Faculty Profiles - HORI Katsutoshi

A new target of multiple lysine methylation in bacteria Reviewed

Inoue, S; Yoshimoto, S; Hori, K

JOURNAL OF BACTERIOLOGY Vol. 207 ( 1 ) page： e0032524 2025.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International coauthorship

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

MICROBIOLOGY RESOURCE ANNOUNCEMENTS Vol. 13 ( 12 ) page： e0056324 2024.12

Language：English Publishing type：Research paper (scientific journal) Publisher：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

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 Reviewed

Size-dependent ability of AtaA to immobilize cells in Acinetobacter sp. Tol 5 Invited Reviewed Open Access

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

SCIENTIFIC REPORTS Vol. 14 ( 1 ) page： 21039 2024.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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

Ishikawa, M; Hori, K

APPLIED AND ENVIRONMENTAL MICROBIOLOGY Vol. 90 ( 6 ) page： e0040024 2024.6

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International coauthorship International journal Open Access

S. Yoshimoto, S. Ishii, A. Kawashiri, T. Matsushita, D. Linke, S. Gottig, VAJ. Kempf, M. Takai, and K. Hori

Frontiers in Bioengineering and Biotechnolgoy Vol. 12 page： 1342418 2024.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

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 Reviewed International journal

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

Electrochemistry Vol. 92 ( 2 ) page： 022007 2024.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

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 Reviewed Open Access

Takahashi, S; Hori, K

FRONTIERS IN MICROBIOLOGY Vol. 14 page： 1298323 2023.11

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal Open Access

S. Takahashi, F. Taguchi and K. Hori

Frontiers in Environmental Science Vol. 11 2023.6

Simple method for the creation of a bacteria-sized unilamellar liposome with different proteins localized to the respective sides of the membrane Reviewed International journal

K. Noba, S. Yoshimoto, Y. Tanaka, T. Yokoyama, T. Matsuura ,and K. Hori

ACS Synthetic Biology Vol. 12 ( 5 ) page： 1437 - 1446 2023.5

Growth phase-dependent production of the adhesive nanofiber protein AtaA in Acinetobacter sp. Tol 5 Reviewed International journal Open Access

Ishikawa Masahito, Nakatani Hajime, Hori Katsutoshi

JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 135 ( 3 ) page： 224 - 231 2023.3

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal Open Access

Chen Yan-Yu, Ishikawa Masahito, Hori Katsutoshi

BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS Vol. 16 ( 1 ) page： 16 2023.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Biotechnology for Biofuels and Bioproducts

ackground: 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 Reviewed International coauthorship International journal Open Access

Yoshimoto Shogo, Aoki Sota, Ohara Yuki, Ishikawa Masahito, Suzuki Atsuo, Linke Dirk, Lupas Andrei N., Hori Katsutoshi

FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY Vol. 10 page： 1095057 2023.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal

Satoshi Ishii, Shogo Yoshimoto, Katsutoshi Hori

JOURNAL OF COLLOID AND INTERFACE SCIENCE Vol. 606 ( Pt 1 ) page： 628 - 634 2022.1

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：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 Reviewed International journal Open Access

Masahito Ishikawa, Takaaki Kojima, Katsutoshi Hori

MICROBIOLOGY SPECTRUM Vol. 9 ( 1 ) page： 1 - 11 2021.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

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 Reviewed International journal Open Access

Yan-Yu Chen, Yuki Soma, Masahito Ishikawa, Masatomo Takahashi, Yoshihiro Izumi, Takeshi Bamba, Katsutoshi Hori

BIORESOURCE TECHNOLOGY Vol. 330 page： 125002 2021.6

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier

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 Reviewed International coauthorship International journal

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

J. Biosci. Bioeng Vol. 129 ( 4 ) page： 412 - 417 2020.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

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 (E)-geranic acid by metabolically engineered Acinetobacter sp. Tol 5 Reviewed International journal

Atsushi Usami, Masahito Ishikawa, Katsutoshi Hori

Green Chemistry Vol. 22 ( 4 ) page： 1258 - 1268 2020.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of 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 Reviewed International journal Open Access

Furuichi Yoshihide, Yoshimoto Shogo, Inaba Tomohiro, Nomura Nobuhiko, Hori Katsutoshi

Environmental Science & Technology Vol. 54 ( 4 ) page： 2520 - 2529 2020.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ACS Publications

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 Reviewed International journal Open Access

Yan-Yu Chen, Masahito Ishikawa, Risa Suzuki, Hidehiro Ito, Toshiaki Kamachi, Katsutoshi Hori

Biochemical Engineering Journal Vol. 154 page： 107441 2020.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier

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

Bottom-up Creation of an Artificial Cell Covered with the Adhesive Bacterionanofiber Protein AtaA Reviewed International journal Open Access

Kosaku Noba, Masahito Ishikawa, Atsuko Uyeda, Takayoshi Watanabe, Takahiro Hohsaka, Shogo Yoshimoto, Tomoaki Matsuura, Katsutoshi Hori

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Vol. 141 ( 48 ) page： 19058 - 19066 2019.12

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ACS Publications

The bacterial cell surface structure has important roles for various cellular functions. However, research on reconstituting bacterial cell surface structures is limited. This study aimed to bottom-up create a cell-sized liposome covered with AtaA, the adhesive bacterionanofiber protein localized on the cell surface of Acinetobacter sp. Tol 5, without the use of the protein secretion and assembly machineries. Liposomes containing a benzylguanine derivative-modified phospholipid were decorated with a truncated AtaA protein fused to a SNAP-tag expressed in a soluble fraction in Escherichia coli. The obtained liposome showed a similar surface structure and function to that of native Tol 5 cells and adhered to both hydrophobic and hydrophilic solid surfaces. Furthermore, this artificial cell was able to drive an enzymatic reaction in the adhesive state. The developed artificial cellular system will allow for analysis of not only AtaA, but also other cell surface proteins under a cell-mimicking environment. In addition, AtaA-decorated artificial cells may inspire the development of biotechnological applications that require immobilization of cells onto a variety of solid surfaces, in particular, in environments where the use of genetically modified organisms is prohibited.

