Cleavage cascade of the sigma regulator FecR orchestrates TonB-dependent signal transduction

TonB-dependent signal transduction is a versatile mechanism observed in gram-negative bacteria, integrating energy-dependent substrate transport with signal relay. In Escherichia coli, the TonB-ExbBD motor complex energizes the TonB-dependent transporter FecA, facilitating ferric citrate import. FecA also functions as a sensor, transmitting signals to the cytoplasmic membrane protein FecR. We previously demonstrated that FecR undergoes a three-step cleavage process, culminating in the activation of the cytoplasmic sigma factor FecI, which drives fec gene transcription. Here, we describe the complete mechanism of FecR cleavage-mediated ferric citrate signaling involving FecA and TonB. The cleavage cascade begins with FecR autoproteolysis prior to membrane integration. The soluble C-terminal domain (CTD) fragment of FecR is co-translocated with the N-terminal domain (NTD) fragment through a Tat system-mediated process. In the periplasm, the interaction between the CTD and NTD fragments prevents further cleavage. This inhibition is lifted by TonB-mediated motor function, which releases the CTD, allowing the cleavage cascade to proceed. This process is essential for ferric citrate signal-induced activation of fec gene expression. Our findings reveal that the regulation of FecR cleavage, relying on the TonB-FecA axis, plays a central role in bacterial response to ferric citrate signals.journal articl

Construction of a Cyclic Regular-Triangle Trimer of Cytochrome c555 with a Central Hole Using Sortase A

Protein-based supramolecules require precise arrangement of building blocks. A regular-triangle trimer (cp-c555)3 has been constructed using an α-helix-inserted-circular permutant (cp-c555) of Aquifex aeolicus cytochrome (cyt) c555, where the trimers may dissociate to monomers. In this study, we stabilized the regular-triangle structure by constructing a cyclic regular-triangle of three α-helix-linked cyt c555 molecules using sortase-mediated ligation (SML). Comparing SML using sortase A for six cp-c555 variant trimers, the variant with GGG at the N-terminus and LPETG at the C-terminus reacted most efficiently. OP-(c555)3 was designed, in which two cyt c555 molecules were fused using an α-helix, generating a dimer. The cyt c555 C-terminal region was attached to the N-terminus of the dimer, and the cyt c555 N-terminal region was attached to the C-terminus of the dimer using the same α-helix. OP-(c555)3 was expressed in Escherichia coli cells, and the termini were connected by SML, forming a cyclic regular-triangle, CL-(c555)3. CL-(c555)3 showed higher thermostability than (cp-c555)3 and OP-(c555)3. CL-(c555)3 structural stability was confirmed using high-speed atomic force microscopy. The X-ray crystal structure of CL-(c555)3 showed a cyclic structure and a nanoporous supramolecular assembly. These results demonstrate that a nanoporous supramolecular assembly can be constructed by designing a cyclic molecule with a central hole using SML.journal articl

CGLA ニ ヨル ダイキボ ゲンゴ モデル ノ ジッソウ ト ヒョウカ

奈良先端科学技術大学院大学修士（工学）master thesi

コウソク セルソーター ノ タメ ノ フェムトビョウレーザー マルチ パルス ショウシャ ギジュツ ノ カイハツ

奈良先端科学技術大学院大学修士（工学）master thesi

Biocontrol of Bacterial Wilt Disease Using Plant-Associated Bacterial Communities in Tomato

Host-protective or disease-suppressive microorganisms are emerging as sustainable solutions for controlling crop diseases, such as bacterial wilt. However, the efficacy of biocontrol strategies is often constrained by limited resilience under varying environmental conditions and interactions with native microbial communities in the field. One major challenge is that introduced biocontrol microbes often face suppression by indigenous microbes due to competitive interactions. Synthetic communities (SynComs) offer a promising alternative strategy. However, conventional approaches to assembling SynComs by combining different microbial isolates often result in antagonism and competition among strains, leading to ineffective and inconsistent outcomes. In this study, we assembled a bacterial wilt-suppressive SynCom for tomato, composed of bacterial isolates derived from co-cultured microbial complexes associated with healthy plants. This SynCom demonstrates significant disease-suppressive effects against Ralstonia pseudosolanacearum in tomato seedlings under both axenic and soil conditions. Additionally, our findings suggest the presence of an optimal SynCom colonization level in plants, which is crucial for effective disease suppression. The SynCom also exhibits direct antibiotic activity and modulates the plant-associated microbiome. Our results provide an effective approach to constructing SynComs with consistent and effective disease-suppressive properties within microbial community contexts.journal articl

