J. Sasahara, S. Yoshimoto, Z. Peng, T. Hwang, I. Kobayashi, R. Kawano, K. Hori
Construction and characterization of a nanopore derived from the transmembrane domain of a trimeric autotransporter adhesin
Front. Bioeng. Biotechnol. 14, 1764864 (2026).

• Protein secretion systems in Gram-negative bacteria are essential biological processes responsible for surface protein presentation and environmental interactions. Because their transmembrane architectures can potentially function as nanoscale pores, these systems have recently attracted attention for applications in biosensing and nanobiotechnology. Trimeric autotransporter adhesins (TAAs) are proteins transported to the cell surface via the type Vc secretion system, and their C-terminal transmembrane β-barrel domains serve as secretion conduits. However, the lumen of this β-barrel is normally occupied by the translocating polypeptide chain, and its potential as a molecular permeation structure has remained largely unexplored. In this study, we focused on the transmembrane β-barrel domain of AtaA, a TAA from Acinetobacter, and engineered a novel nanopore termed “AtaApore” by removing the coiled-coil region that occupies the barrel interior, thereby creating an open pore structure. By combining cell-free protein synthesis, reconstitution into lipid bilayers, electrophysiological measurements, and molecular dynamics simulations, we demonstrated that AtaApore forms a stable nanopore in membranes and permits ion conduction. Furthermore, we identified specific arginine residues within the pore lumen that transiently capture anions and modulate ion permeation properties. These results strongly suggest that the TAA transmembrane domain can be repurposed not only as a secretion apparatus but also as a controllable nanostructure for molecular transport. This study represents the first electrophysiological functional characterization of a nanopore derived from the type Vc secretion system. Beyond advancing our understanding of bacterial secretion mechanisms, it provides a new scaffold for nanopore engineering with potential applications in molecular sensing, artificial cells, and synthetic biology. This work was conducted in collaboration with the Kawano Laboratory at Tokyo University of Agriculture and Technology.

H. Nakatani, N. Suetake, K. Hori
Antimicrobial-producing bacteria from fish epidermal mucus alter the fish epidermal bacterial flora and host resistance to infection
Appl. Environ. Microbiol. 91, e01450-25 (2025).

• In this study, numerous bacterial strains were isolated from the skin of zebrafish, among which an antimicrobial compound–producing bacterium was identified. This strain was classified as Pseudomonas mosselii and designated KH-ZF1. Isolation and structural identification of the antimicrobial compound produced by KH-ZF1 revealed it to be Fluviol C. KH-ZF1 was shown to transiently colonize the fish epidermis and to alter the composition of the epidermal microbiota in a manner that enhances resistance to waterborne pathogens. This protective effect was dependent on the timing and method of bacterial administration. In addition to directly inhibiting pathogen growth, Fluviol C contributes to host protection by reshaping the epidermal microbial community. The excessive use of antibiotics in aquaculture has led to the serious emergence of antimicrobial-resistant bacteria, highlighting the urgent need for alternative strategies to prevent infectious diseases in farmed fish. One promising approach is the use of naturally occurring bacteria that colonize fish skin and suppress harmful microorganisms as probiotics. This study is the first to demonstrate that antimicrobial compounds produced by probiotics can modify the epidermal microbiota of a vertebrate host and thereby reduce infection risk. These findings suggest that manipulating the fish skin microbiota using beneficial bacteria or their bioactive compounds represents a viable strategy for disease prevention in aquaculture.

S. Yoshimoto, J. Sasahara, A. Suzuki, J. Kanie, K. Koiwai, A. N. Lupas, K. Hori
Insights into the complex formation of a trimeric autotransporter adhesin with a peptidoglycan-binding periplasmic protein
Cell Surf. 14, 100155 (2025).

