Biology subjects

Suzuki, S.

Publications and source records attributed to Suzuki, S..

At least 19 recordsLinked to original sources

bioRxiv2026

De novo design of mini-proteins as specific binders to PACAP-38, a key mediator of migraine

Pituitary adenylate cyclase-activating peptide 38 (PACAP-38) is a vasoactive peptide that is attracting attention as a potential target for next-generation migraine treatments. Antibodies against PACAP-38 have recently shown promising results as a ligand trap medicine in clinical trials. In this study, we investigated the de novo design of miniproteins to evaluate the potential for developing a medium-molecular-weight drug that binds to PACAP-38. Using Bindcraft, we designed binders against PACAP-38 and identified ICLIST-2, a 63-residue peptide with affinity for PACAP-38 (KD = 26.5 nM). Furthermore, by alanine scanning and structural analysis of ICLIST-2, we determined residues that were suitable for further optimization using ProteinMPNN. This resulted in the design of ICLIST-11, which incorporates nine amino acid substitutions relative to ICLIST-2, resulting in a KD of 4.51 nM. Interaction analysis based on a structural model suggested that three mutations in ICLIST-11 may be important for enhancing affinity. Moreover, ICLIST-11 exhibits a Tm value that is 14 {degrees}C higher, and MD simulations suggest that the substitution of three residues with hydrophobic residues (E30F, M31L, and R54W) is one reason for this. In cell-based functional assays, ICLIST-11 demonstrated potent, concentration-dependent neutralizing activity against PACAP-38-induced PAC1R activation (IC50 = 10.6 nM), significantly outperforming the parental ICLIST-2. Taken together, these results provide a framework for protein drug discovery targeting PACAP-38.

bioengineering↗

bioRxiv2026

Screening of Stereochemically Defined 2,5-Diketopiperazines Identifies Autophagy Inducers without mTORC1 Suppression

Modulating autophagy has emerged as a potential strategy for treating age-related diseases. However, commonly used pharmacological approaches to induce autophagy, particularly inhibition of mechanistic target of rapamycin complex 1 (mTORC1), can be associated with adverse effects, including immunosuppression and insulin resistance. This has prompted interest in autophagy modulators that act without directly inhibiting mTORC1. 2,5-Diketopiperazines (DKPs) are bioactive cyclic dipeptide scaffolds with diverse biological activities. However, systematic evaluation of their structure-activity relationships has been hindered by racemization during conventional chemical synthesis, leaving the contribution of stereochemistry to autophagy regulation poorly understood. Here, we used a stereoselective one-pot chemoenzymatic synthesis based on the adenylation domain of tyrocidine synthetase A to generate a DKP library with defined stereochemistry. Phenotypic screening in Caco-2 cells stably expressing the GFP-LC3-RFP autophagic flux probe identified four DKPs that increased autophagic flux: c(DW-DP), c(DW-LP), c(DF-DP), and c(DM-LP). Structure-activity analysis revealed stereochemistry-dependent effects associated with amino acid side-chain properties: D-configured residues were favored among DKPs containing aromatic amino acids or methionine, whereas L-configured residues were favored among those containing branched-chain amino acids. Substitution of the proline residue further altered activity, with glycine substitution tending to increase autophagic flux in some DKP scaffolds. Importantly, the active DKPs did not detectably reduce the phosphorylation of the mTORC1 downstream targets p70 S6K and 4EBP1, indicating that their autophagy-inducing effects do not require detectable suppression of canonical mTORC1 signaling. These findings establish stereochemically defined DKPs as candidate scaffolds for the development of autophagy inducers that act through mechanisms distinct from direct mTORC1 inhibition.

biochemistry↗

bioRxiv2026

Conjugation dynamics and persistence of a carbapenem resistance gene blaOXA-72 from Acinetobacter pittii to Acinetobacter baumannii

