Details of a Researcher - Arakawa, Kazuharu

Papers - Arakawa, Kazuharu

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Genetic mutation in Escherichia coli genome during adaptation to the murine intestine is optimized for the host diet

Tsukimi T., Obana N., Shigemori S., Arakawa K., Miyauchi E., Yang J., Song I., Ashino Y., Wakayama M., Soga T., Tomita M., Ohno H., Mori H., Fukuda S.

Msystems 9 ( 2 ) 2024.02

Mammalian gut microbes colonize the intestinal tract of their host and adapt to establish a microbial ecosystem. The host diet changes the nutrient profile of the intestine and has a high impact on microbiota composition. Genetic mutations in Escherichia coli, a prevalent species in the human gut, allow for adaptation to the mammalian intestine, as reported in previous studies. However, the extent of colonization fitness in the intestine elevated by genetic mutation and the effects of diet change on these mutations in E. coli are still poorly known. Here, we show that notable mutations in sugar metabolism-related genes (gatC, araC, and malI) were detected in the E. coli K-12 genome just 2 weeks after colonization in the germ-free mouse intestine. In addition to elevated fitness by deletion of gatC, as previously reported, deletion of araC and malI also elevated E. coli fitness in the murine intestine in a host diet-dependent manner. In vitro cultures of medium containing nutrients abundant in the intestine (e.g., galactose, N-acetylglucosamine, and asparagine) also showed increased E. coli fitness after deletion of the genes-of-interest associated with their metabolism. Furthermore, the host diet was found to influence the developmental trajectory of gene mutations in E. coli. Taken together, we suggest that genetic mutations in E. coli are selected in response to the intestinal environment, which facilitates efficient utilization of nutrients abundant in the intestine under laboratory conditions. Our study offers some insight into the possible adaptation mechanisms of gut microbes.

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The Evolution of Temperature and Desiccation-Related Protein Families in Tardigrada Reveals a Complex Acquisition of Extremotolerance

Fleming J.F., Pisani D., Arakawa K.

Genome Biology and Evolution 16 ( 1 ) 2024.01

Tardigrada is an ecdysozoan lineage famed for its resilience. Tardigrades can tolerate high doses of radiation, low-oxygen environments, desiccation, and both high and low temperatures under a dormant state called "anhydrobiosis", which is a reversible halt of metabolism upon almost complete desiccation. A large amount of research has focused on the genetic pathways related to these capabilities, and a number of genes have been identified and linked to the extremotolerant response of tardigrades. However, the history of these genes is unclear, and the origins and history of extremotolerant genes within Tardigrada remain a mystery. Here, we generate the first phylogenies of six separate protein families linked with desiccation and radiation tolerance in Tardigrada: cytosolic abundant heat-soluble protein, mitochondrial abundant heat-soluble protein, secretory abundant heat-soluble protein, meiotic recombination 11 homolog, and the newly discovered Echiniscus testudo abundant heat-soluble proteins (alpha and beta). The high number of independent gene duplications found amongst the six gene families studied suggests that tardigrades have a complex history with numerous independent adaptations to cope with aridity within the limnoterrestrial environment. Our results suggest that tardigrades likely transitioned from a marine environment to a limnoterrestrial environment only twice, once in stem Eutardigrada and once in Heterotardigrada, which explains the unique adaptations to anhydrobiosis present in both classes.

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Complete genome sequence of Limnobacter thiooxidans CS-K2T, isolated from freshwater lake sediments in Bavaria, Germany

Naruki M., Watanabe A., Warashina T., Morita T., Arakawa K.

Microbiology Resource Announcements 13 ( 1 ) 2024.01

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Reliability of Time-Series Plasma Metabolome Data over 6 Years in a Large-Scale Cohort Study

Miyake A., Harada S., Sugiyama D., Matsumoto M., Hirata A., Miyagawa N., Toki R., Edagawa S., Kuwabara K., Okamura T., Sato A., Amano K., Hirayama A., Sugimoto M., Soga T., Tomita M., Arakawa K., Takebayashi T., Iida M.

