Biology subjects

Yoshimoto, S.

Publications and source records attributed to Yoshimoto, S..

13 recordsLinked to original sources

bioRxiv2026

AtaA as a Self-Immobilizing Scaffold for Enzymatically Active Surfaces through Oxidative Tyrosine Coupling

Protein nanofibers are hold great promise as genetically programmable scaffolds for constructing functional material interfaces. AtaA, a giant trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, forms an elongated architecture from the bacterial cell surface, enabling the cells to adhere directly to a wide variety of abiotic surfaces. However, the potential use of AtaA as a scaffold with additional biological functions remains largely unexplored. In this study, we investigated the use of AtaA as a self-immobilizing scaffold for constructing enzymatically active surfaces via horseradish peroxidase (HRP)-catalyzed tyrosine coupling. We prepared a recombinant AtaA nanofiber fragment (NhNs-AtaA) and its derivative containing two tyrosine-containing loop structures (YL-NhNs-AtaA). Both recombinant proteins retained their adhesive properties. Treatment with HRP and H2O2 generated high-molecular-weight products from both proteins, and dityrosine fluorescence emission indicated the formation of tyrosine-derived covalent cross-links. Y-Loop insertion further increased the consumption of monomeric AtaA and promoted the formation of high-molecular-weight products. The removal of arginine, which was included during purification, further increased the loss of monomeric AtaA in both constructs, indicating that intermolecular protein association influenced productive AtaA cross-linking. NhNs-AtaA and YL-NhNs-AtaA were combined with Y-tagged bacterial alkaline phosphatase (BAP-Y). HRP-mediated coupling generated high-molecular-weight products consistent with the cross-linked AtaA-BAP-Y conjugates and enabled the retention of BAP activity on AtaA-coated surfaces without prior chemical modification. Although Y-Loop insertion enhanced polymerization in solution, it reduced AtaA adhesion and consequently lowered surface-bound phosphatase activity, revealing a trade-off between cross-linking reactivity and native adhesive function. These findings establish AtaA as a self-immobilizing protein nanofiber scaffold for constructing enzymatically active surfaces, and suggest the potential extension of this strategy to other AtaA-based interfaces.

bioengineering↗

bioRxiv2026

Characterization of central carbon metabolism of Acinetobacter sp. Tol 5 based on 13C-metabolic flux analysis

Acinetobacter sp. strain Tol 5 is an attractive microorganism because of its ability to utilize various aromatic compounds, including toluene. However, because Tol 5 is a non-model microorganism, the utilization of carbon sources in central carbon metabolism remains unclear. In this study, the activity of the central carbon metabolism was characterized using 13C-metabolic flux analysis (13C-MFA). Tol 5 was cultured in synthetic medium using ethanol or acetate as the sole carbon source. In addition, because toluene is degraded into pyruvate and acetyl-CoA via the toluene dioxygenase pathway, this situation was mimicked by culturing with ethanol and lactate as co-substrates. The 13C-MFA confirmed flux through the glyoxylate shunt, which bypasses part of the TCA cycle when either ethanol or acetate was used as the sole carbon source. In ethanol, the flux ratio of isocitrate dehydrogenase at the isocitrate branch point decreased markedly. Unlike acetate, the conversion of ethanol to acetyl-CoA is accompanied by NADH production. Therefore, the observed change in this flux ratio was influenced by the excess NADH generated. Under ethanol-lactate conditions, Tol 5 simultaneously utilized ethanol and lactate. The ethanol consumption rate decreased to less than half of that observed when ethanol was the sole carbon source. Most of the lactate uptake was converted to pyruvate and metabolized via the TCA cycle. The glyoxylate shunt flux was lower than that in ethanol. These metabolic characteristics provide valuable insights into the use of Tol 5 as a next-generation microbial cell factory.

microbiology↗

bioRxiv2026

Gas-phase environment activates an alternative catabolic route in toluene-degrading Acinetobacter