Carbapenem-resistant Acinetobacter (CRA) has been associated with increased morbidity and mortality in clinical settings. In this study, we explored the transfer potential of a mobilizable plasmid-harboring blaOXA-72 gene between Acinetobacter species originating from patient, municipal wastewater, and pig farm wastewater. PCR-based evidence suggested putative transfer of blaOXA-72 from Acinetobacter pittii to Acinetobacter baumannii. In this pair, the apparent frequency of PCR-marker-positive putative transconjugants varied depending on temperature and meropenem supplementation, with higher number observed at 27{degrees}C compared to 17{degrees}C and 37{degrees}C. Likewise, the presence of antibiotic pressure yields to higher apparent conjugation frequency, however this observation was limited to a singled donor-recipient pair. Further, we revealed a phenotypic conversion in terms of meropenem susceptibility and a fitness cost in the putative transconjugants. While whole genome sequencing did not conclusively verify the presence of blaOXA-72 or fully resolved plasmid configuration, Oxford Nanopore read mapping consistently detected the chromosomal strA gene in all isolates. In contrast, only a limited number of reads aligned with blaOXA-72 gene, traC, or the complete plasmid sequences. Comparative analyses further revealed variations in the surface-associated factors and defense systems composition of the recipient strains, which could be considered as barriers in conjugation. Lastly, the persistence of PCR-detectable marker genes in putative transconjugants was variable and generally unstable over a 30-day period. Overall, these findings provide preliminary insights into the factors that may influence horizontal gene transfer and short-term maintenance of blaOXA-72. IMPORTANCECarbapenem-resistant Acinetobacter (CRA) is a serious clinical concern linked to elevated morbidity and mortality. This study shed light on how the carbapenemase gene blaOXA_72 spreads in the environment via horizontal gene transfer, Using isolates of Acinetobacter pittii and Acinetobacter baumannii from municipal wastewater treatment plant, we show that conjugation efficiency depends on temperature and antibiotic exposure. The process also incurs fitness costs, and the transferred gene persists only briefly in recipient cells. These results emphasize the importance of environmental sources as reservoirs for antimicrobial resistance gene exchange. Additionally, variations in surface structures and defense systems may act as potential barriers to conjugation. Overall, the work improves our understanding of AMR movement and supports the need for better integrated surveillance under a One Health approach.

microbiology↗

bioRxiv2026

EIF3H Sustains Translational Programs Essential for Proliferation in Human Primed Pluripotency

Pluripotent stem cells (PSCs) have remarkable capacity for unlimited self-renewal and differentiation into all somatic lineages. Although translational regulation has been implicated in the maintenance of PSC identity, the specific mechanisms involved remain poorly understood. Here, we identified EIF3H, a conserved subunit of the eIF3 translation initiation complex, as an essential regulator of human primed PSC proliferation and differentiation. CRISPR interference-mediated knockdown of EIF3H markedly reduced colony size, impaired proliferation, and diminished differentiation potential in all three germ layers. Integrated transcriptomic and translatomic profiling revealed that EIF3H loss decreased the translation of metal ion-related genes. Notably, the targeted suppression of metallothionein genes encoding metal-binding proteins recapitulated the proliferative defects observed in EIF3H-deficient PSCs, demonstrating a functional requirement for EIF3H-mediated translation of this gene family. Taken together, these findings establish EIF3H as a critical translational regulator that sustains PSC self-renewal and differentiation by maintaining the expression of key metabolic and stress-response genes, providing new insights into the molecular basis of pluripotency.

cell biology↗

bioRxiv2026

ConforFlux: Particle-Guided Trunk Repulsion for Diverse Protein Conformations

Deep-learning protein structure predictors achieve near-experimental accuracy on individual folds, yet their default inference samples concentrate around a single dominant conformation. We introduce ConforFlux, an inference-time procedure for Boltz-2 that couples multiple structure-prediction trajectories through a pair-wise C-RMSD repulsion gradient on the trunks single and pair embeddings. Because the trunk conditions every block of the diffusion module, this update propagates to every subsequent denoising step. On four conformational-change categories, ConforFlux improves the per-state success rate over default Boltz-2 by 3-17 percentage points. On twelve transporter pairs with at least one alternate-state reference released after the Boltz-2 cutoff, ConforFlux raises the 2[A] success rate from 33% to 75%. Under an extended bandwidth sweep, ConforFlux samples reach the inward, occluded, and outward states of the human dopamine transporter alternating-access cycle, while default samples cluster between them.

