研究者詳細 - 堀　克敏

論文 - 堀　克敏

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Complete genome sequence of Chryseobacterium sp. strain KCF3-3, isolated from the body surface of channel catfish, Ictalurus punctatus 査読有り国際共著

Kojima, M; Tobioka, K; Okazaki, M; Yokota, K; Anggorowati, DA; Nakatani, H; Hori, K; Tamaru, Y; Okazaki, F

MICROBIOLOGY RESOURCE ANNOUNCEMENTS 14 巻 ( 2 ) 頁： e0105824 2025年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Microbiology Resource Announcements

Here, we report the complete genome sequence of Chryseobacterium sp. strain KCF3-3, isolated from the body surface of channel catfish, Ictalurus punctatus. The de novo assembly revealed a chromosome size of 5,623,437 bp with an estimated 4,939 open reading frames.

DOI： 10.1128/mra.01058-24

A new target of multiple lysine methylation in bacteria 査読有り

Inoue, S; Yoshimoto, S; Hori, K

JOURNAL OF BACTERIOLOGY 207 巻 ( 1 ) 頁： e0032524 2025年1月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bacteriology

The methylation of ε-amino groups in protein lysine residues is an important posttranslational modification in eukaryotes. This modification plays a pivotal role in the regulation of diverse biological processes, including epigenetics, transcriptional control, and cellular signaling. Recent research has begun to reveal the potential role of methylation in modulating bacterial immune evasion and adherence to host cells. In this study, we analyzed the cell surface proteins of the toluene-degrading bacterium Acinetobacter sp. Tol 5 by label-free liquid chromatography-mass spectrometry and found multiple lysine methylation in its trimeric autotransporter adhesin (TAA), AtaA. Over 130 lysine residues of AtaA, consisting of 3,630 amino acids and containing 234 lysine residues, were methylated. We identified that the outer membrane protein lysine methyltransferase (OM PKMT) of Tol 5, KmtA, specifically methylates the lysine residues of AtaA. In the KmtA-deficient mutant, most lysine methylations on AtaA were absent, indicating that KmtA is responsible for the methylation of multiple lysine residues throughout AtaA. Bioinformatic analysis revealed that the OM PKMT genes were widely distributed among Gram-negative bacteria, including pathogens with TAAs that promote infectivity, such as Burkholderia mallei and Haemophilus influenzae. Although KmtA has sequence similarities to the OM PKMTs of Rickettsia involved in infectivity, KmtA-like PKMTs formed a distinct cluster from those of the Rickettsia type according to the clustering analysis, suggesting that they are new types of OM PKMTs. Furthermore, the deletion of Tol 5 KmtA led to an increase in AtaA on the cell surface and enhanced bacterial adhesion, resulting in slower growth. IMPORTANCE Lysine methylation has been underexplored in prokaryotes, and information on it is limited to some pathogens. Our finding is the second case of multiple lysine methylation of bacterial outer membrane (OM) proteins, following that of OmpB of Rickettsia. The newly found target of methylation, AtaA, a trimeric autotransporter adhesin family protein of Acinetobacter sp. Tol 5 isolated from activated sludge, extends our understanding of OM protein methylation to non-pathogenic environmental strains. The newly identified enzyme KmtA shows higher specificity than rickettsial lysin methylases, protein lysine methyltransferases, and methylates more lysine residues of the target, which raises interest in the mechanism underlying its biological specificity. The widespread presence of KmtA-like PKMTs throughout Gram-negative bacteria suggests that lysine methylation functions more extensively in bacterial physiology than previously recognized.

DOI： 10.1128/jb.00325-24

Complete genome sequence of Flavobacterium sp. strain CFS9, a potential fish probiotic isolated from the body surface of Silurus asotus 査読有り国際共著

Kojima, M; Tobioka, K; Okazaki, M; Yokota, K; Anggorowati, DA; Nakatani, H; Hori, K; Tamaru, Y; Okazaki, F

MICROBIOLOGY RESOURCE ANNOUNCEMENTS 13 巻 ( 12 ) 頁： e0056324 2024年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Microbiology Resource Announcements

Here, we report the complete genome sequence of Flavobacterium sp. strain CFS9, a potential fish probiotic isolated from the body surface of Silurus asotus. The de novo assembly revealed a chromosome size of 5,370,016 bp with an estimated 4,374 open reading frames.