Metabolites 14 ( 1 ) 2024.01

Studies examining long-term longitudinal metabolomic data and their reliability in large-scale populations are limited. Therefore, we aimed to evaluate the reliability of repeated measurements of plasma metabolites in a prospective cohort setting and to explore intra-individual concentration changes at three time points over a 6-year period. The study participants included 2999 individuals (1317 men and 1682 women) from the Tsuruoka Metabolomics Cohort Study, who participated in all three surveys—at baseline, 3 years, and 6 years. In each survey, 94 plasma metabolites were quantified for each individual and quality control (QC) sample. The coefficients of variation of QC, intraclass correlation coefficients, and change rates of QC were calculated for each metabolite, and their reliability was classified into three categories: excellent, fair to good, and poor. Seventy-six percent (71/94) of metabolites were classified as fair to good or better. Of the 39 metabolites grouped as excellent, 29 (74%) in men and 26 (67%) in women showed significant intra-individual changes over 6 years. Overall, our study demonstrated a high degree of reliability for repeated metabolome measurements. Many highly reliable metabolites showed significant changes over the 6-year period, suggesting that repeated longitudinal metabolome measurements are useful for epidemiological studies.

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Possible roles of CAHS proteins from Tardigrade in osmotic stress tolerance in mammalian cells

Bino T., Goto Y., Maryu G., Arakawa K., Aoki K.

Cell Structure and Function 49 ( 2 ) 123 - 133 2024

ISSN 03867196

Anhydrobiosis, a phenomenon in which organisms survive extreme dehydration by entering a reversible ametabolic state, is a remarkable example of survival strategies. This study focuses on anhydrobiosis in tardigrades, which are known for their resilience to severe environmental conditions. Tardigrades utilize several protective mechanisms against desiccation, notably the constitutive expression of cytoplasmic abundant heat soluble (CAHS) proteins in Ramazzottius varieornatus. These proteins share similarities in their amphiphatic alpha helices with late embryogenesis abundant (LEA) proteins, but differ significantly in their amino acid sequences. In this study, we further explored the functionality of CAHS proteins by analyzing their role in aggregation and tolerance to hyperosmotic stress in mammalian cells. Using live cell imaging, we examined the subcellular localization of several CAHS and LEA proteins in response to hyperosmotic stress. The expression of CAHS1, CAHS3, and CAHS8 tended to enhance the resilience to the hyperosmotic conditions. These findings not only deepen our understanding of the molecular mechanisms of anhydrobiosis but also highlight the potential of CAHS proteins as cryoprotectants.

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Transcriptome analysis of the tardigrade Hypsibius exemplaris exposed to the DNA-damaging agent bleomycin

Yoshida Y., Hirayama A., Arakawa K.

Proceedings of the Japan Academy Series B Physical and Biological Sciences 100 ( 7 ) 414 - 428 2024

ISSN 03862208

Tardigrades are microscopic animals that are renowned for their capabilities of tolerating near-complete desiccation by entering an ametabolic state called anhydrobiosis. However, many species also show high tolerance against radiation in the active state as well, suggesting cross-tolerance via the anhydrobiosis mechanism. Previous studies utilized indirect DNA damaging agents to identify core components of the cross-tolerance machinery in species with high anhydrobiosis capacities. However, it was difficult to distinguish whether transcriptomic changes were specific to DNA damage or mutual with anhydrobiosis. To this end, we performed transcriptome analysis on bleomycin-exposed Hypsibius exemplaris. We observed induction of several tardigrade-specific gene families, including a previously identified novel antioxidative stress family, which may be a core component of the cross-tolerance mechanism. We also identified enrichment of the tryptophan metabolism pathway, for which metabolomic analysis suggested engagement of this pathway in stress tolerance. These results provide several candidates for the core component of cross-tolerance, as well as possible anhydrobiosis machinery.

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Complete genome sequence of PETase type IIa-harboring Marinobacter nanhaiticus D15-8W, isolated from a South China Sea sediment

Suzuki Y., Fukazawa A., Sugawara K., Galipon J., Arakawa K.

Microbiology Resource Announcements 12 ( 12 ) 2023.12

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Complete genome sequence of Actinoplanes sichuanensis strain 03-723T

Kawashima T., Watanabe Y., Arakawa K.

Microbiology Resource Announcements 12 ( 12 ) 2023.12

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Two new species in Leptocheliidae (Crustacea: Peracarida: Tanaidacea) from Japan, with notes on their phylogenetic position and aspects of morphology

Sato A., Arakawa K., Kakui K.