molecular biology↗

bioRxiv2026

Comparative benchmarking of CRISPRi and CasRx in standardized pluripotent stem cell platforms reveals context-dependent knockdown performance

Advances in transcriptome profiling have revealed transcriptomic differences across different cellular states. However, functional interpretation requires precise perturbation tools and experimental frameworks. This study benchmarked two widely used modalities: CRISPR interference (CRISPRi) and Cas13d/CasRx. A standardized workflow was established to generate human pluripotent stem cells (PSCs) with inducible ZIM3-dCas9 or CasRx expression. The cell lines were subjected to flow cytometry, copy number, and immunocytochemical analyses. The knockdown performance was validated via robust OCT4 suppression and the expected downstream effects on pluripotency genes. Time-course measurements indicated that CRISPRi produced faster and stronger repression but slower recovery after inducer withdrawal. In contrast, CasRx yielded slower and typically weaker knockdown with rapid reversibility. Furthermore, a key limitation of CRISPRi was demonstrated using the ATF5-NUP62 locus, wherein CRISPRi could co-repress genes with overlapping promoter regions. In contrast, CasRx avoids these limitations and supports isoform-resolved targeting of circular and alternatively spliced transcripts, albeit with variable efficiency. These results provide practical guidance for selecting complementary knockdown tools to improve the interpretability of transcriptomic function studies. MOTIVATIONAdvances in transcriptome profiling have enabled the detection of subtle cell type-specific differences. However, mechanistic interpretation still depends on perturbation tools that can modulate transcripts with high precision and efficiency. Recent CRISPR-based modalities, CRISPRi and Cas13/CasRx, function as robust and orthogonal methods to achieve the knockdown of specific gene targets. However, a standardized approach for cell line preparation and comparative studies on their relative performances and limitations remains unclear. Consequently, this study presents a standardized workflow for generating cell lines that support high-efficiency knockdown using CRISPRi and CasRx. Moreover, it compares the trade-offs in potency, reversibility, and isoform resolution, along with a practical overview of method-specific pitfalls to guide tool selection and data interpretation in future studies. HIGHLIGHTSO_LIDoxycycline-inducible AAVS1 knock-in human PSC platforms for CRISPRi (ZIM3-dCas9) and CasRx (RfxCas13d) were generated to enable standardized RNA perturbation experiments. C_LIO_LIThe prepared cell lines demonstrated strong OCT4 knockdown, with expected downstream effects on the expression of another pluripotency gene, NANOG. C_LIO_LIA comparison of knockdown characteristics and their reversibility revealed rapid and sustained repression with CRISPRi, whereas slow but rapid recovery was observed with CasRx. C_LIO_LIA CRISPRi-specific off-target effect arising from TSS proximity/overlap (ATF5-NUP62) was identified, whereas CasRx achieved ATF5 knockdown without collateral repression of the neighboring NUP62 gene. C_LIO_LICasRx enables isoform-resolved knockdown of structural isoforms (circHIPK3 vs. linear HIPK3 mRNA) and splice isoforms (RAB6A-iso1 vs. RAB6A-iso2). C_LI

cell biology↗

bioRxiv2026

Structural basis for site-specific histone H3 acetylation-dependent regulation of RNAPII transcription through nucleosomes

Acetylation of histone H3 regulates chromatin dynamics and transcription, but how specific acetylation sites affect RNA polymerase II (RNAPII) transcription through nucleosomes remains unclear. Here, we found that H3 acetylation at Lys56 and Lys122 markedly enhances RNAPII transcription through nucleosomes, whereas acetylation at Lys64 has little effect. To elucidate the structural basis for these functional differences, we determined cryo-electron microscopy (cryo-EM) structures of nucleosomes bearing site-specific acetylation at H3K56, H3K64, or H3K122. The cryo-EM structures revealed that H3K56ac and H3K122ac locally weaken histone-DNA interactions at the DNA entry/exit region and near the dyad, respectively, while H3K64ac induces no detectable structural changes. These structural differences correlate with the observed transcriptional outcomes, indicating that acetylation at H3K56 and H3K122, but not H3K64, alleviates the nucleosomal barrier to RNAPII progression. Our findings provide direct structural evidence that specific acetylations within the histone fold domain of H3 finetune nucleosome dynamics to facilitate RNAPII transcription.