DOI： 10.1128/mra.00563-24

Size-dependent ability of AtaA to immobilize cells in Acinetobacter sp. Tol 5 招待有り査読有り Open Access

Yoshimoto, S; Aoki, S; Ishikawa, M; Suzuki, A; Hori, K

SCIENTIFIC REPORTS 14 巻 ( 1 ) 頁： 21039 2024年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Microbial cells serve as efficient and environmentally friendly biocatalysts, but their stability and reusability in practical applications must often be improved through immobilization. Acinetobacter sp. Tol 5 shows high adhesiveness to materials due to its large cell surface protein AtaA, which consists of 3630 amino acids (aa). Previously, we developed a method for immobilizing bacteria using AtaA. Herein, we investigated the cell immobilization ability of in-frame deletion (IFD) mutants of AtaA with different sizes in Tol 5. Mini-AtaA, which consists of 775 aa and is functional in Escherichia coli, was produced and present on the cell surface; however, mini-AtaA showed no immobilization ability in Tol 5. A cell immobilization assay was performed with cells expressing 16 IFD mutants of AtaA with different sizes, revealing that a length of at least 1417 aa was required for the sufficient immobilization of Tol 5 cells; thus, the minimum length needed to achieve the adhesive function of AtaA varies among bacterial species. The constructed mutant library of AtaA ranging from 3630 to 775 aa will allow researchers to quickly and easily explore the optimal size of AtaA, even for bacteria newly introduced to AtaA.

DOI： 10.1038/s41598-024-71920-3

The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5 査読有り

Ishikawa, M; Hori, K

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 90 巻 ( 6 ) 頁： e0040024 2024年6月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Applied and Environmental Microbiology

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent capabilities, which include efficiently converting a wide range of substrates into valuable products and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5, an environmentally isolated Gram-negative bacterium, possesses intriguing properties for use in synthetic biology applications. Despite the previous development of genetic tools for the engineering of strain Tol 5, its genetic manipulation has been hindered by low transformation efficiency via electroporation, rendering the process laborious and time-consuming. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding type I and type III restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA without reliance on intermediate Escherichia coli constructs. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our findings provide genetic modification strategies that allow for the domestication of environmentally isolated bacteria, streamlining their utilization in synthetic biology applications. IMPORTANCE Recent synthetic biology has sought diverse bacterial chassis from environmental sources to circumvent the limitations of laboratory Escherichia coli strains for industrial and environmental applications. One of the critical barriers in cell engineering of bacterial chassis is their inherent resistance to recombinant DNA, propagated either in vitro or within E. coli cells. Environmental bacteria have evolved defense mechanisms against foreign DNA as a response to the constant threat of phage infection. The ubiquity of phages in natural settings accounts for the genetic intractability of environmental isolates. The significance of our research is in demonstrating genetic modification strategies for the cell engineering of such genetically intractable bacteria. This research marks a pivotal step in the domestication of environmentally isolated bacteria, promising candidates for emerging synthetic biology chassis. Our work thus significantly contributes to advancing their applications across industrial, environmental, and biomedical fields.

DOI： 10.1128/aem.00400-24

Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5 査読有り国際共著国際誌 Open Access

Yoshimoto, S; Ishii, S; Kawashiri, A; Matsushita, T; Linke, D; Göttig, S; Kempf, VAJ; Takai, M; Hori, K

FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY 12 巻頁： 1342418 2024年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Bioengineering and Biotechnology

Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.