DOI： 10.1021/jacs.9b09340

細菌の接着機構と界面微生物工学プロセスの創出に関する研究 Invited

堀　克敏

生物工学会誌 Vol. 第102巻 ( 第2号 ) page： 54 - 60 2024

微生物飼料を活用した次世代陸上養殖の展開

田丸　浩、堀　克敏

月刊バイオインダストリー Vol. 40 ( 1 ) page： 51 - 57 2023.1

小型化接着タンパク質による大腸菌の固定化と微生物反応への応用; バイオサイエンスとインダストリー

吉本　将悟、堀　克敏

バイオサイエンスとインダストリー Vol. 40 page： 51 - 57 2023

Perturbation by Antimicrobial Bacteria of the Epidermal Bacterial Flora of Rainbow Trout in Flow-Through Aquaculture Reviewed International journal Open Access

Nakatani Hajime, Yamada Naoki, Hashimoto Naoki, Okazaki Fumiyoshi, Arakawa Tomoko, Tamaru Yutaka, Hori Katsutoshi

BIOLOGY-BASEL Vol. 11 ( 8 ) page： 1249 2022.8

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：MDPI

The bacterial flora of the epidermal mucus of fish is closely associated with the host’s health and susceptibility to pathogenic infections. In this study, we analyzed the epidermal mucus bacteria of rainbow trout (Oncorhynchus mykiss) reared in flow-through aquaculture under environmental perturbations. Over ~2 years, the bacteria present in the skin mucus and water were analyzed based on the 16S rDNA sequences. The composition of the mucus bacterial community showed significant monthly fluctuations, with frequent changes in the dominant bacterial species. Analysis of the beta- and alpha-diversity of the mucus bacterial flora showed the fluctuations of the composition of the flora were caused by the genera Pseudomonas, Yersinia, and Flavobacterium, and some species of Pseudomonas and Yersinia in the mucus were identified as antimicrobial bacteria. Examination of the antimicrobial bacteria in the lab aquarium showed that the natural presence of antimicrobial bacteria in the mucus and water, or the purposeful addition of them to the rearing water, caused a transition in the mucus bacteria community composition. These results demonstrate that specific antimicrobial bacteria in the water or in epidermal mucus comprise one of the causes of changes in fish epidermal mucus microflora.

DOI： 10.3390/biology11081249

The extracellular juncture domains of Type 5 autotransporters Reviewed International coauthorship

Weikum, J; Kulakova, A; Tesei, G; Yoshimoto, S; Jægerum, LV; Schütz, M; Hori, K; Skepö, M; Harris, P; Leo, JC; Morth, JP

ACTA CRYSTALLOGRAPHICA A-FOUNDATION AND ADVANCES Vol. 78 page： E5 - E5 2022.8

Fabrication of a highly protective 3D-printed mask and evaluation of its viral filtration efficiency using a human head mannequin Reviewed International journal Open Access

Ohara Yuki, Kanie Junichi, Hori Katsutoshi

HARDWAREX Vol. 11 page： e00314 2022.4

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier

Facemasks are one of the most effective and low-cost prophylactics for COVID-19. In the spring 2020, when a severe shortage of facemasks occurred worldwide, various types of 3D-printed masks were designed and proposed. However, the protective effects conferred by most of these masks were not experimentally evaluated. Here, we provide a new simple design of 3D-printed mask and evaluate its protective effect in a viral filtration test using a human head mannequin. The developed mask can be constructed with a low-cost 3D printer, with an approximate production cost of US $4. This mask has three parts: the main part, wearing parts, and a piece of non-woven fabric filter. The volume of the filter, which needs to be changed daily, was reduced to approximately 1/10 of that of commercially available surgical masks used in this study. The developed mask is fabricated from polylactic acid, a biodegradable plastic, and its surface contour contacting the face may be adjusted after softening the material with hot water at 60–80 °C. The viral filtration efficiency of the developed mask was found to be over 80%. This performance is better than that of commercially available facemasks, such as surgical masks and cloth masks, and equal to those of KN95 and KF94.