molecular biology↗

bioRxiv2026

A densely sampled fMRI dataset for investigating food valuation

Subjective valuation of food rewards guides our dietary choices and is fundamental to human health and well-being. Extensive literature in human functional magnetic resonance imaging (fMRI) studies has consistently shown that a network of reward-processing brain regions, including the ventromedial prefrontal cortex (vmPFC) and ventral striatum, encodes the subjective values of food rewards. However, the representational geometry of value signals and the mechanisms by which they are constructed in the brain remain poorly understood. This is partly because most fMRI studies on food valuation rely on small stimulus sets, yielding datasets too shallow for advanced analyses such as multi-voxel pattern analysis, and deep neural network modeling. Here, we present a densely sampled fMRI dataset wherein 31 participants provided subjective value ratings for over 500 food images across three separate days. We validate the dataset by replicating the well-established findings regarding the neural encoding of subjective value in the vmPFC and ventral striatum. We anticipate this resource will facilitate diverse studies on neural food valuation using advanced analytical methods.

neuroscience↗

bioRxiv2026

Steering Conformational Sampling in Boltz-2 via Pair Representation Scaling

Deep learning has transformed protein structure prediction, yet most systems return a single dominant conformation with little control over alternative functional states. We introduce pair representation scaling, an inference-time method that biases conformational sampling in diffusion-based structure predictors by multiplying the latent pair representation by a single scalar before the Pairformer trunk, without retraining, an auxiliary model, or a second forward pass. On 86 two-state targets spanning domain motions and membrane transporters, scaling broadens the conformational ensembles of both AlphaFold 3 and Boltz-2 and recovers alternative states that default inference misses, most strongly in AlphaFold 3, where the gains extend even to targets deposited after the training cutoff. It approaches the alternative-state recovery of alignment-based sampling methods, and the benefit persists even without a multiple sequence alignment. The predicted distance distributions show that scaling shifts the encoded two-state distribution toward the experimentally observed alternative state, a directed modulation rather than arbitrary perturbation. Pair representation scaling is an interpretable, low-cost handle on the conformational ensembles of deep learning structure predictors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/701250v3_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@5e52dorg.highwire.dtl.DTLVardef@1090910org.highwire.dtl.DTLVardef@3218beorg.highwire.dtl.DTLVardef@f67068_HPS_FORMAT_FIGEXP M_FIG C_FIG Pair representation scalingA query sequence and its multiple sequence alignment enter the Pairformer trunk, where the latent pair representation z is rescaled by a single global scalar to (1 + {beta}) z and the diffusion module then generates a structure. The alignment and the trained weights are unchanged, and the same operation applies in AlphaFold 3 and Boltz-2. The scalar {beta} is a single explicit handle: sweeping it broadens the sampled ensemble toward alternative states, and its effect can be read out directly inside the network.

bioinformatics↗

bioRxiv2025

The evolution lifecycle of ribosome hibernation factors

Bacteria defend against hostile environments through a variety of molecular mechanisms, including ribosome hibernation. Previously, bacteria were shown to initiate ribosome hibernation by activating protective proteins known as hibernation factors. It was demonstrated that hibernation factors prevent ribosome degradation by nucleases, which allows bacteria to safely store their inactive ribosomes and survive under starvation or persistent stress. Because homologs of hibernation factors were found in diverse lineages of bacteria, it is currently assumed that the mechanism of ribosome hibernation is highly conserved across species. Here, we assess 46,015 complete bacterial genomes to reveal the principles underlying the origin and evolution of these essential proteins in bacterial cells. We find that hibernation factors emerged in ancient bacteria as relatively large proteins that then gradually reduced in size and have undergone complete extinction in over 10% of studied bacteria. We then demonstrate that the degeneration of ancient hibernation factors is often accompanied by "borrowing" hibernation factors from other species via gene transfers, de novo gene birth or fusion of truncated hibernation factors with fragments from other stress-response proteins. These findings reveal a unique evolutionary pathway in which bacteria respond to the reductive evolution of hibernation machinery by inventing novel hibernation mechanisms, thus restoring their capacity to survive starvation and stress. This model implies that most ribosome hibernation factors are yet to be discovered and predicts the organisms that rely on currently unknown hibernation mechanisms.