DOI： 10.3389/fbioe.2024.1342418

Electrochemical monnitoring of metabolic activity of methsne/methanol conversing Methylococcus capsulatus (Bath) cells based on extrancellular electron transfer 査読有り国際誌

K. Sugimoto, K. Hori, M. Ishikawa, H. Ito, T. Kamachi, K. Tanaka, Y-Y. Chen, and S. Nakanishi

Electrochemistry 92 巻 ( 2 ) 頁： 022007 2024年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）

Bioconversion of methane to methanol by methanotrophs under mild conditions is a promising approach for efficiently utilizing methane. Here, we present an electrochemical technique based on open-circuit potential (OCP) measurements to monitor the metabolic activity of Methylococcus capsulatus (Bath), a representative methanotrophic model. This technique
is based on the extracellular electron transfer (EET) mechanism, in which intracellular electrons in living cells are exchanged across the cell membrane with an extracellular electrode. Without using artificial electron mediators in our study, we observed that OCP shifted to negative when methane metabolism was activated. By manipulating the culture conditions with the absence or presence of copper supplement to regulate the expression of outer membrane cytochromes (OMCs), the cells with a high OMC expression level, known to serve as conduits for EET, responded with increased sensitivity to stimulation with excess NADH compared to the cells with a low OMC expression level. We, therefore, used the instinctive EET capacity of M. capsulatus (Bath) for real-time OCP measurement to monitor the bioconversion of methane to methanol. Our measurements showed that the OCP levels change with
intracellular redox variations and reflect methanol production rates. Our findings may facilitate the development of a methanotrophic bioprocess that allows more effective and efficient control of intracellular redox status using OCP monitoring based on EET.

DOI： https://doi.org/10.5796/electrochemistry.23-68120

Long-term continuous degradation of carbon nanotubes by a bacteria-driven Fenton reaction 査読有り Open Access

Takahashi, S; Hori, K

FRONTIERS IN MICROBIOLOGY 14 巻頁： 1298323 2023年11月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Microbiology

Very few bacteria are known that can degrade carbon nanotubes (CNTs), and the only known degradation mechanism is a Fenton reaction driven by Labrys sp. WJW with siderophores, which only occurs under iron-deficient conditions. No useful information is available on the degradation rates or long-term stability and continuity of the degradation reaction although several months or more are needed for CNT degradation. In this study, we investigated long-term continuous degradation of oxidized (carboxylated) single-walled CNTs (O-SWCNTs) using bacteria of the genus Shewanella. These bacteria are widely present in the environment and can drive the Fenton reaction by alternating anaerobic-aerobic growth conditions under more general environmental conditions. We first examined the effect of O-SWCNTs on the growth of S. oneidensis MR-1, and it was revealed that O-SWCNTs promote growth up to 30 μg/mL but inhibit growth at 40 μg/mL and above. Then, S. oneidensis MR-1 was subjected to incubation cycles consisting of 21-h anaerobic and 3-h aerobic periods in the presence of 30 μg/mL O-SWCNTs and 10 mM Fe(III) citrate. We determined key factors that help prolong the bacteria-driven Fenton reaction and finally achieved long-term continuous degradation of O-SWCNTs over 90 d. By maintaining a near neutral pH and replenishing Fe(III) citrate at 60 d, a degraded fraction of 56.3% was reached. S. oneidensis MR-1 produces Fe(II) from Fe(III) citrate, a final electron acceptor for anaerobic respiration during the anaerobic period. Then, ·OH is generated through the Fenton reaction by Fe(II) and H2O2 produced by MR-1 during the aerobic period. ·OH was responsible for O-SWCNT degradation, which was inhibited by scavengers of H2O2 and ·OH. Raman spectroscopy and X-ray photoelectron spectroscopy showed that the graphitic structure in O-SWCNTs was oxidized, and electron microscopy showed that long CNT fibers initially aggregated and became short and isolated during degradation. Since Shewanella spp. and iron are ubiquitous in the environment, this study suggests that a Fenton reaction driven by this genus is applicable to the degradation of CNTs under a wide range of conditions and will help researchers develop novel methods for waste treatment and environmental bioremediation against CNTs.