DOI： 10.1016/j.ohx.2022.e00314

細菌接着タンパク質と微生物固定化への利用 Invited

堀　克敏

バイオインダストリー Vol. 39 ( 3 ) page： 11 - 18 2022.3

Recent advances in research on biointerfaces: From cell surfaces to artificial interfaces Invited Reviewed International journal

K. Hori, S. Yoshimoto, T. Yoshino, T. Zako, G. Hirao, S. Fujita, C. Nakamura, A. Yamagishi, and N. Kamiya

Journal of Bioscience and Bioengineering Vol. 133 ( 3 ) page： 195 - 207 2022.1

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Biointerfaces are regions where biomolecules, cells, and organic materials are exposed to environmental media or come in contact with other biomaterials, cells, and inorganic/organic materials. In this review article, six research topics on biointerfaces are described to show examples of state-of-art research approaches. First, biointerface design of nanoparticles for molecular detection is described. Functionalized gold nanoparticles can be used for sensitive detection of various target molecules, including chemical compounds and biomolecules, such as DNA, proteins, cells, and viruses. Second, the interaction between bacterial cell surfaces and material surfaces, including the introduction of advances in analytical methods and theoretical calculations, are explained as well as their applications to bioprocesses. Third, bioconjugation technologies for localizing functional proteins at biointerfaces are introduced, in particular, by focusing the potential of enzymes as a catalytic tool for designing different types of bioconjugates that function at biointerfaces. Forth topics is focusing on lipideprotein interaction in cell membranes as natural biointerfaces. Examples of membrane lipid engineering are introduced, and it is mentioned how their compositional profiles affect membrane protein functions. Fifth topic is the physical method for molecular delivery across the biointerface being developed currently, such as highly efficient nanoinjection, electroporation, and nanoneedle devices, in which the key is how to perforate the cell membrane. Final topic is the chemical design of lipid- or polymer-based RNA delivery carriers and their behavior on the cell interface, which are currently attracting attention as RNA vaccine technologies targeting COVID-19. Finally, future directions of biointerface studies are presented.

ウイルス除去効果の高い樹脂製立体マスクの開発 Invited

堀　克敏

繊維機械学会誌月刊せんい Vol. 74 ( 11 ) page： 593 - 598 2021.11

Complete Genome Sequence of the Highly Adhesive Bacterium Acinetobacter sp. Strain Tol 5 Reviewed International journal Open Access

Masahito Ishikawa, Katsutoshi Hori

Microbiology Resource Announcements Vol. 10 ( 35 ) page： e0056721 2021.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

Acinetobacter sp. strain Tol 5 is a nonpathogenic Gram-negative bacterium with biotechnological and environmental applications. Here, we report the complete genome sequence of Acinetobacter sp. strain Tol 5, which has a genome size of 4,799,506 bp and a G1C content of 38.1%.

DOI： 10.1128/MRA.00567-21

界面微生物工学微生物固定化・細胞表層工学から膜タンパク質利用技術まで Invited

堀　克敏

化学と生物 Vol. 59 ( 8 ) page： 393 - 400 2021.8

Switching between methanol accumulation and cell growth by expression control of methanol dehydrogenase in Methylosinus trichosporium OB3b mutant Reviewed International journal Open Access

Hidehiro Ito, Kosei Yoshimori, Masahito Ishikawa, Katsutoshi Hori, Toshiaki Kamachi

Frontiers in Microbiology Vol. 12 page： 639266 2021.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers

Methanotrophs have been used to convert methane to methanol at ambient temperature and pressure. In order to accumulate methanol using methanotrophs, methanol dehydrogenase (MDH) must be downregulated as it consumes methanol. Here, we describe a methanol production system wherein MDH expression is controlled by using methanotroph mutants. We used the MxaF knockout mutant of Methylosinus trichosporium OB3b. It could only grow with MDH (XoxF) which has a cerium ion in its active site and is only expressed by bacteria in media containing cerium ions. In the presence of 0 μM copper ion and 25 μM cerium ion, the mutant grew normally. Under conditions conducive to methanol production (10 μM copper ion and 0 μM cerium ion), cell growth was inhibited and methanol accumulated (2.6 μmol·mg−1 dry cell weight·h−1). The conversion efficiency of the accumulated methanol to the total amount of methane added to the reaction system was ~0.3%. The aforementioned conditions were repeatedly alternated by modulating the metal ion composition of the bacterial growth medium.

DOI： 10.3389/fmicb.2021.639266

微生物接着タンパク質AtaAの無限の可能性 Invited

堀　克敏

バイオインダストリー Vol. 38 ( 2 ) page： 8 - 17 2021.2

Establishing a Percutaneous Infection Model Using Zebrafish and a Salmon Pathogen Reviewed Open Access

Nakatani, H; Hori, K

BIOLOGY-BASEL Vol. 10 ( 2 ) page： 1 - 15 2021.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Biology

To uncover the relationship between skin bacterial flora and pathogen infection, we developed a percutaneous infection model using zebrafish and Yersinia ruckeri, a pathogen causing enteric redmouth disease in salmon and in trout. Pathogen challenge, either alone or together with pricking by a small needle, did not cause infection of the fish. However, cold stress given by water temperature shift from the optimum 28°C for zebrafish to 20°C caused fatal infection of injured fish following pathogen challenge. We investigated the effects of cold stress, injury, and pathogen challenge, alone and in combination, on fish skin bacterial flora using 16S rDNA metagenomics. We found that cold stress drastically altered the skin bacterial flora, which was dominated by Y. ruckeri on infected fish. In addition, fish whose intrinsic skin bacterial flora was disrupted by antibiotics had their skin occupied by Y. ruckeri following a challenge with this pathogen, although the fish survived without injury to create a route for invasion into the fish body. Our results suggest that the intrinsic skin bacterial flora of fish protects them from pathogen colonization, and that its disruption by stress allows pathogens to colonize and dominate their skin.