Co-option and neofunctionalization of stomatal executors for defence against herbivores in Brassicales

Co-option of gene regulatory networks leads to the acquisition of new cell types and tissues. Stomata, valves formed by guard cells (GCs), are present in most land plants and regulate CO2 exchange. The transcription factor (TF) FAMA globally regulates GC differentiation. In the Brassicales, FAMA also promotes the development of idioblast myrosin cells (MCs), another type of specialized cell along the vasculature essential for Brassicales-specific chemical defences. Here we show that in Arabidopsis thaliana, FAMA directly induces the TF gene WASABI MAKER (WSB), which triggers MC differentiation. WSB and STOMATAL CARPENTER 1 (SCAP1, a stomatal lineage-specific direct FAMA target), synergistically promote GC differentiation. wsb mutants lacked MCs and the wsb scap1 double mutant lacked normal GCs. Evolutionary analyses revealed that WSB is conserved across stomatous angiosperms. We propose that the conserved and reduced transcriptional FAMA–WSB module was co-opted before evolving to induce MC differentiation.journal articl

Evaluating and Enhancing Japanese Large Language Models for Genetic Counseling Support: Comparative Study of Domain Adaptation and the Development of an Expert-Evaluated Dataset

Background: Advances in genetics have underscored a strong association between genetic factors and health outcomes, leading to an increased demand for genetic counseling services. However, a shortage of qualified genetic counselors poses a significant challenge. Large language models (LLMs) have emerged as a potential solution for augmenting support in genetic counseling tasks. Despite the potential, Japanese genetic counseling LLMs (JGCLLMs) are underexplored. To advance a JGCLLM-based dialogue system for genetic counseling, effective domain adaptation methods require investigation. Objective: This study aims to evaluate the current capabilities and identify challenges in developing a JGCLLM-based dialogue system for genetic counseling. The primary focus is to assess the effectiveness of prompt engineering, retrieval-augmented generation (RAG), and instruction tuning within the context of genetic counseling. Furthermore, we will establish an experts-evaluated dataset of responses generated by LLMs adapted to Japanese genetic counseling for the future development of JGCLLMs. Methods: Two primary datasets were used in this study: (1) a question-answer (QA) dataset for LLM adaptation and (2) a genetic counseling question dataset for evaluation. The QA dataset included 899 QA pairs covering medical and genetic counseling topics, while the evaluation dataset contained 120 curated questions across 6 genetic counseling categories. Three enhancement techniques of LLMs---instruction tuning, RAG, and prompt engineering---were applied to a lightweight Japanese LLM to enhance its ability for genetic counseling. The performance of the adapted LLM was evaluated on the 120-question dataset by 2 certified genetic counselors and 1 ophthalmologist (SK, YU, and AY). Evaluation focused on four metrics: (1) inappropriateness of information, (2) sufficiency of information, (3) severity of harm, and (4) alignment with medical consensus. Results: The evaluation by certified genetic counselors and an ophthalmologist revealed varied outcomes across different methods. RAG showed potential, particularly in enhancing critical aspects of genetic counseling. In contrast, instruction tuning and prompt engineering produced less favorable outcomes. This evaluation process facilitated the creation an expert-evaluated dataset of responses generated by LLMs adapted with different combinations of these methods. Error analysis identified key ethical concerns, including inappropriate promotion of prenatal testing, criticism of relatives, and inaccurate probability statements. Conclusions: RAG demonstrated notable improvements across all evaluation metrics, suggesting potential for further enhancement through the expansion of RAG data. The expert-evaluated dataset developed in this study provides valuable insights for future optimization efforts. However, the ethical issues observed in JGCLLM responses underscore the critical need for ongoing refinement and thorough ethical evaluation before these systems can be implemented in health care settings.journal articl

Deep Learning Based Beamforming and Metasurface Design for Reconfigurable Intelligent Surface

奈良先端科学技術大学院大学博士（工学）doctoral thesi

IC ノ ニュウシュツリョク カイロ カラノ バックスキャッタ オ モチイタ リサイクル IC ケンシュツ ノ コウソクカ ニ カンスル ケンキュウ

奈良先端科学技術大学院大学修士（工学）master thesi

Human-AI collaboration ニ オケル タイジン スキル ノ エイキョウ チョウサ

奈良先端科学技術大学院大学修士（工学）master thesi