadal sediment cores collected from three trench regions in the northwest Pacific Ocean: the Japan, Izu-Ogasawara, and Mariana Trenches. Tag-sequencing analyses showed specific distribution patterns of several phyla associated with oxygen and nitrate. The community structure was distinct between abyssal and hadal sediments, following geographic locations and factors represented by sediment depth. Co-occurrence network revealed six potential prokaryotic consortiums that covaried across regions. Our results further support that the endogenous OM cycle is driven by hadal currents and/or rapid burial shapes microbial community structures at trench bottom sites, in addition to vertical deposition from the surface ocean. Our trans-trench analysis highlights intra- and inter-trench distributions of microbial assemblages and geochemistry in surface seafloor sediments, providing novel insights into ultra-deep-sea microbial ecology, one of the last frontiers on our planet.

microbiology↗

bioRxiv2018

Metaepigenomic analysis reveals the unexplored diversity of DNA methylations in an environmental prokaryotic community

DNA methylation plays important roles in prokaryotes, such as in defense mechanisms against phage infection, and the corresponding genomic landscapes--prokaryotic epigenomes--have recently begun to be disclosed. However, our knowledge of prokaryote methylation systems has been severely limited to those of culturable prokaryotes, whereas environmental communities are in fact dominated by uncultured members that must harbor much more diverse DNA methyltransferases. Here, using single-molecule real-time and circular consensus sequencing techniques, we revealed the metaepigenomes of an environmental prokaryotic community in the largest lake in Japan, Lake Biwa. A total of 19 draft genomes from phylogenetically diverse groups, most of which are yet to be cultured, were successfully reconstructed. The analysis of DNA chemical modifications identified 29 methylated motifs in those genomes, among which 14 motifs were novel.\n\nFurthermore, we searched for the methyltransferase genes responsible for the methylation of the detected novel motifs and confirmed their catalytic specificities via transformation experiments involving artificially synthesized genes. Finally, we found that genomes without DNA methylation tended to exhibit higher phage infection levels than those with methylation. In summary, this study proves that metaepigenomics is a powerful approach for revealing the vast unexplored variety of prokaryotic DNA methylation systems in nature.

microbiology↗

bioRxiv2017

Collaborative environmental DNA sampling from petal surfaces of flowering cherry Cerasus ×- yedoensis 'Somei-yoshino' across the Japanese archipelago

Recent studies have shown that environmental DNA is found almost everywhere. Flower petal surfaces are an attractive tissue to use for investigation of the dispersal of environmental DNA in nature as they are isolated from the external environment until the bud opens and only then can the petal surface accumulate environmental DNA. Here, we performed a crowdsourced experiment, the \"Ohanami Project\", to obtain environmental DNA samples from petal surfaces of Cerasus x yedoensis Somei-yoshino across the Japanese archipelago during spring 2015. C. x yedoensis is the most popular garden cherry species in Japan and clones of this cultivar bloom simultaneously every spring. Data collection spanned almost every prefecture and totaled 577 DNA samples from 149 collaborators. Preliminary amplicon-sequencing analysis showed the rapid attachment of environmental DNA onto the petal surfaces. Notably, we found DNA of other common plant species in samples obtained across a wide distribution; this DNA likely originated from pollen of the Japanese cedar. Our analysis supports our belief that petal surfaces after blossoming are a promising target to reveal the dynamics of environmental DNA in nature. The success of our experiment also shows that crowdsourced environmental DNA analyses have considerable value in ecological studies.

microbiology↗