DOI： 10.3390/biology10020166

The extracellular juncture domains in the Intimin passenger adopt a constitutively extended conformation inducing restrains to its sphere of action Reviewed International coauthorship International journal Open Access

J. Weikum, A. Kulakova, G. Tesei, S. Yoshimoto, L. V. Jægerum, M. Schütz, K. Hori, M. Skepö, P. Harris, J. C. Leo, and J. P. Morth

Scientific Reports Vol. 10 ( 1 ) page： 21249 2020.12

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Nature

Enterohemorrhagic and enteropathogenic Escherichia coli are among the most important food-borne pathogens, posing a global health threat. The virulence factor intimin is essential for the attachment of pathogenic E. coli to the intestinal host cell. Intimin consists of four extracellular bacterial immunoglobulin-like (Big) domains, D00–D2, extending into the fifth lectin subdomain (D3) that binds to the Tir-receptor on the host cell. Here, we present the crystal structures of the elusive D00–D0 domains at 1.5 Å and D0–D1 at 1.8 Å resolution, which confirms that the passenger of intimin has five distinct domains. We describe that D00–D0 exhibits a higher degree of rigidity and D00 likely functions as a juncture domain at the outer membrane-extracellular medium interface. We conclude that D00 is a unique Big domain with a specific topology likely found in a broad range of other inverse autotransporters. The accumulated data allows us to model the complete passenger of intimin and propose functionality to the Big domains, D00–D0–D1, extending directly from the membrane.

DOI： 10.1038/s41598-020-77706-7

人工細胞と固体表面をつなぐ接着ナノファイバー～新規生体触媒の開発を目指して～ Invited

石川　聖人,堀　克敏

バイオサイエンスとインダストリー Vol. 78 ( 5 ) page： 408 - 409 2020.9

Extracellular electron transfer mediated by a cytocompatible redox polymer to study the crosstalk among the mammalian circadian clock, cellular metabolism, and cellular redox state Reviewed International journal Open Access

Masahito Ishikawa, Kazuki Kawai, Masahiro Kaneko, Kenya Tanaka, Shuji Nakanishi, Katsutoshi Hori

RSC ADVANCES Vol. 10 ( 3 ) page： 1648 - 1657 2020.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry

The circadian clock is an endogenous biological timekeeping system that controls various physiological and cellular processes with a 24 h rhythm. The crosstalk among the circadian clock, cellular metabolism, and cellular redox state has attracted much attention. To elucidate this crosstalk, chemical compounds have been used to perturb cellular metabolism and the redox state. However, an electron mediator that facilitates extracellular electron transfer (EET) has not been used to study the mammalian circadian clock due to potential cytotoxic effects of the mediator. Here, we report evidence that a cytocompatible redox polymer pMFc (2-methacryloyloxyethyl phosphorylcholine-co-vinyl ferrocene) can be used as the mediator to study the mammalian circadian clock. EET mediated by oxidized pMFc (ox-pMFc) extracted intracellular electrons from human U2OS cells, resulting in a longer circadian period. Analyses of the metabolome and intracellular redox species imply that ox-pMFc receives an electron from glutathione, thereby inducing pentose phosphate pathway activation. These results suggest novel crosstalk among the circadian clock, metabolism, and redox state. We anticipate that EET mediated by a redox cytocompatible polymer will provide new insights into the mammalian circadian clock system, which may lead to the development of new treatments for circadian clock disorders.

DOI： 10.1039/c9ra10023g

接着タンパク質・接着微生物を利用した効率的バイオプロセスの構築 Invited

石川　聖人，堀　克敏

酵素工学ニュース Vol. 84 page： 13 - 17 2020

接着性人工細胞触媒のボトムアップ創製 Invited

石川　聖人，堀　克敏

生物工学 Vol. 98 page： 651 - 654 2020

Control of AtaA-mediated bacterial immobilization by casein hydrolysates. Reviewed International journal

Y. Ohara, S. Yoshimoto, K. Hori

Journal of bioscience and bioengineering Vol. 128 ( 5 ) page： 544 - 550 2019.11

Language：English Publishing type：Research paper (scientific journal)

Acinetobacter sp. Tol 5 exhibits an autoagglutinating nature and high adhesiveness to various abiotic surfaces through its bacterionanofiber protein AtaA. We have developed new bacterial immobilization methods utilizing the high adhesiveness of AtaA. We previously reported that salt is essential for the adhesiveness of AtaA. In the current study, we unexpectedly found that Tol 5 cells were not immobilized onto polyurethane foam support during growth in LB medium although AtaA was properly expressed and displayed onto the cell surface. The adhesion of Tol 5 resting cells was not affected by sugars but drastically inhibited by yeast extract and casein hydrolysates such as tryptone and casamino acids technical grade (CA-T). Some amino acids, which are major components of CA-T, partially inhibited the adhesion of Tol 5 cells. Experimental results suggested that oligopeptides might effectively inhibit the cell adhesion. Immobilized cells onto the support through AtaA were detached in CA-T solution. Also, the detached cells could be re-immobilized onto the support without impairing of their adhesiveness by replacing CA-T solution to a basal salt medium. Microscopic observation revealed that breaking of AtaA-mediated cell–cell interaction is important for the detachment of Tol 5 cells from the support. CA-T also inhibited AtaA-mediated autoagglutination and dispersed cell clumps through AtaA. This is the first report on adhesion inhibitors against AtaA and suggests that casein hydrolysates like CA-T would be a powerful tool for controlling AtaA-mediated bacterial immobilization.

