Faculty Profiles - HORI Katsutoshi

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

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

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

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

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

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 Open Access

堀　克敏

生物工学会誌 Vol. 第102巻 ( 第2号 ) page： 54 - 60 2024.2

Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5 Reviewed International coauthorship International journal

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