microbiology↗

bioRxiv2025

Generation of Region-specific Airway Basal Stem Cells from Human Pluripotent Stem Cells via Regulation of BMP-NOGGIN Axis

Basal cells (BCs) are the primary stem cell population of adult human airways and a key target for hPSC-based models of airway development, disease and regenerative medicine. Recent studies have revealed substantial regional differences between human proximal and distal airway cell types, including BCs. Here, we show that the NOGGIN-BMP signaling axis governs proximal-distal patterning of hPSC-derived lung progenitors leading to the generation of region-specific induced BCs (iBCs). Continuous BMP inhibition through NOGGIN, coupled with tapered WNT activation, generates proximal iBCs that molecularly and functionally resemble human proximal airway BCs and differentiate in vitro and in vivo into the full repertoire of specialized proximal airway cell types, including ionocytes and pulmonary neuroendocrine cells. In sharp contrast, BMP activation with tapered WNT generates distal airway-like cells and distal BCs with limited ionocyte differentiation potential. Progeny of proximal iBCs derived from G551D CFTR mutant hiPSCs also recapitulate a cystic fibrosis-associated ionocyte phenotype.

cell biology↗

bioRxiv2025

Clonal Dynamics in the Male Germline from Development to Next Generation

Germ cells secure the continuity and evolutionary potential of species. Yet how reproductive success is allocated among the founder cells of the mammalian male germline, the primordial germ cells (PGCs), remains unknown. Here, we quantitatively traced individual PGCs, non-invasively barcoded in mouse embryos, across their establishment, lifelong maintenance within the testis, spermatogenesis, and transmission to the next generation. We found that highly skewed clonal contributions arise from a very early stochastic bottleneck during migration to the developing testes and remain stable throughout adult reproductive life. Reproductive success during adulthood was proportional to the embryonically established PGC clone size. Mathematical modeling shows that the patchy compartmentalization of PGC clones within the extended thin seminiferous tubules maintain clonal diversity and safeguard against the expansion of clones that gain a selective advantage. These findings uncover fundamental principles governing the development, stability, and evolutionary transmission of the mammalian germline.

developmental biology↗

bioRxiv2025

Construction of a novel 3D urinary bladder mucosa model and its application in toxicity assessment of arsenicals

The urinary bladder is a major target organ for environmental toxicants, including arsenic. The objects of this study were two-fold. First, we constructed a novel 3D urinary bladder mucosa model that incorporated an overlying epithelium and a supporting subepithelial layer, referred to as the 3D-UBMM. Primary human bladder urothelial and fibroblast cells were immortalized by introducing the human CDK4R24C and TERT genes. The subsequent construction of the 3D-UBMM involved incorporating the immortalized fibroblast cells into a collagen raft, while the immortalized urothelial cells were cultured at the air-liquid interface of the raft. This 3D-UBMM closely resembles the human urinary bladder epithelium in terms of morphology and marker protein expression, including uroplakin 1b, P63, and cytokeratin 5. Second, using the 3D-UBMM we investigated the cytotoxicity of sodium arsenite (iAsIII) and dimethylarsenic acid (DMAV). Exposure to iAsIII and DMAV resulted in increased urothelial cell necrosis and increased {gamma}-H2AX-positive cells along with a reduction of P63-positive cells, and each of these effects was induced in a dose-response manner. These findings affirm that this novel 3D-UBMM closely resembles the human urinary bladder epithelial layer, offering a practical in vitro model for the evaluation of the toxicity of arsenic and other bladder carcinogens and the role of cancer-related genes in bladder carcinogenicity. In addition, by identifying mechanisms of carcinogenesis this model will aid in hazard identification and risk assessment of potential bladder carcinogens.

pharmacology and toxicology↗

bioRxiv2025

Developing a Novel In Vitro Toxicity Assay for Predicting Inhalation Toxicity in Rats