DOI： 10.1016/j.jbiosc.2019.04.019

接着性バクテリオナノファイバーによる微生物のプロセスの革新 Invited Reviewed

堀　克敏

環境バイオテクノロジー学会誌 Vol. 19 page： 21 - 28 2019.3

On-fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin Reviewed International journal

Hajime Nakatani, Junichi Kanie, Katsutoshi Hori

BIOTECHNOLOGY AND BIOENGINEERING Vol. 116 ( 2 ) page： 239 - 249 2019.2

Language：English Publishing type：Research paper (scientific journal) Publisher：Wiley

In the cell surface display system, the distance of a surface-displayed molecule from the cell surface should influence its functionality due to the interference by other surface structures. For the purpose of developing this distance-variable surface display system, we utilized a long fibrous adhesin, Acinetobacter trimeric autotransporter adhesin (AtaA) of the strain Tol 5. We constructed His-tagged full-length and shorter AtaA fibers designed by N-terminal deletion and expressed them in the ΔataA mutant. Immunoelectron microscopy clearly showed that they formed fibers on the cell surface and the His-tag was displayed on the fiber tip located at fixed distances from the cell surface. N-terminal deletion of AtaA shortened the distance between the His-tag and the cell surface, as designed. Time-course analyses of the cell-to-Ni-Sepharose beads binding revealed that cells producing the longer fibers bound more rapidly to the beads. The His-tagged AtaA derivatives were also displayed on Escherichia coli cells, and a similar tendency was shown; the His-tag on the longer fiber was more functional than that on the shorter one. Thus, we developed an on-fiber display system of a functional peptide using a long trimeric autotransporter adhesin (TAA) fiber, which can vary the distance between the displayed molecule and the cell surface.

DOI： 10.1002/bit.26857

The Acinetobacter trimeric autotransporter adhesin Ata controls key virulence traits of Acinetobacter baumannii Reviewed International coauthorship International journal Open Access

Marko Weidensdorfer, Masahito Ishikawa, Katsutoshi Hori, Dirk Linke, Bardya Djahanschiri, Ruben Iruegas, Ingo Ebersberger, Sara Riedel-Christ, Giulia Enders, Laura Leukert, Peter Kraiczy, Florian Rothweiler, Jindrich Cinatl, Juergen Berger, Katharina Hipp, Volkhard A. J. Kempf, Stephan Goettig

VIRULENCE Vol. 10 ( 1 ) page： 68 - 81 2019.1

Language：English Publishing type：Research paper (scientific journal)

Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.

DOI： 10.1080/21505594.2018.1558693

Efficient Counterselection for Methylococcus capsulatus (Bath) by Using a Mutated pheS Gene Reviewed International journal Open Access

Masahito Ishikawa, Sho Yokoe, Souichiro Kato, Katsutoshi Hori

APPLIED AND ENVIRONMENTAL MICROBIOLOGY Vol. 84 ( 23 ) page： e01875-18 2018.12

Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology

Methylococcus capsulatus (Bath) is a representative gammaproteobacterial methanotroph that has been studied extensively in diverse research fields. The sacB gene, which encodes levansucrase, causing cell death in the presence of sucrose, is widely used as a counterselectable marker for disruption of a target gene in Gram-negative bacteria. However, sacB is not applicable to all Gram-negative bacteria, and its efficiency for the counterselection of M. capsulatus (Bath) is low. Here, we report the construction of an alternative counterselectable marker, pheS*, by introduction of two point mutations (A306G and T252A) into the pheS gene from M. capsulatus (Bath), which encodes the α-subunit of phenylalanyl-tRNA synthetase. The transformant harboring pheS* on an expression plasmid showed sensitivity to 10 mM p-chloro-phenylalanine, whereas the transformant harboring an empty plasmid showed no sensitivity, indicating the availability of pheS* as a counterselectable marker in M. capsulatus (Bath). To validate the utility of the pheS* marker in counterselection, we attempted to obtain an unmarked mutant of xoxF, a gene encoding the major subunit of Xox methanol dehydrogenase, which we failed to obtain by counterselection using the sacB marker. PCR, immunodetection using an anti-XoxF antiserum, and a cell growth assay in the absence of calcium demonstrated successful disruption of the xoxF gene in M. capsulatus (Bath). The difference in counterselection efficiencies of the markers indicated that pheS* is more suitable than sacB for counterselection in M. capsulatus (Bath). This study provides a new genetic tool enabling efficient counterselection in M. capsulatus (Bath).