Volatile aromatic compounds are important industrial feedstocks but also major environmental pollutants, highlighting the need for bioprocesses for their removal and valorization. Although gas-phase bioprocesses offer practical advantages for handling poorly water-soluble and highly volatile substrates, how gas-phase environments alter microbial metabolism remains poorly understood. Here, we investigated the effect of gas-phase conditions on toluene metabolism in the highly adhesive aromatic hydrocarbon-degrading bacterium Acinetobacter sp. Tol 5. A mutant lacking todC1, which encodes an essential component of the toluene dioxygenase, failed to grow on toluene in liquid culture but retained the ability to grow on solid media under a toluene atmosphere. Consistent with this phenotype, the mutant showed no detectable toluene degradation in the liquid phase, whereas it degraded toluene under gas-phase conditions after a prolonged lag phase. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the accumulation of o-cresol and p-cresol specifically in the mutant under toluene vapor, indicating that toluene metabolism had shifted to an alternative route involving cresol intermediates. In addition, transcriptome analysis identified strong induction of the mph operon encoding phenol monooxygenase (PMO), suggesting that PMO is a likely candidate enzyme mediating TDO-independent toluene oxidation under gas-phase conditions. Together, these results demonstrate that the gas-phase environment can activate an alternative catabolic route in Tol 5 that is not active during conventional liquid cultivation. Our findings highlight the importance of direct metabolic analysis under gas-phase conditions for understanding and designing bioprocesses using highly volatile substrates.

microbiology↗

bioRxiv2026

Integrated omics analysis reveals reorganization of nitrogen and lipids metabolism in a toluene-degrading bacterium

Gas-phase bioprocesses that immobilize microbial cells on solid carriers enable the efficient conversion of poorly water-soluble gaseous substrates, thereby offering significant potential to advance bioremediation and bioproduction. However, microorganisms in the gas phase are exposed to various environmental stresses, mainly due to the absence of bulk water. While survival strategies of microorganisms in gaseous environments have been studied in environmental microbiology, the metabolic adaptations that sustain bacterial cell activity remain poorly understood. In this study, we elucidated the comprehensive metabolic alterations of a highly adhesive bacterium Acinetobacter sp. Tol 5 degrading toluene under gas- and aqueous-phase conditions. An integrated approach combining metabolomics, lipidomics, and transcriptomics revealed significant differences in metabolic profiles between cells under these conditions. Under the gas-phase condition, the degradation of amino acids and nucleic acids was significantly promoted, and the intracellular glutamate pool was maintained at high levels. Notably, citrulline was found to accumulate specifically under the gas-phase condition, representing a stress response similar to that reported in Cucurbitaceae plants during drought. Furthermore, lipidomics revealed the lipid composition of Tol 5 and demonstrated a shift in response to environmental conditions. Specifically, the degradation of intracellular storage lipids was promoted under gas-phase conditions, suggesting a crucial link to bacterial survival in water-limited environments. These findings provide critical insights into the adaptation strategies of bacteria adapting to gaseous environments, offering fundamental information for the rational design of robust gas-phase bioprocesses and a deeper understanding of environmental microbiology.

microbiology↗

bioRxiv2026

Clinical and molecular characterization of an outbreak of leptospirosis in dogs from Los Angeles County, California, USA, 2021

BackgroundIn 2021, the Los Angeles County (LAC) Department of Public Health suspected a leptospirosis outbreak in LAC affecting over 200 client-owned dogs. ObjectiveTo characterize the outbreak and describe microbiologic findings, risk factors, diagnostic test performance, and outcomes in dogs diagnosed with leptospirosis at two specialty practices. MethodsLeptospira culture isolates from four cases were subjected to serotyping and whole genome sequencing (WGS); WGS was also performed on one enriched genome isolate. After the outbreak, data were gathered on 59 cases through record review and compared to the background hospital population (controls, n=15,536). ResultsAll isolates were Leptospira interrogans serovar Canicola, but each was distinct based on WGS. Cases clustered in space and in time. Cases evaluated during the outbreak peak had increased odds of exposure to indoor congregate facilities (ICFs). None of 47 dogs with known leptospirosis vaccination history were completely vaccinated. Leptospira real-time PCR on blood and urine and initial serologic testing using the microscopic agglutination test and point-of-care tests were positive in 15/56 (27%), 49/54 (91%) dogs, 22/29 (76%), and 27/35 (77%) dogs respectively. Fifty-four (92%) of 59 dogs survived to discharge; some remained azotemic. No associated human cases were identified. Conclusions and Clinical ImportanceL. interrogans serovar Canicola was associated with a leptospirosis outbreak in unvaccinated dogs from LAC, which had public health implications given widespread dog ownership rates. Data analysis suggested multiple infection sources, including ICFs. Urine PCR was the most sensitive diagnostic test. Such outbreaks might be prevented through more widespread vaccination.