Zoologischer Anzeiger 306 51 - 68 2023.09

ISSN 00445231

We describe two new species of leptocheliid tanaidaceans, Chondrochelia sublitoralis sp. nov. and Neoleptochelia japonica sp. nov., from Japan. Chondrochelia individuals were collected from 113 to 185 m depth in the Kumano Sea. Chondrochelia sublitoralis sp. nov. resembles Chondrochelia africana, Chondrochelia corsica, Chondrochelia taitungensis, and Chondrochelia tanykeraia in having females with slender chelipeds but differs from them in the length ratio of antennular articles and the setal pattern on maxillipeds, cheliped, pereopods 4–6, and pleopods. Males differ from Chondrochelia durbanensis, for which information on females is lacking, in having pereopod-1 propodus lacking ventrodistal spiniform setae and a uniarticulate uropodal exopod. Chondrochelia sublitoralis sp. nov. varies intraspecifically in the number of female antennular articles, five in larger and four in smaller females. Neoleptochelia japonica sp. nov., collected from the coast of Okinawa Island, differ from Neoleptochelia javaensis (previously the sole species in Neoleptochelia) in the setal pattern on antennae, maxillipeds, cheliped, and pleopods in females; and that on the cheliped in males. Male pereopods 1–3 show a unique subchelate condition that we speculate may function for clasping during copulation. Several females in both species had the second thoracomere demarcated on the carapace, whereas others did not. Cytochrome c oxidase subunit I (COI) gene sequences determined for C. sublitoralis sp. nov. and N. japonica sp. nov. showed intraspecific K2P distances of 0–0.4% and 0–0.5%, respectively. We inferred the phylogenetic positions of the two species in Leptocheliidae based on COI data.

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Nondomain biopolymers: Flexible molecular strategies to acquire biological functions

Arakawa K., Hirose T., Inada T., Ito T., Kai T., Oyama M., Tomari Y., Yoda T., Nakagawa S.

Genes to Cells 28 ( 8 ) 539 - 552 2023.08

ISSN 13569597

A long-standing assumption in molecular biology posits that the conservation of protein and nucleic acid sequences emphasizes the functional significance of biomolecules. These conserved sequences fold into distinct secondary and tertiary structures, enable highly specific molecular interactions, and regulate complex yet organized molecular processes within living cells. However, recent evidence suggests that biomolecules can also function through primary sequence regions that lack conservation across species or gene families. These regions typically do not form rigid structures, and their inherent flexibility is critical for their functional roles. This review examines the emerging roles and molecular mechanisms of “nondomain biomolecules,” whose functions are not easily predicted due to the absence of conserved functional domains. We propose the hypothesis that both domain- and nondomain-type molecules work together to enable flexible and efficient molecular processes within the highly crowded intracellular environment.

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Composition of Minor Ampullate Silk Makes Its Properties Different from Those of Major Ampullate Silk

Nakamura H., Kono N., Mori M., Masunaga H., Numata K., Arakawa K.

Biomacromolecules 24 ( 5 ) 2042 - 2051 2023.05

ISSN 15257797

Spider’s minor ampullate silk, or MI-silk, exhibits distinct mechanical properties and water resistance compared to its major ampullate counterpart (MA-silk). The principal protein constituent of MI-silk is known as minor ampullate spidroin, or MiSp, and while its sequence has been deciphered and is thought to underlie the differences in properties with MA-silk, the composition of MI-silk and the relationship between its composition and properties remain elusive. In this study, we set out to investigate the mechanical properties, water resistance, and proteome of MA-silk and MI-silk from Araneus ventricosus and Trichonephila clavata. We also synthesized artificial fibers from major ampullate spidroin, MaSp1 and 2, and MiSp to compare their properties. Our proteomic analysis reveals that the MI-silk of both araneids is composed of MiSp, MaSp1, and spidroin constituting elements (SpiCEs). The absence of MaSp2 in the MI-silk proteome and the comparison of the water resistance of artificial fibers suggest that the presence of MaSp2 is the reason for the disparity in water resistance between MI-silk and MA-silk.

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In vivo expression vector derived from anhydrobiotic tardigrade genome enables live imaging in Eutardigrada

Tanaka S., Aoki K., Arakawa K.