The development of alternative in vitro methods for assessing acute inhalation toxicity is a critical step toward reducing animal testing and aligns with the principles of the 3Rs (replacement, reduction, and refinement). In this study, we developed and optimized a neutral red uptake (NRU) assay using human lung adenocarcinoma cells (A549) as a predictive model (A549-NRU) for acute inhalation toxicity. To improve assay efficiency and robustness, we introduced two key modifications: the incubation time was reduced to 15 minutes to enable rapid and high-throughput screening, and for chemicals reactive with polystyrene 6-well glass plates were used to prevent chemical-induced degradation and ensure assay consistency. LC50 values were determined for 49 chemicals and compared with reported LC50 values from 4-hour rat inhalation studies. A significant positive correlation was observed between A549-NRU-derived LC50 values and in vivo LC50 values for water-soluble compounds and chemicals containing aldehyde, ketone, alcohol, ether, and epoxide functional groups, suggesting that in vivo LC50 values may be predictable using the A549-NRU assay. Additionally, A549-NRU LC50 values showed significant negative correlations with molecular weight and octanol-water partition coefficients, indicating that chemicals with higher values tended to be less cytotoxic in vitro. Importantly, the A549-NRU assay demonstrated stronger correlation with in vivo LC50 values than the conventional NRU assay using mouse 3T3 fibroblast cells. These findings support the use of the A549-NRU assay to estimate starting doses for in vivo studies, and potentially as an in vitro alternative for predicting acute inhalation toxicity. Impact statementThe optimized A549-NRU assay demonstrates predictive potential for inhalation toxicity while reducing reliance on animal testing. This model serves as a human-relevant alternative for estimating starting doses for in vivo inhalation toxicity studies.

pharmacology and toxicology↗

bioRxiv2025

When word order matters: human brains represent sentence meaning differently from large language models

Large language models based on the transformer architecture are now capable of producing human-like language. But do they encode and process linguistic meaning in a human-like way? Here, we address this question by analysing 7T fMRI data from 30 participants reading 108 sentences each. These sentences are carefully designed to disentangle sentence structure from word meaning, thereby testing whether transformer representations of sentence meaning resemble those formed by the brain. We found that while transformer models match brain representations better than models that completely ignore word order, all transformer models performed poorly overall. Further, transformers were significantly inferior to models explicitly designed to encode the structural relations between words. Our results provide insight into the nature of sentence representation in the brain, highlighting the critical role of sentence structure. They also cast doubt on the claim that transformers represent sentence meaning similarly to the human brain.

neuroscience↗

bioRxiv2025

Neural mechanisms underlying reward processing and social cognition: a replication study with a Japanese sample

Neural functions underlying reward processing and social cognition play a critical role in everyday decision-making. Given that these processes may be shaped by cultural factors, it is essential to examine their cross-cultural generalizability. In this study, we used functional MRI to scan native Japanese speakers as they performed two well-established experimental paradigms: the Monetary Incentive Delay (MID) task for reward processing and the Theory of Mind (ToM) task for social cognition. We successfully replicated previous findings. Specifically, in the MID task, reward expectation and reward outcome were associated with neural activity in the ventral striatum and ventromedial prefrontal cortex. In the ToM task, social cognition was linked to activation in the temporoparietal junction. Notably, the posterior cingulate cortex was engaged in both tasks, suggesting its integrative role across cognitive domains. Together, these results replicate and extend earlier work, supporting the cross-cultural generalizability of the neural mechanisms underlying reward and social cognition, and further validating our fMRI protocol for future research.