DOI： 10.3389/fmicb.2023.1298323

Contribution of the Fenton reaction to the degradation of carbon nanotubes by enzymes 査読有り国際誌 Open Access

Takahashi, S; Taguchi, F; Hori, K

FRONTIERS IN ENVIRONMENTAL SCIENCE 11 巻 2023年6月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Environmental Science

The widespread use of carbon nanotubes (CNTs) has raised concerns about the human health and ecological effects of CNTs released into the environment. Bacteria play an important role in bioremediation and waste treatment, and their enzymes are mostly responsible for the degradation of contaminants. However, there are still only a few reports about the bacterial degradation of CNTs, and evidence showing the involvement of bacterial enzymes in CNT degradation with their mechanisms has never been reported. The purpose of this study is to clarify whether CNTs can be degraded by bacterial enzymes. In this study, the degradation of oxidized (carboxylated) single-walled CNTs (O-SWCNTs) by mt2DyP, a dye-decolorizing peroxidase of Pseudomonas putida mt-2, a common soil bacterium, was investigated. After incubation of O-SWCNTs with recombinant mt2DyP and its substrate H2O2 for 30 d, the optical absorbance and Raman spectra revealed the degradation of O-SWCNTs. However, inactivation of the enzyme was observed within 60 min of the start of incubation, suggesting that the degradation of O-SWCNTs occurred nonenzymatically. The inactivation of mt2DyP was accompanied by the release of iron, the active center metal, and degradation of O-SWCNTs was significantly inhibited in the presence of diethylenetriamine pentaacetic acid, a chelating agent, indicating that O-SWCNTs were degraded by the Fenton reaction with iron released from mt2DyP and H2O2. The same phenomenon was observed with P450, which is also a heme enzyme. Furthermore, we investigated the contribution of the Fenton reaction to the O-SWCNT degradation by horseradish peroxidase (HRP), which was reported to enzymatically and rapidly degrade O-SWCNTs. Our results revealed that the degradation of O-SWCNTs in the presence of HRP is also mainly due to the Fenton reaction, with negligible enzymatic degradation. This contradicts the report showing enzymatic degradation of O-SWCNTs by HRP but supports the subsequent report quantitatively showing very slow transformation of O-SWCNTs by HRP. The current results emphasize that the Fenton reaction, which has received little attention in CNT degradation by heme enzymes, must be taken into consideration and will contribute to the development of a simple disposal method for CNTs, utilizing the Fenton reaction with bacteria/bacterial enzymes and H2O2.

DOI： 10.3389/fenvs.2023.1184257

Simple Method for the Creation of a Bacteria-Sized Unilamellar Liposome with Different Proteins Localized to the Respective Sides of the Membrane 査読有り国際誌

Noba, K; Yoshimoto, S; Tanaka, Y; Yokoyama, T; Matsuura, T; Hori, K

ACS SYNTHETIC BIOLOGY 12 巻 ( 5 ) 頁： 1437 - 1446 2023年5月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACS Synthetic Biology

Artificial cells are membrane vesicles mimicking cellular functions. To date, giant unilamellar vesicles made from a single lipid membrane with a diameter of 10 μm or more have been used to create artificial cells. However, the creation of artificial cells that mimic the membrane structure and size of bacteria has been limited due to technical restrictions of conventional liposome preparation methods. Here, we created bacteria-sized large unilamellar vesicles (LUVs) with proteins localized asymmetrically to the lipid bilayer. Liposomes containing benzylguanine-modified phospholipids were prepared by combining the conventional water-in-oil emulsion method and the extruder method, and green fluorescent protein fused with SNAP-tag was localized to the inner leaflet of the lipid bilayer. Biotinylated lipid molecules were then inserted externally, and the outer leaflet was modified with streptavidin. The resulting liposomes had a size distribution in the range of 500-2000 nm with a peak at 841 nm (the coefficient of variation was 10.3%), which was similar to that of spherical bacterial cells. Fluorescence microscopy, quantitative evaluation using flow cytometry, and western blotting proved the intended localization of different proteins on the lipid membrane. Cryogenic electron microscopy and quantitative evaluation by α-hemolysin insertion revealed that most of the created liposomes were unilamellar. Our simple method for the preparation of bacteria-sized LUVs with asymmetrically localized proteins will contribute to the creation of artificial bacterial cells for investigating functions and the significance of their surface structure and size.