Proceedings of the National Academy of Sciences of the United States of America 120 ( 5 ) 2023.01

ISSN 00278424

Water is essential for life, but anhydrobiotic tardigrades can survive almost complete dehydration. Anhydrobiosis has been a biological enigma for more than a century with respect to how organisms sustain life without water, but the few choices of genetic toolkits available in tardigrade research have been a challenging circumstance. Here, we report the development of an in vivo expression system for tardigrades. This transient transgenic technique is based on a plasmid vector (TardiVec) with promoters that originated from an anhydrobiotic tardigrade Ramazzottius varieornatus. It enables the introduction of GFP-fused proteins and genetically encoded indicators such as the Ca<sup>2+</sup> indicator GCaMP into tardigrade cells; consequently, the dynamics of proteins and cells in tardigrades may be observed by fluorescence live imaging. This system is applicable for several tardigrades in the class Eutardigrada: the promoters of anhydrobiosis-related genes showed tissue-specific expression in this work. Surprisingly, promoters functioned similarly between multiple species, even for species with different modes of expression of anhydrobiosis-related genes, such as Hypsibius exemplaris, in which these genes are highly induced upon facing desiccation, and Thulinius ruffoi, which lacks anhydrobiotic capability. These results suggest that the highly dynamic expression changes in desiccation-induced species are regulated in trans. Tissue-specific expression of tardigrade-unique unstructured proteins also suggests differing anhydrobiosis machinery depending on the cell types. We believe that tardigrade transgenic technology opens up various experimental possibilities in tardigrade research, especially to explore anhydrobiosis mechanisms.

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Molecular mechanisms of the high performance of spider silks revealed through multi-omics analysis

Watanabe Y., Arakawa K.

Biophysics and Physicobiology 20 ( 1 ) 2023

Spider silk is considered a promising next-generation biomaterial due to its exceptional toughness, coupled with its renewability and biodegradability. Contrary to the conventional view that spider silk is mainly composed of two types of silk proteins (spidroins), MaSp1 and MaSp2, multi-omics strategies are increasingly revealing that the inclusion of complex components confers the higher mechanical properties to the material. In this review, we focus on several recent findings that report essential components and mechanisms that are necessary to reproduce the properties of natural spider silk. First, we discuss the discovery of MaSp3, a newly identified spidroin that is a major component in the composition of spider silk, in addition to the previously understood MaSp1 and MaSp2. Moreover, the role of the Spider-silk Constituting Element (SpiCE), which is present in trace amounts but has been found to significantly increase the tensile strength of artificial spider silk, is explored. We also delve into the process of spidroin fibril formation through liquid-liquid phase separation (LLPS) that forms the hierarchical structure of spider silk. In addition, we review the correlation between amino acid sequences and mechanical properties such as toughness and supercontraction, as revealed by an analysis of 1,000 spiders. In conclusion, these recent findings contribute to the comprehensive understanding of the mechanisms that give spider silk its high mechanical properties and help to improve artificial spider silk production.

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A Nanopore Sequencing Course for Graduate School Curriculum

Arakawa K.

Methods in Molecular Biology Clifton N J 2632 113 - 127 2023

The high-throughput long-read sequencing has become affordable enough for any molecular biology lab to utilize genome sequencing in their research. Complete genome sequencing and assembly of bacterial genomes is one such application which is powerful yet simple enough for anyone without advanced molecular biology or bioinformatics skills to conduct on his/her own. High-throughput sequencing will eventually become a basic routine tool in molecular biology labs just like polymerase chain reaction and electrophoresis in a near future. To assist the use of such nanopore sequencing technologies, we designed a graduate school course to learn both the experimental and bioinformatic skills of complete bacterial genome sequencing and assembly.

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The Method of Eliminating the Wolbachia Endosymbiont Genomes from Insect Samples Prior to a Long-Read Sequencing

Takasuka K., Arakawa K.

Methods in Molecular Biology Clifton N J 2632 101 - 112 2023

When extracting DNA of invertebrates for long-read sequencing, not only enough quantity and size of the DNA but, depending on the species, elimination of contamination of endosymbiotic Wolbachia genome also has to be achieved. These requirements become troublesome, especially in small-sized species with a limited number of individuals available for the experiment. In this chapter, using tiny parasitoid wasps (Reclinervellus nielseni) parasitizing spiders as hosts, we developed a method of eliminating the Wolbachia genomes by means of an antibiotic administration to adult wasps via honey solution. Twenty days of rifampicin treatment since their emergence from cocoons resulted in a significant decrease in the Wolbachia genomes while keeping good DNA conditions for nanopore sequencing. An adequate quantity of DNA was then gained by pooling several individuals. The method could be applied to other insects or invertebrates that can be maintained by laboratory feeding with liquid food.

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Ultralow-Input Genome Library Preparation for Nanopore Sequencing with Droplet MDA

Arakawa K.