• Trimeric autotransporter adhesins (TAAs) of Gram-negative bacteria are important adhesion factors secreted to the cell surface via the type Vc secretion system. AtaA (Acinetobacter TAA), a representative TAA, was previously shown to interact with TpgA, a periplasmic protein that binds peptidoglycan; however, the interaction interface and molecular mechanism remained unclear. In this study, we combined recombinant protein interaction assays, X-ray crystallography, molecular dynamics simulations, and site-directed mutagenesis to demonstrate that the C-terminal transmembrane domain of AtaA assembles with the N-terminal domain of TpgA into an A3B3-type heterohexameric complex. We further identified key residues essential for stabilization of this complex. Comprehensive searches for tpgA-like genes and sequence clustering analysis revealed that a gene cassette in which tpgA is located immediately downstream of taa, together with conserved critical residues, is widely preserved across diverse Pseudomonadota (Proteobacteria), strongly suggesting that the TAA–TpgA complex is evolutionarily conserved across species. These findings indicate that, following surface presentation, TAAs interact with periplasmic proteins and, through them, with peptidoglycan. This study provides important insights into the interplay between the type Vc secretion system and cell envelope architecture, and offers useful implications for the development of anti-adhesion strategies and protein engineering applications. This work was conducted in international collaboration with Professor Andrei N. Lupas at the Max Planck Institute (Germany).

Y. Ohara, S. Yoshimoto, K. Hori
Grid partitioning image analysis of highly aggregative bacterium Acinetobacter sp. Tol 5
Front. Microbiol. 16, 1637462 (2025).

• Bacteria frequently form aggregates and biofilms in diverse environments such as soil, aquatic systems, and infected hosts. Microscopic observation of these structures to analyze their morphology, developmental processes, and species composition is a key approach in microbial ecology. However, quantitative methods to evaluate which bacterial cell types interact with each other have been limited. In this study, we propose a method termed Grid Partitioning Image Analysis (GPIA), in which microscopic images of microbial aggregates are divided into 2-μm square grids, and the proportions of bacterial species within each micro-area are quantified and accumulated to evaluate interspecies interaction frequencies. GPIA enables quantitative assessment of both homo-aggregation (aggregation between the same cell type) and hetero-aggregation (aggregation between two different cell types). This approach allows straightforward determination of which bacterial interactions are relatively strong or weak. Importantly, GPIA can be performed without programming skills, using only the freely available image analysis software ImageJ and Microsoft Excel, thereby lowering the technical barrier to implementation. The findings of this study are expected to contribute to research on bacterial aggregation and biofilm ecology, as well as functional analyses of surface proteins that mediate cell–cell interactions. This work was conducted in collaboration with Friend Microbe Inc.

S. Yoshimoto, M. Hattori, S. Inoue, S. Mori, Y. Ohara and K. Hori
Identification of toluene degradation genes in Acinetobacter sp. Tol 5
J. Biosci. Bioeng. 140, 284-289 (2025).

• Microbial degradation of aromatic compounds is an important process for environmental remediation and biotransformation; however, the toluene metabolic pathway in Acinetobacter sp. Tol 5 had not been elucidated. In this study, we identified a gene cluster in the Tol 5 genome that shows high homology to the tod operon, which is known to be involved in toluene degradation in Pseudomonas putida. Transcriptional analysis revealed that todC1, encoding the large subunit of a putative dioxygenase presumed to function as the initial enzyme, is co-transcribed with the downstream gene todF as part of the same operon, whereas the upstream putative outer membrane transporter gene fadL2 is transcribed independently. Growth assays using gene deletion mutants demonstrated that todC1 is essential for growth on toluene and benzene as carbon sources, while fadL2 is not required. Collectively, these results indicate that the TOD pathway is the principal degradation pathway for toluene and benzene in Tol 5. Combined with the strong adhesion capability of Tol 5, enabling rapid immobilization, this finding is expected to contribute to improved efficiency in environmental remediation and biomanufacturing processes utilizing aromatic compounds as substrates. This work was conducted in collaboration with Friend Microbe inc.

S. Inoue, S. Yoshimoto, K. Hori
A new target of multiple lysine methylation in bacteria
J. Bacteriol. 207, e00325-24 (2025).