neuroscience↗

bioRxiv2025

A Novel Insertion Site for Group I Introns in tRNA Genes of Patescibacteria

Patescibacteria is a bacterial phylum with small genomes, frequent loss of essential genes, and the presence of introns. While many aspects of Patescibacteria remain enigmatic, an intriguing feature is the widespread occurrence of introns within their compact genomes. To better understand the diversity, roles, and evolution of bacterial introns, we focused on tRNA introns and analyzed Patescibacteria complete genome. Notably, 20% of these genomes lacked at least one tRNA gene for a canonical amino acid, primarily tRNAAsn and tRNAAsp, whereas other tRNA genes were readily detected. This observation led us to conduct further analyses, resulting in the discovery of a novel group I intron insertion site at position 35/36 within the anticodon loop that likely prevented detection by conventional annotation tools. Splicing assays demonstrated that these bacterial introns are catalytically active and capable of self-splicing. To assess the broader distribution of this insertion site across bacteria, we analyzed 4,934 bacterial genomes and identified 269 group I introns within tRNA genes across 14 phyla. Nearly 70% of introns at position 35/36 originate from Patescibacteria, indicating that this feature is largely confined to the phylum. Subgroup classification showed that 79% of all tRNA introns belonged to the IC subgroup, whereas almost all Patescibacteria introns were assigned to IA, suggesting a distinct evolutionary origin. As most tRNA introns lacked homing endonuclease genes, horizontal transfer appears limited. Collectively, these findings advance our understanding of the phylogenetic distribution and evolutionary history of bacterial group I introns in tRNAs, with particular emphasis on Patescibacteria. IMPORTANCEGroup I introns in bacterial tRNA genes were previously known only in a limited number of phyla. Our study expands this knowledge by identifying a novel insertion position in tRNA genes of phylum Patescibacteria and mapping their phylogenetic distribution across bacterial lineages. Our result revealed that group I introns inserted in tRNA genes differed in subgroups between Patescibacteria and other bacteria, highlighting the evolutionary uniqueness of introns of Patescibacteria. Additionally, we found that group I introns are maintained in 43% of bacterial phyla, with tRNA insertions being the most common. Our findings highlight that even in complete genomes, the presence of group I introns can hinder the detection of all 20 canonical tRNA genes by conventional tRNA annotation tools. This study illustrates the overlooked phylogenetic distribution of group I introns across the bacterial domain.

microbiology↗

bioRxiv2025

Nested mobile genetic elements mediating antimicrobial resistance genes mobility within and between cells in clinical and environmental Acinetobacter isolates

Mobile genetic elements (MGEs) play a central role in the acquisition and dissemination of antibiotic resistance genes (ARGs). This study analyzed the distribution of MGEs using the whole-genome sequences of 38 Acinetobacter isolates from patient, environmental, and pig waste samples. Insertion sequence (IS) elements were identified as the most common MGE type, followed by plasmids and prophages, with significant variations depending on isolation sources. Pig waste isolates exhibited the highest mean number of plasmids, while prophages were more prevalent in environment-associated isolates. Interestingly, we observed a significant positive correlation between the number of plasmids and number of defense systems. In contrast, a significant negative correlation was identified between the number of prophages and number of defense systems. Plasmids were the primary vectors of ARGs. Co-localization of multiple ARGs, such as msr(E) and mph(E); blaOXA-58, tet(Y), mph(B), and sul2 within a single plasmid was observed, suggesting potential for co-resistance and multidrug resistance. ARGs were enriched in pdif modules, with up to 10 distinct ARGs observed within a single module. Moreover, IS elements were more densely concentrated in conjugative elements. The study further highlights the role of nested MGEs in enabling hierarchical ARG transfer processes both within and between bacterial cells. Additionally, putative novel genomic resistance islands (GRIs) were identified in non-baumannii Acinetobacter species, representing the first documentation of GRIs outside the Acinetobacter calcoaceticus-baumannii (Acb) complex. This study provides new insights into the mechanisms of ARG dissemination in Acinetobacter, particularly the role of MGEs in facilitating hierarchical gene transfer processes. IMPORTANCEUnderstanding the mechanisms behind antimicrobial resistance gene (ARG) dissemination in various environments is crucial for combating the growing global problem of antimicrobial resistance. This study investigated the distribution patterns of mobile genetic elements (MGEs) across distinct ecological contexts, analyzing samples from patients, pig wastewater, and environmental surface water. By investigating the genomes of Acinetobacter species from various isolation sources, we revealed that pig waste-associated isolates possessed more plasmid-borne ARGs compared to patient- and environment-associated isolates. Further, we demonstrated that ARG-bearing plasmids possessed higher density of IS elements than ARG-free plasmids. Putative novel genomic islands were also observed in non-baumannii species isolated from pig wastewater. These findings elucidate the hierarchical structure of ARG transfer mechanisms mediated by nested MGEs, providing valuable insights into the emergence of resistance across both clinical and environmental ecosystems.

microbiology↗