DOI： 10.1128/AEM.01875-18

Extracellular Electron Transfer via Outer Membrane Cytochromes in a Methanotrophic Bacterium Methylococcus capsulatus (Bath) Reviewed International journal Open Access

Kenya Tanaka, Sho Vokoe, Kensuke Igarashi, Motoko Takashino, Masahito Ishikawa, Katsutoshi Hori, Shuji Nakanishi, Souichiro Kato

FRONTIERS IN MICROBIOLOGY Vol. 9 ( NOV ) page： 2905 2018.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers

Electron exchange reactions between microbial cells and solid materials, referred to as extracellular electron transfer (EET), have attracted attention in the fields of microbial physiology, microbial ecology, and biotechnology. Studies of model species of iron-reducing, or equivalently, current-generating bacteria such as Geobacter spp. and Shewanella spp. have revealed that redox-active proteins, especially outer membrane c-type cytochromes (OMCs), play a pivotal role in the EET process. Recent (meta)genomic analyses have revealed that diverse microorganisms that have not been demonstrated to have EET ability also harbor OMC-like proteins, indicating that EET via OMCs could be more widely preserved in microorganisms than originally thought. A methanotrophic bacterium Methylococcus capsulatus (Bath) was reported to harbor multiple OMC genes whose expression is elevated by Cu starvation. However, the physiological role of these genes is unknown. Therefore, in this study, we explored whether M. capsulatus (Bath) displays EET abilities via OMCs. In electrochemical analysis, M. capsulatus (Bath) generated anodic current only when electron donors such as formate were available, and could reduce insoluble iron oxides in the presence of electron donor compounds. Furthermore, the current-generating and iron-reducing activities of M. capsulatus (Bath) cells that were cultured in a Cu-deficient medium, which promotes high levels of OMC expression, were higher than those cultured in a Cu-supplemented medium. Anodic current production by the Cu-deficient cells was significantly suppressed by disruption of MCA0421, a highly expressed OMC gene, and by treatment with carbon monoxide (CO) gas (an inhibitor of c-type cytochromes). Our results provide evidence of EET in M. capsulatus (Bath) and demonstrate the pivotal role of OMCs in this process. This study raises the possibility that EET to solid compounds is a novel survival strategy of methanotrophic bacteria.

DOI： 10.3389/fmicb.2018.02905

Heterologous expression of geraniol dehydrogenase for identifying the metabolic pathways involved in the biotransformation of citral by Acinetobacter sp Tol 5 Reviewed International journal Open Access

Usami Atsushi, Masahito Ishikawa, Katsutoshi Hori

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY Vol. 82 ( 11 ) page： 2012 - 2020 2018.11

Language：English Publishing type：Research paper (scientific journal)

The biotransformation of citral, an industrially important monoterpenoid, has been extensively studied using many microbial biocatalysts. However, the metabolic pathways involved in its biotransformation are still unclear, because citral is a mixture of the trans-isomer geranial and the cis-isomer neral. Here, we applied the heterologous expression of geoA, a gene encoding geraniol dehydrogenase that specifically converts geraniol to geranial and nerol to neral, to identify the metabolic pathways involved in the biotransformation of citral. Acinetobacter sp. Tol 5 was employed in order to demonstrate the utility of this methodology. Tol 5 transformed citral to (1R,3R,4R)-1-methyl-4-(1-methylethenyl)-1,3-cyclohexanediol and geranic acid. Biotransformation of citral precursors (geraniol and nerol) by Tol 5 transformant cells expressing geoA revealed that these compounds were transformed specifically from geranial. Our methodology is expected to facilitate a better understanding of the metabolic pathways involved in the biotransformation of substrates that are unstable and include geometric isomers.

DOI： 10.1080/09168451.2018.1501263

Cell behavior of the highly sticky bacterium Acinetobacter sp Tol 5 during adhesion in laminar flows Reviewed International journal Open Access

Yoshihide Furuichi, Keita Iwasaki, Katsutoshi Hori

SCIENTIFIC REPORTS Vol. 8 ( 1 ) page： 8285 2018.5

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Nature

It is important to characterize how medically, industrially, or environmentally important bacteria adhere to surfaces in liquid flows in order to control their cell adhesion and subsequent biofilm formation. Acinetobacter sp. Tol 5 is a remarkably sticky bacterium that autoagglutinates through the adhesive nanofiber protein AtaA, which is applicable to cell immobilization in bioprocesses. In this study, the adhesion and behavior of Tol 5 cells in laminar flows were investigated using flow cell systems. Tol 5 cells autoagglutinated through AtaA and formed cell clumps during flowing. The cell clumps rather than single cells went downward due to gravity and adhered to the bottom surface. Under appropriate shear stress, a twin vortex was caused by a separated flow generated at the rear of the pre-immobilized cell clumps and carried the small cell clumps to this location, resulting in their stacking there. The rearward immobilized cell clumps developed into a large, stable aggregate with a streamlined shape, independent of cell growth. Cell clumps hardly ever developed under weak shear stress that could not generate a twin vortex and were broken up under excessively strong shear stress. These cell behaviors including the importance of clumping are interesting features in the bacterial adhesion processes.

DOI： 10.1038/s41598-018-26699-5

Immobilization of Enterobacter aerogenes by a Trimeric Autotransporter Adhesin, AtaA, and Its Application to Biohydrogen Production Reviewed International journal Open Access

Hajime Nakatani, Ding Nan, Yuki Ohara, Katsutoshi Hori

CATALYSTS Vol. 8 ( 4 ) page： 159 2018.4

Language：English Publishing type：Research paper (scientific journal)

Biological hydrogen production by microbial cells has been extensively researched as an energy-efficient and environmentally-friendly process. In this study, we propose a fast, easy method for immobilizing Enterobacter aerogenes by expressing ataA, which encodes the adhesive protein of Acinetobacter sp. Tol 5. AtaA protein on the E. aerogenes cells carrying the ataA gene was demonstrated by immunoblotting and flow cytometry. The AtaA-producing cells exhibited stronger adherence and auto-agglutination characteristics than wild-type cells, and were successfully immobilized (at approximately 2.5 mg/cm3) on polyurethane foam. Hydrogen production from the cell-immobilized polyurethane foams was monitored in repetitive batch reactions and flow reactor studies. The total hydrogen production in triple-repetitive batch reactions reached 0.6 mol/mol glucose, and the hydrogen production rate in the flow reactor was 42 mL·h−1·L−1. The AtaA production achieved simple and immediate immobilization of E. aerogenes on the foam, enabling repetitive and continuous hydrogen production. This report newly demonstrates the production of AtaA on the cell surfaces of bacterial genera other than Acinetobacter, and can simplify and accelerate the immobilization of whole-cell catalysts.