• The methylation of protein lysine residues is an important posttranslational modification in eukaryotes. In contrast, this modification has been underexplored in prokaryotes. In this study, we analyzed the cell surface proteins of the toluene-degrading bacterium Acinetobacter sp. Tol 5 using label-free liquid chromatography‒mass spectrometry (LC‒MS) and found extensive lysine methylation in its trimeric autotransporter adhesin (TAA), AtaA. Over 130 lysine residues of AtaA, which consists of 3,630 amino acids and contains 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. The lack of KmtA also led to increased AtaA levels on the cell surface, enhanced bacterial adhesion, and slower growth, suggesting that KmtA is essential for maintaining optimal cellular adhesion. Bioinformatic analysis revealed that genes similar to KmtA are widely distributed across various pathogenic and environmental bacteria. The widespread presence of KmtA-like PKMTs throughout gram-negative bacteria suggests that lysine methylation plays a more extensive role in bacterial physiology than previously recognized.
  This study was selected as an Editor's Pick in Journal of Bacteriology, published by the American Society for Microbiology.

S. Yoshimoto, S. Ishii, A. Kawashiri, T. Matsushita, D. Linke, S. Göttig, VAJ. Kempf, M. Takai and K. Hori
Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5
Front. Bioeng. Biotechnol. 12, 1342418 (2024).

• The bacterium Acinetobacter sp. Tol 5, discovered in the Hori Laboratory, demonstrates high adhesiveness to various material surfaces, from hydrophobic plastics to hydrophilic glass and even metals, via a trimeric autotransporter adhesin (TAA) protein known as AtaA. This paper investigates the adhesive behavior of Tol 5 and other bacteria possessing TAAs on surfaces that are traditionally considered difficult for cell and biomolecule adhesion. Tol 5 was found to adhere to surfaces with low surface free energy such as polytetrafluoroethylene (Teflon), hydrophilic polymer brushes, and even atomically flat mica surfaces. Furthermore, single-cell measurements using atomic force microscopy (AFM) revealed the strong cell adhesion force of Tol 5 to these challenging surfaces. Conversely, it was found that Tol 5 hardly adheres to 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer surfaces. These results contribute to the understanding and control of bacterial adhesion mechanisms mediated by TAAs, including AtaA, which are of interest in biochemical engineering and medical fields. This research is the result of an international collaboration with Professor Madoka Takai of the University of Tokyo, Professor Dirk Linke of the University of Oslo (Norway), Professor Stephan Göttig and Professor Volkhard A. J. Kempf of Goethe University (Germany).

K. Noba, S. Yoshimoto, Y. Tanaka, T. Yokoyama, T. Matsuura, and K. Hori
Simple Method for the Creation of a Bacteria-Sized Unilamellar Liposome with Different Proteins Localized to the Respective Sides of the Membrane
ACS Synth. Biol. 12, (2023) 1437-1446

• In artificial cell creation, liposomes, consisting of a single lipid membrane with a diameter of over 10 μm, have typically been used. However, there have been technical constraints in creating artificial cells that mimic the size and membrane structure of bacterial cells, which are about 1 μm in diameter. In this study, by combining the interface transfer method, a universal technique for liposome production, with the extruder method, we developed a simple method to produce asymmetric liposomes with different proteins localized on the inner and outer layers of the lipid bilayer. This research represents a step towards the creation of artificial bacterial cells and is expected to contribute to the analysis of bacterial membrane structures and the functions of proteins therein. This research is a collaborative effort with the Matsuura Laboratory at Tokyo Institute of Technology and the Tanaka Laboratory at Tohoku University.

S. Yoshimoto, S. Aoki, Y. Ohara, M. Ishikawa, A. Suzuki, D. Linke, A. Lupas, and K. Hori
Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli
Front. Bioeng. Biotechnol. 10, 1095057 (2023)

• In our laboratory, we have developed a simple, powerful, and reversible method for microbial immobilization by growing a unique nanofiber protein, AtaA, on bacterial cells. However, due to AtaA's large size, formed by a poly-peptide chain of 3630 amino acid residues that assemble into a homotrimer, it could only be grown on a limited range of bacteria. In this study, we successfully identified the adhesive sites of AtaA and downsized it to 775 amino acid residues while retaining its function. This enabled us to grow the downsized AtaA in industrially useful bacteria such as E. coli without decreasing their growth rate or other enzyme activities, making it applicable for immobilized microbial reactions. This research is expected to accelerate the creation of environmentally friendly bioprocesses and contribute to achieving carbon neutrality. This study is an international collaboration with Professor Andrei N. Lupas from the Max Planck Institute in Germany and Professor Dirk Linke from the University of Oslo in Norway.