Methods in Molecular Biology Clifton N J 2632 91 - 100 2023

Genome sequencing of small species, such as those of meinofauna, can be challenging due to the extremely low input of genomic DNA. While nanopore sequencing is a promising technology for genome assembly due to its limitless long reads, recommended input of 1 μg for the Ligation Sequencing Kit often precludes the use of this technology. Here, I detail an unbiased droplet-based multiple displacement amplification of picogram order of DNA to realize nanopore sequencing with ultralow input of genomic DNA. For this purpose, a microfluidic chip of 10X Genomics Chromium Controller is utilized. With this method, over 10 μg of unbiased amplicons around 10 kbp in length can be obtained from as low as 50 μg of input DNA, which is enough for the construction of multiple sequencing libraries, or for the size selection of longer DNA fragments.

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Time-series transcriptomic screening of factors contributing to the cross-tolerance to UV radiation and anhydrobiosis in tardigrades

Yoshida Y., Satoh T., Ota C., Tanaka S., Horikawa D.D., Tomita M., Kato K., Arakawa K.

BMC Genomics 23 ( 1 ) 2022.12

Background: Tardigrades are microscopic animals that are capable of tolerating extreme environments by entering a desiccated state of suspended animation known as anhydrobiosis. While antioxidative stress proteins, antiapoptotic pathways and tardigrade-specific intrinsically disordered proteins have been implicated in the anhydrobiotic machinery, conservation of these mechanisms is not universal within the phylum Tardigrada, suggesting the existence of overlooked components. Results: Here, we show that a novel Mn-dependent peroxidase is an important factor in tardigrade anhydrobiosis. Through time-series transcriptome analysis of Ramazzottius varieornatus specimens exposed to ultraviolet light and comparison with anhydrobiosis entry, we first identified several novel gene families without similarity to existing sequences that are induced rapidly after stress exposure. Among these, a single gene family with multiple orthologs that is highly conserved within the phylum Tardigrada and enhances oxidative stress tolerance when expressed in human cells was identified. Crystallographic study of this protein suggested Zn or Mn binding at the active site, and we further confirmed that this protein has Mn-dependent peroxidase activity in vitro. Conclusions: Our results demonstrated novel mechanisms for coping with oxidative stress that may be a fundamental mechanism of anhydrobiosis in tardigrades. Furthermore, localization of these sets of proteins mainly in the Golgi apparatus suggests an indispensable role of the Golgi stress response in desiccation tolerance.

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1000 spider silkomes: Linking sequences to silk physical properties

Arakawa K., Kono N., Malay A.D., Tateishi A., Ifuku N., Masunaga H., Sato R., Tsuchiya K., Ohtoshi R., Pedrazzoli D., Shinohara A., Ito Y., Nakamura H., Tanikawa A., Suzuki Y., Ichikawa T., Fujita S., Fujiwara M., Tomita M., Blamires S.J., Chuah J.A., Craig H., Foong C.P., Greco G., Guan J., Holland C., Kaplan D.L., Sudesh K., Mandal B.B., Norma-Rashid Y., Oktaviani N.A., Preda R.C., Pugno N.M., Rajkhowa R., Wang X., Yazawa K., Zheng Z., Numata K.

Science Advances 8 ( 41 ) 2022.10

Spider silks are among the toughest known materials and thus provide models for renewable, biodegradable, and sustainable biopolymers. However, the entirety of their diversity still remains elusive, and silks that exceed the performance limits of industrial fibers are constantly being found. We obtained transcriptome assemblies from 1098 species of spiders to comprehensively catalog silk gene sequences and measured the mechanical, thermal, structural, and hydration properties of the dragline silks of 446 species. The combination of these silk protein genotype-phenotype data revealed essential contributions of multicomponent structures with major ampullate spidroin 1 to 3 paralogs in high-performance dragline silks and numerous amino acid motifs contributing to each of the measured properties. We hope that our global sampling, comprehensive testing, integrated analysis, and open data will provide a solid starting point for future biomaterial designs.

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Complete Genome Sequence of Streptomyces albus Strain G153

Takeda T., Fukumitsu N., Yuzawa S., Arakawa K.

Microbiology Resource Announcements 11 ( 7 ) 2022.07

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Complete Genome Sequences of Four Bacteria Isolated from the Gut of a Spiny Ant (Polyrhachis lamellidens)

Ishikawa S., Huang M., Tomita A., Kurihara Y., Watanabe R., Iwai H., Arakawa K.

Microbiology Resource Announcements 11 ( 7 ) 2022.07

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