DOI： 10.3390/catal8040159

Open Access

Real-time monitoring of intracellular redox changes in Methylococcus capsulatus (Bath) for efficient bioconversion of methane to methanol Reviewed International journal Open Access

Masahito Ishikawa, Yuya Tanaka, Risa Suzuki, Kota Kimura, Kenya Tanaka, Kazuhide Kamiya, Hidehiro Ito, Souichiro Kato, Toshiaki Kamachi, Katsutoshi Hori, Shuji Nakanishi

BIORESOURCE TECHNOLOGY Vol. 241 page： 1157 - 1161 2017.10

Language：English Publishing type：Research paper (scientific journal)

This study aimed to develop a novel method for real-time monitoring of the intracellular redox states in a methanotroph Methylococcus capsulatus, using Peredox as a genetically encoded fluorescent sensor of the NADH:NAD+ ratio. As expected, the fluorescence derived from the Peredox-expressing M. capsulatus transformant increased by supplementation of electron donor compounds (methane and formate), while it decreased by specifically inhibiting the methanol oxidation reaction. Electrochemical measurements confirmed that the Peredox fluorescence reliably represents the intracellular redox changes. This study is the first to construct a reliable redox-monitoring method for methanotrophs, which will facilitate to develop more efficient methane-to-methanol bioconversion processes.

DOI： 10.1016/j.biortech.2017.05.107

Reversible bacterial immobilization based on the salt-dependent adhesion of the bacterionanofiber protein AtaA Reviewed International journal Open Access

Shogo Yoshimoto, Yuki Ohara, Hajime Nakatani, Katsutoshi Hori

MICROBIAL CELL FACTORIES Vol. 16 ( 1 ) page： 123 2017.7

Language：English Publishing type：Research paper (scientific journal)

Background: Immobilization of microbial cells is an important strategy for the efficient use of whole-cell catalysts because it simplifies product separation, enables the cell concentration to be increased, stabilizes enzymatic activity, and permits repeated or continuous biocatalyst use. However, conventional immobilization methods have practical limitations, such as limited mass transfer in the inner part of a gel, gel fragility, cell leakage from the support matrix, and adverse effects on cell viability and catalytic activity. We previously showed a new method for bacterial cell immobilization using AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5. This approach is expected to solve the drawbacks of conventional immobilization methods. However, similar to all other immobilization methods, the use of support materials increases the cost of bioprocesses and subsequent waste materials. Results: We found that the stickiness of the AtaA molecule isolated from Tol 5 cells is drastically diminished at ionic strengths lower than 10 mM and that it cannot adhere in deionized water, which also inhibits cell adhesion mediated by AtaA. Cells immobilized on well plates and polyurethane foam in a salt solution were detached in deionized water by rinsing and shaking, respectively. The detached cells regained their adhesiveness in a salt solution and could rapidly be re-immobilized. The cells expressing the ataA gene maintained their adhesiveness throughout four repeated immobilization and detachment cycles and could be repeatedly immobilized to polyurethane foam by a 10-min shake in a flask. We also demonstrated that both bacterial cells and a support used in a reaction could be reused for a different type of reaction after detachment of the initially immobilized cells from the support and a subsequent immobilization step. Conclusions: We invented a unique reversible immobilization method based on the salt-dependent adhesion of the AtaA molecule that allows us to reuse bacterial cells and supports by a simple manipulation involving a deionized water wash. This mitigates problems caused by the use of support materials and greatly helps to enhance the efficiency and productivity of microbial production processes.

DOI： 10.1186/s12934-017-0740-7

バイオコントロールによる高濃度油脂含有排水のハイパフォーマンス処理 Invited Reviewed

堀　克敏

環境バイオテクノロジー学会誌 Vol. 17 page： 3-8 2017.3

Cell surface protein engineering for high-performance whole-cell catalysts Reviewed International journal

Nakatani Hajime, Hori Katsutoshi

FRONTIERS OF CHEMICAL SCIENCE AND ENGINEERING Vol. 11 ( 1 ) page： 46 - 57 2017.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers of Chemical Science and Engineering

Cell surface protein engineering facilitated by accumulation of information on genome and protein structure involves heterologous production and modification of cell surface proteins using genetic engineering, and is important for the development of high-performance whole-cell catalysts. In this field, cell surface display is a major technology by exposing target proteins, such as enzymes, on the cell surface using a carrier protein. The target proteins are fused to the carrier proteins that transport and tether them to the cell surface, as well as to a secretion signal. This paper reviews cell surface display systems for prokaryotic and eukaryotic cells from the perspective of carrier proteins, which determine the number of displayed molecules, and the localization, size, and direction (N- or C-terminal anchoring) of the passengers. We also discuss advanced methods for displaying multiple enzymes and a new method for the immobilization of whole-cell catalysts using adhesive surface proteins.

