研究者詳細 - 堀　克敏

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招待有り査読有り

堀　克敏

環境バイオテクノロジー学会誌 19 巻頁： 21 - 28 2019年3月

On-fiber display of a functional peptide at sites distant from the cell surface using a long bacterionanofiber of a trimeric autotransporter adhesin 査読有り国際誌

Nakatani, H; Kanie, J; Hori, K

BIOTECHNOLOGY AND BIOENGINEERING 116 巻 ( 2 ) 頁： 239 - 249 2019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：Biotechnology and Bioengineering

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 査読有り国際共著国際誌 Open Access

Weidensdorfer, M; Ishikawa, M; Hori, K; Linke, D; Djahanschiri, B; Iruegas, R; Ebersberger, I; Riedel-Christ, S; Enders, G; Leukert, L; Kraiczy, P; Rothweiler, F; Cinatl, J; Berger, J; Hipp, K; Kempf, VAJ; Göttig, S

VIRULENCE 10 巻 ( 1 ) 頁： 68 - 81 2019年1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：Virulence

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 査読有り国際誌 Open Access

Ishikawa, M; Yokoe, S; Kato, S; Hori, K

APPLIED AND ENVIRONMENTAL MICROBIOLOGY 84 巻 ( 23 ) 頁： e01875-18 2018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：Applied and Environmental 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) 査読有り国際誌 Open Access

Tanaka, K; Vokoe, S; Igarashi, K; Takashino, M; Ishikawa, M; Hori, K; Nakanishi, S; Kato, S

FRONTIERS IN MICROBIOLOGY 9 巻 ( NOV ) 頁： 2905 2018年11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：Frontiers in Microbiology

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 査読有り国際誌 Open Access

Usami, A; Ishikawa, M; Hori, K

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 82 巻 ( 11 ) 頁： 2012 - 2020 2018年11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：Bioscience, Biotechnology and Biochemistry

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 査読有り国際誌 Open Access

Furuichi, Y; Iwasaki, K; Hori, K

SCIENTIFIC REPORTS 8 巻 ( 1 ) 頁： 8285 2018年5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：Scientific Reports

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 査読有り国際誌 Open Access

Nakatani, H; Ding, N; Ohara, Y; Hori, K

CATALYSTS 8 巻 ( 4 ) 頁： 159 2018年4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：Catalysts

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 査読有り国際誌 Open Access

Ishikawa, M; Tanaka, Y; Suzuki, R; Kimura, K; Tanaka, K; Kamiya, K; Ito, H; Kato, S; Kamachi, T; Hori, K; Nakanishi, S

BIORESOURCE TECHNOLOGY 241 巻頁： 1157 - 1161 2017年10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：Bioresource Technology

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 査読有り国際誌 Open Access

Yoshimoto, S; Ohara, Y; Nakatani, H; Hori, K

MICROBIAL CELL FACTORIES 16 巻 ( 1 ) 頁： 123 2017年7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：Microbial Cell Factories

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招待有り査読有り

堀　克敏

環境バイオテクノロジー学会誌 17 巻頁： 3-8 2017年3月

Cell surface protein engineering for high-performance whole-cell catalysts 査読有り国際誌

Nakatani, H; Hori, K

FRONTIERS OF CHEMICAL SCIENCE AND ENGINEERING 11 巻 ( 1 ) 頁： 46 - 57 2017年3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：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.[Figure not available: see fulltext.]

DOI： 10.1007/s11705-017-1609-3