DOI： 10.1021/acssynbio.2c00564

DOI： 10.1021/acssynbio.2c00564

Growth phase-dependent production of the adhesive nanofiber protein AtaA in Acinetobacter sp. Tol 5 査読有り国際誌 Open Access

Ishikawa, M; Nakatani, H; Hori, K

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 135 巻 ( 3 ) 頁： 224 - 231 2023年3月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bioscience and Bioengineering

AtaA, the sticky, long, and peritrichate nanofiber protein from Acinetobacter sp. Tol 5, mediates autoagglutination and is highly adhesive to various material surfaces, resulting in a biofilm. Although the production of the adhesive nanofiber protein is likely to require a large amount of energy and material sources, the relationship between AtaA fiber production and cell growth remains unknown. Here, we report the growth phase-dependent AtaA fiber production in Tol 5. We examined the ataA gene expression in different growth phases using a reporter gene assay with an originally developed reporter plasmid and using reverse transcription-quantitative polymerase chain reaction. Bacterial cells with surface-displayed AtaA at different growth phases were immunostained and analyzed using fluorescence flow cytometry and confocal laser scanning microscopy. The results indicate that Tol 5 modulated the amount of surface-displayed AtaA at the transcriptional level. AtaA production was low in the early growth phase but remarkably increased in the late growth phase, covering the whole bacterial cell with AtaA fibers in the stationary phase. Tol 5 displayed AtaA fibers poorly in the early growth phase and showed less autoagglutination and adhesiveness than those in the stationary phase. Although Tol 5 grew as fast as its ataA-deficient mutant in the early growth phase, the optical density of Tol 5 culture was slightly lower than that of the ataA-deficient mutant in the late growth phase. Based on these experimental results, we propose the growth-phase-dependent production of AtaA fiber for efficient and fast cell growth.

DOI： 10.1016/j.jbiosc.2022.12.012

A novel inverse membrane bioreactor for efficient bioconversion from methane gas to liquid methanol using a microbial gas-phase reaction 査読有り国際誌 Open Access

Chen, YY; Ishikawa, M; Hori, K

BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS 16 巻 ( 1 ) 頁： 16 2023年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biotechnology for Biofuels and Bioproducts

Background: Methane (CH4), as one of the major energy sources, easily escapes from the supply chain into the atmosphere, because it exists in a gaseous state under ambient conditions. Compared to carbon dioxide (CO2), CH4 is 25 times more potent at trapping radiation; thus, the emission of CH4 to the atmosphere causes severe global warming and climate change. To mitigate CH4 emissions and utilize them effectively, the direct biological conversion of CH4 into liquid fuels, such as methanol (CH3OH), using methanotrophs is a promising strategy. However, supplying biocatalysts in an aqueous medium with CH4 involves high energy consumption due to vigorous agitation and/or bubbling, which is a serious concern in methanotrophic processes, because the aqueous phase causes a very large barrier to the delivery of slightly soluble gases. Results: An inverse membrane bioreactor (IMBR), which combines the advantages of gas-phase bioreactors and membrane bioreactors, was designed and constructed for the bioconversion of CH4 into CH3OH in this study. In contrast to the conventional membrane bioreactor with bacterial cells that are immersed in an aqueous phase, the filtered cells were placed to face a gas phase in the IMBR to supply CH4 directly from the gas phase to bacterial cells. Methylococcus capsulatus (Bath), a representative methanotroph, was used to demonstrate the bioconversion of CH4 to CH3OH in the IMBR. Cyclopropanol was supplied from the aqueous phase as a selective inhibitor of methanol dehydrogenase, preventing further CH3OH oxidation. Sodium formate was added as an electron donor to generate NADH, which is necessary for CH3OH production. After optimizing the inlet concentration of CH4, the mass of cells, the cyclopropanol concentration, and the gas flow rate, continuous CH3OH production can be achieved over 72 h with productivity at 0.88 mmol L−1 h−1 in the IMBR, achieving a longer operation period and higher productivity than those using other types of membrane bioreactors reported in the literature. Conclusions: The IMBR can facilitate the development of gas-to-liquid (GTL) technologies via microbial processes, allowing highly efficient mass transfer of substrates from the gas phase to microbial cells in the gas phase and having the supplement of soluble chemicals convenient.