DOI： 10.1007/s11705-017-1609-3

バイオフィルム・微生物付着の工学利用

堀　克敏

膜 Vol. 42 page： 54-59 2017

細胞表層タンパク質の合成生物学 Invited

石川聖人、堀　克敏

生物工学会誌 Vol. 94 page： 701-703 2016.11

細菌の持つ接着ナノファイバータンパク質AtaA

堀　克敏

バイオサイエンスとインダストリー Vol. 74 page： 380-381 2016.11

Secretion of the intimin passenger domain is driven by protein folding Reviewed

J. C. Leo, P. Oberhettinger, S. Yoshimoto, D. B. R. K. G. Udatha, J. P. Morth, M. Schütz, K. Hori, and D. Linke

J. Biol. Chem Vol. 291 page： 20096 2016.7

An Acinetobacter trimeric autotranspoter adhesin reaped from cells exhibits its nonspecific stickiness via a highly stable 3D strucuture Reviewed

S. Yoshimoto, H. Nakatani, K.Iwasaki and K.Hori

Sci Rep 2016.6

Discovery of a novel periplasmic protein that forms acomplex with a trimeric autotransporter adhesin and Peptidoglycan Reviewed

M. Ishikawa, S. Yoshimoto, A. Hayashi, J. Kanie and K.Hori

Mol Microbiol 2016.5

細菌接着タンパク質AtaA分子の特性と応用 Invited

堀　克敏

バイオサイエンスとインダストリー Vol. 74 page： 417-419 2016.5

Structural basis for toughness and flexibility in the C-terminal Passenger domain of an Acinetobactor trimeric autotranspoter Adhesin Reviewed

K. Koiwai, M.D.Hartmann, D. Linke, A.N. Lupas and K.Hori

J. Biol. Chem. Vol. 291 page： 3705-3724 2016.2

Effectiveness of direct immobilization of bacterial cells onto material surfaces using the bacterionanofiber protein AtaA. Reviewed

Hori K, Ohara Y, Ishikawa M, Nakatani H.

Applied Microbiology and Biotechnology 2015.4

Language：English Publishing type：Research paper (scientific journal)

The bacterionanofiber protein AtaA, a member of the trimeric autotransporter adhesin family found in Acinetobacter sp. Tol 5, is responsible for the nonspecific, high adhesiveness and autoagglutination of this strain. Previously, we introduced the ataA gene into the nonadhesive Acinetobacter strain ST-550, which conferred high adhesiveness to this strain, immobilized its cells, and improved indigo productivity due to enhanced tolerance to the toxic substrate. In this study, we again demonstrated the effectiveness of this new microbial immobilization method using AtaA in a number of conditions. AtaA enabled the effective immobilization of growing, resting, and lyophilized cells of a type strain of Acinetobacter, ADP1, which is also intrinsically nonadhesive, onto the surface of several kinds of support ranging from artificial to natural materials and from hydrophobic polyurethane to hydrophilic glass. Immobilization with AtaA enabled exclusive cell growth in the support space and only a few cells existed in the bulk medium. Immobilization of resting cells drastically increased cell concentration, depending on the support material; dry cells of approximately 110 g/L could be immobilized onto glass wool. Finally, we demonstrated that ADP1 cells immobilized on polyurethane foam can undergo at least 10 repetitive reactions without inactivation during a 5-h period. Even after drying and storing for 3 days, the immobilized cells showed enzymatic activity and an ester hydrolysis reaction was repeated by simply transferring the support with the cells into a fresh reaction buffer.

DOI： 10.1007/s00253-015-6554-9

Definition of the transcription factor TdIF1 consensus-binding sequence through genomewide mapping of its binding sites Reviewed

K. Koiwai,T. Kubota, N. Watanabe, K. Hori, O. Koiwai and H. Masai

Genes to Cells Vol. 20 ( 3 ) page： 242-254 2015.1

Language：English Publishing type：Research paper (scientific journal)

TdIF1 was originally identified as a protein that directly binds to terminal deoxynucleotidyltransferase, TdT. Through in vitro selection assays (SELEX), we recently showed that TdIF1 recognizes both AT-tract and a specific DNA sequence motif, 5'-TGCATG-3', and can up-regulate the expression of RAB20 through the latter motif. However, whether TdIF1 binds to these sequences in the cells has not been clear and its other target genes remain to be identified. Here, we determined in vivo TdIF1-binding sequences (TdIF1-invivoBMs) on the human chromosomes through ChIP-seq analyses. The result showed a 160-base pair cassette containing 'AT-tract~palindrome (inverted repeat)~AT-tract' as a likely target sequence of TdIF1. Interestingly, the core sequence of the palindrome in the TdIF1-invivoBMs shares significant similarity to the above 5'-TGCATG-3' motif determined by SELEX in vitro. Furthermore, spacer sequences between AT-tract and the palindrome contain many potential transcription factor binding sites. In luciferase assays, TdIF1 can up-regulate transcription activity of the promoters containing the TdIF1-invivoBM, and this effect is mainly through the palindrome. Clusters of this motif were found in the potential target genes. Gene ontology analysis and RT-qPCR showed the enrichment of some candidate targets of TdIF1 among the genes involved in the regulation of ossification. Potential modes of transcription activation by TdIF1 are discussed.

DOI： 10.1111/gtc.12216