S. Ishii, S. Yoshimoto and K. Hori
Single-cell adhesion force mapping of a highly sticky bacterium in liquid
J Colloid Interface Sci., 606, (2022) 628-634

• Acinetobacter sp. Tol 5, discovered in the Hori lab, has unique properties that allow it to adhere to various material surfaces via the nanofiber protein AtaA. However, because Tol 5 is also self-agglomerating, it has not been possible to evaluate the pure interaction between Tol 5 and the surface of materials using the conventional adhesion test method, which measures the number of bacteria attached to the surface. In this study, the adhesion force between Tol 5 and the material surface was measured at the single-cell level in liquid using an atomic force microscope (AFM), which can measure nanometer-sized irregularities and picoNewton-level forces scanning the sample surface with a fine tip. As a result, it was found that Tol 5 exhibited significantly stronger adhesion than other bacteria. Furthermore, it was found that the low ionic strength environment and the casamino acid solution, which were known to reduce the adhesion of Tol 5, acted through different mechanisms. As a result of this study, a better understanding of AtaA-mediated adhesion of Tol 5 is expected to develop AtaA-based bacterial immobilization technology further.

M. Ishikawa and K. Hori
Complete Genome Sequence of the Highly Adhesive Bacterium Acinetobacter sp. Strain Tol 5
Microbiol Resour Announc, 10, (2021) e00567-21

• We have sequenced the complete genome of the highly adhesive bacterium Acinetobacter sp. Tol 5, which was discovered in the Hori lab. Because the genome of Tol 5 contains an extensive repeat sequence, next-generation sequencers (NGS), which can only analyze short reads, have not been able to determine the full-length genome. In this study, we determined the full-length genome with high accuracy by using the MinION nanopore sequencer, which can analyze long reads, and the iSeq 100, the top model NGS in the lab. In the past, outsourced analysis was done genome sequencing, but the Hori laboratory, equipped with MinION and iSeq 100, can determine the genome within a few days. The defined genome sequence is expected to advance the research on Tol 5 further.

Y.-Y. Chen, Y. Soma, M. Ishikawa, M. Takahashi, Y. Izumi, T. Bamba and K. Hori
Metabolic alteration of Methylococcus capsulatus str. Bath during a microbial gas-phase reaction
Biores. Technol., 330, (2021) 125002

• The gas-phase microbial reaction is an innovative bioprocess developed in the Hori laboratory. By removing the liquid phase from the reactor, the diffusion rate of gaseous molecules can be increased, dramatically improving the performance of the microbial reaction. Under such reaction conditions, microorganisms are expected to have different metabolic kinetics from conventional microbial reaction systems (i.e., those with a liquid phase). In this study, we used metabolomic analysis of Methylococcus capsulatus (Bath), a representative methanogen, to clarify the changes in metabolic dynamics during gas-phase microbial reactions. We found that the metabolic state of the gas-phase reaction was significantly different from that of the liquid-phase reaction, suggesting that the metabolic state was more suitable for material production. The results of this study provide hints for further improving the efficiency of gas-phase microbial reactions and the production of new substances. This research was conducted in collaboration with Bamba Laboratory, Kyushu University.

M. Ishikawa, T. Kojima, and K. Hori
Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection
Microbiol Spectr., 9, (2021) e00259-21

• Genetically engineered microorganisms (GEMs) are strictly restricted from leaking into the environment due to the risk of disturbing the ecosystem. Therefore, although many valuable GEMs have been developed through genetic modification technology, we have not used GEMs for environmental cleanup. Biological containment" is a technology that makes GEMs live only in a specific environment so that they do not affect the ecosystem. We thought that if we could create GEMs that degrade pollutants but can only live in the environment where the contaminants are present, we could use GEMs for environmental conservation (GEMs die after degrading pollutants). We developed a GEM that would not increase after degrading toluene in this research. The GEM we set significantly limited the growth of toluene after it was contaminated, but we could not suppress it entirely. To find the cause of this, we conducted a genetic analysis using the nanopore sequencer MinION and discovered and proposed improvements. The results obtained are essential for developing biological containment technology for environmental protection.