DOI： 10.1186/s13068-023-02267-6

Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli 査読有り国際共著国際誌 Open Access

Yoshimoto, S; Aoki, S; Ohara, Y; Ishikawa, M; Suzuki, A; Linke, D; Lupas, AN; Hori, K

FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY 10 巻頁： 1095057 2023年1月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Frontiers in Bioengineering and Biotechnology

Cell immobilization is an important technique for efficiently utilizing whole-cell biocatalysts. We previously invented a method for bacterial cell immobilization using AtaA, a trimeric autotransporter adhesin from the highly sticky bacterium Acinetobacter sp. Tol 5. However, except for Acinetobacter species, only one bacterium has been successfully immobilized using AtaA. This is probably because the heterologous expression of large AtaA (1 MDa), that is a homotrimer of polypeptide chains composed of 3,630 amino acids, is difficult. In this study, we identified the adhesive domain of AtaA and constructed a miniaturized AtaA (mini-AtaA) to improve the heterologous expression of ataA. In-frame deletion mutants were used to perform functional mapping, revealing that the N-terminal head domain is essential for the adhesive feature of AtaA. The mini-AtaA, which contains a homotrimer of polypeptide chains from 775 amino acids and lacks the unnecessary part for its adhesion, was properly expressed in E. coli, and a larger amount of molecules was displayed on the cell surface than that of full-length AtaA (FL-AtaA). The immobilization ratio of E. coli cells expressing mini-AtaA on a polyurethane foam support was significantly higher compared to the cells with or without FL-AtaA expression, respectively. The expression of mini-AtaA in E. coli had little effect on the cell growth and the activity of another enzyme reflecting the production level, and the immobilized E. coli cells could be used for repetitive enzymatic reactions as a whole-cell catalyst.

DOI： 10.3389/fbioe.2022.1095057

Single-cell adhesion force mapping of a highly sticky bacterium in liquid 査読有り国際誌

Ishii, S; Yoshimoto, S; Hori, K

JOURNAL OF COLLOID AND INTERFACE SCIENCE 606 巻 ( Pt 1 ) 頁： 628 - 634 2022年1月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Colloid and Interface Science

The sticky bacterium Acinetobacter sp. Tol 5 adheres to various material surfaces via its cell surface nanofiber protein, AtaA. This adhesiveness has only been evaluated based on the amount of cells adhering to a surface. In this study, the adhesion force mapping of a single Tol 5 cell in liquid using the quantitative imaging mode of atomic force microscopy (AFM) revealed that the adhesion of Tol 5 was near 2 nN, which was 1–2 orders of magnitude higher than that of other adhesive bacteria. The adhesion force of a cell became stronger with the increase in AtaA molecules present on the cell surface. Many fibers of peritrichate AtaA molecules simultaneously interact with a surface, strongly attaching the cell to the surface. The adhesion force of a Tol 5 cell was drastically reduced in the presence of 1% casamino acids but not in deionized water (DW), although both liquids decrease the adhesiveness of Tol 5 cells, suggesting that DW and casamino acids inhibit the cell approaching step and the subsequent direct interaction step of AtaA with surfaces, respectively. Heterologous production of AtaA provided non-adhesive Acinetobacter baylyi ADP1 cells with a strong adhesion force to AFM tip surfaces of silicon and gold.

DOI： 10.1016/j.jcis.2021.08.039

Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection 査読有り国際誌 Open Access

Ishikawa, M; Kojima, T; Hori, K

MICROBIOLOGY SPECTRUM 9 巻 ( 1 ) 頁： 1 - 11 2021年9月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Microbiology Spectrum

Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10−7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection.

DOI： 10.1128/Spectrum.00259-21

Metabolic alteration of Methylococcus capsulatus str. Bath during a microbial gas-phase reaction 査読有り国際誌 Open Access

Chen, YY; Soma, Y; Ishikawa, M; Takahashi, M; Izumi, Y; Bamba, T; Hori, K

BIORESOURCE TECHNOLOGY 330 巻頁： 125002 2021年6月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Bioresource Technology

This study demonstrates the metabolic alteration of Methylococcus capsulatus (Bath), a representative bacterium among methanotrophs, in microbial gas-phase reactions. For comparative metabolome analysis, a bioreactor was designed to be capable of supplying gaseous substrates and liquid nutrients continuously. Methane degradation by M. capsulatus (Bath) was more efficient in a gas-phase reaction operated in the bioreactor than in an aqueous phase reaction operated in a batch reactor. Metabolome analysis revealed remarkable alterations in the metabolism of cells in the gas-phase reaction; in particular, pyruvate, 2-ketoglutarate, some amino acids, xanthine, and hypoxanthine were accumulated, whereas 2,6-diaminopimelate was decreased. Based on the results of metabolome analysis, cells in the gas-phase reaction seemed to alter their metabolism to reduce the excess ATP and NADH generated upon increased availability of methane and oxygen. Our findings will facilitate the development of efficient processes for methane-based bioproduction with low energy consumption.

DOI： 10.1016/j.biortech.2021.125002

Native display of a huge homotrimeric protein fiber on the cell surface after precise domain deletion 査読有り国際共著国際誌

Aoki, S; Yoshimoto, S; Ishikawa, M; Linke, D; Lupas, A; Hori, K

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 129 巻 ( 4 ) 頁： 412 - 417 2020年4月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Bioscience and Bioengineering

AtaA, a trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, exhibits nonspecific, high adhesiveness to abiotic surfaces. For identification of the functional domains of AtaA, precise design of domain-deletion mutants is necessary so as not to cause undesirable structural distortion. Here, we designed and constructed three types of AtaA mutants from which the same domain, FGG1, was deleted. The first mutant was designed to preserve the periodicity of hydrophobic residues in the coiled-coil segments sandwiching the deleted region. After the deletion, the protein was properly displayed on the cell surface and had the same adhesive function as the wild type. Transmission electron microscopy (TEM) imaging and circular dichroism (CD) spectroscopy showed that its isolated passenger domain had the same fiber structure as in the AtaA wild type. In contrast, a mutant designed to disturb the coiled-coil periodicity at the deletion site failed to reach the cell surface. Although secretion occurred for the mutant designed with a flexible connector between the coiled coils, the cells exhibited a decrease in adhesiveness. Furthermore, TEM imaging of the mutant fibers showed bending at the fiber tip and changes in their CD spectrum indicated a decrease in secondary structure content. Thus, we succeeded to natively display the huge homotrimeric fiber structure of AtaA on the cell surface after precise deletion of a domain, maintaining the proper folding state and adhesive function by preserving its coiled-coil periodicity. This strategy enables us to construct various domain-deletion mutants of AtaA without structural distortion for complete functional mapping.

DOI： 10.1016/j.jbiosc.2019.09.022

Gas-phase bioproduction of a high-value-added monoterpenoid (<i>E</i>)-geranic acid by metabolically engineered <i>Acinetobacter</i> sp. Tol 5 査読有り国際誌

Usami, A; Ishikawa, M; Hori, K

GREEN CHEMISTRY 22 巻 ( 4 ) 頁： 1258 - 1268 2020年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Green Chemistry

Gas-phase bioproduction, in which immobilized biocatalysts are employed and chemical reactions are performed in a gas phase, has attracted researchers' attention as a green process. However, there is difficulty in the employment of whole cell catalysts for gas-phase bioproduction due to the lack of a suitable cell immobilization method. Acinetobacter sp. Tol 5 is a unique bacterium, which is remarkably sticky and can be easily immobilized onto various material surfaces through the adhesive bacterionanofiber protein AtaA. In this study, we demonstrate the gas-phase bioproduction of (E)-geranic acid (GA), a high-value-added monoterpenoid, from geraniol using immobilized Tol 5 transformant cells, into which a gene involved in a (E)-GA synthetic pathway was introduced. Time course analysis of the liquid-phase bioproduction of (E)-GA revealed the inherent metabolism of Tol 5 involved in the degradation of (E)-GA. By disrupting the fadD4-ortholog gene, which encodes a key enzyme of the (E)-GA degradation, we successfully generated a (E)-GA-accumulating strain, Tol 5 ΔfadD4 (pGeoA). The immobilized cells of this mutant strain on a polyurethane support enabled the production of (E)-GA with a passive supply of gaseous geraniol in a batch gas-phase reaction. A major fraction of the (E)-GA, which was produced, was adsorbed onto the polyurethane support but easily extracted into ethanol, a safe solvent without environmental impact. This is the first example of gas-phase bioproduction of a complex and high-value-added compound. Tol 5 is a highly promising platform for gas-phase bioproduction.

DOI： 10.1039/c9gc03478a

Process Description of an Unconventional Biofilm Formation by Bacterial Cells Autoagglutinating through Sticky, Long, and Peritrichate Nanofibers 査読有り国際誌 Open Access

Furuichi, Y; Yoshimoto, S; Inaba, T; Nomura, N; Hori, K

ENVIRONMENTAL SCIENCE & TECHNOLOGY 54 巻 ( 4 ) 頁： 2520 - 2529 2020年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Environmental Science and Technology

In this study, we elucidated the formation process of an unconventional biofilm formed by a bacterium autoagglutinating through sticky, long, and peritrichate nanofibers. Understanding the mechanisms of biofilm formation is essential to control microbial behavior and improve environmental biotechnologies. Acinetobacter sp. Tol 5 autoagglutinate through the interaction of the long, peritrichate nanofiber protein AtaA, a trimeric autotransporter adhesin. Using AtaA, without cell growth or extracellular polymeric substances production, Tol 5 cells quickly form an unconventional biofilm. The process forming this unconventional biofilm started with cell-cell interactions, proceeded to cell clumping, and led to the formation of large cell aggregates. The cell-cell interaction was described by Derjaguin-Landau-Verwey-Overbeek (DLVO) theory based on a new concept, which considers two independent interactions between two cell bodies and between two AtaA fiber tips forming a discontinuous surface. If cell bodies cannot collide owing to an energy barrier at low ionic strengths but approach within the interactive distance of AtaA fibers, cells can agglutinate through their contact. Cell clumping proceeds following the cluster-cluster aggregation model, and an unconventional biofilm containing void spaces and a fractal nature develops. Understanding its formation process would extend the utilization of various types of biofilms, enhancing environmental biotechnologies.

DOI： 10.1021/acs.est.9b06577

Evaluation of methane degradation performance in microbial gas-phase reactions using effectively immobilized methanotrophs 査読有り国際誌 Open Access

Chen, YY; Ishikawa, M; Suzuki, R; Ito, H; Kamachi, T; Hori, K

BIOCHEMICAL ENGINEERING JOURNAL 154 巻頁： 107441 2020年2月

担当区分：最終著者,　責任著者記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical Engineering Journal

The removal of atmospheric methane by methanotrophs is a potential strategy for mitigating global warming. However, due to the low mass transfer of methane to methanotrophic cells, the conventional bioprocesses require active gas-supply with high energy consumption. This study aimed to evaluate methane degradation performance in microbial gas-phase reactions using effectively immobilized Methylococcus capsulatus (Bath) cells. Cell immobilization was achieved by freeze-thaw treatment and a simple filtration process. The methane degradation rate was much faster in the gas-phase reaction than in the aqueous-phase reaction with stirring. The efficiency of methane degradation was almost the same between the gas-phase reaction using gel-entrapped cells and the static aqueous reaction using a cell suspension. The results of this study will facilitate the development of a high-efficiency and low-energy bioprocess for methane removal.

DOI： 10.1016/j.bej.2019.107441

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