microbiology↗

bioRxiv2025

Desiccation-tolerant Acinetobacter as a robust chassis for gas-phase bioprocesses

BackgroundGas-phase bioprocesses that immobilize microbial cells on solid carriers enable efficient conversion of volatile or poorly water-soluble substrates, but they also expose cells to fluctuating humidity and transient desiccation that can compromise viability and catalytic performance. In Gram-negative bacteria, especially in pathogens, desiccation is known to impose multifactorial stress, including loss of cellular water and energetics, damage to DNA and proteins, oxidative stress, and disruption of the cell envelope. However, for chassis strains used in or being considered for gas-phase bioprocesses, their desiccation tolerance and responses to desiccation stress remain incompletely defined. ResultsHere, we evaluated the viability, energy status, and gas-phase toluene degradation of Acinetobacter sp. Tol 5 as a chassis for gas-phase processes after controlled desiccation (8%, 52%, and >95% RH), in comparison with Acinetobacter baylyi ADP1, Pseudomonas putida, and Escherichia coli. To minimize adhesion-related bias in post-desiccation measurements, we used an ataA-deficient Tol 5 mutant (Tol 5 {Delta}ataA) for the assays. Acinetobacter strains maintained high viability during desiccation for 16 days, whereas P. putida and E. coli showed significant loss of viability at 8% RH and 52% RH. Intracellular ATP measurements further indicated that desiccation reduced intracellular ATP in all strains, but E. coli rapidly exhausted ATP at 8% and 52% RH, whereas Acinetobacter strains and P. putida retained intracellular ATP. In a gas-phase toluene degradation assay, immobilized Tol 5 retained higher toluene-degrading activity after desiccation than P. putida mt-2. Transcriptome profiling revealed a multilayered Tol 5 response involving DNA and RNA maintenance, cell envelope trafficking and remodeling, redox-responsive functions, and broad repression of growth-associated metabolism. ConclusionsOur results highlight Acinetobacter, particularly Tol 5, as a promising chassis strain for gas-phase bioprocesses and suggest potential mechanistic targets for stabilizing bacterial cells under low-water-activity conditions.

microbiology↗

bioRxiv2025

Metabolic pathway analysis of an Acinetobacter strain capable of assimilating diverse hydrocarbons

Sustainable bioproduction requires developing robust microbial chassis with broad metabolic versatility and suitability for industrial applications. Acinetobacter sp. Tol 5 is a highly adhesive bacterium capable of utilizing various hydrocarbons, making it a promising chassis candidate for immobilized whole-cell catalysis. In this study, we characterized the carbon metabolism of Tol 5 by reconstructing metabolic pathway maps from its genomic data and analyzing the transcriptomes of cells grown on ethanol, hexadecane, toluene, and phenol. Genomic analysis revealed that Tol 5 has limited capacity for sugar utilization but possesses a wide range of metabolic pathways for alkane and aromatic compounds, including five distinct aromatic degradation routes that expand the known metabolic diversity of the genus Acinetobacter. Transcriptome analysis identified the specific pathway genes induced in response to each carbon source and revealed substrate-dependent cross-regulation between aromatic degradation pathways. Gene disruption experiments further demonstrated that toluene dioxygenase facilitates rapid entry into exponential growth on phenol but reduces carbon assimilation efficiency, while phenol monooxygenase serves as the primary and indispensable route for phenol assimilation, revealing a different physiological role for toluene dioxygenase in Tol 5 compared with Pseudomonas putida strains. These findings provide a comprehensive view of the carbon metabolism of Tol 5 and highlight its potential as a microbial chassis for bioprocesses utilizing non-sugar feedstocks, while revealing new aspects of metabolic versatility in the genus Acinetobacter.

microbiology↗

bioRxiv2025

Heterologous expression of carbonic anhydrase in Acinetobacter sp. Tol 5 for whole-cell biocatalysis

Carbonic anhydrase accelerates the hydration of carbon dioxide (CO2) and is an attractive biocatalyst for carbon capture and utilization. Acinetobacter sp. Tol 5 shows high adhesiveness via its cell-surface protein AtaA. We previously demonstrated its application to bacterial immobilization and gas-phase bioproduction. Here, we developed Tol 5 cells expressing carbonic anhydrase and evaluated CO2 conversion ability as whole-cell biocatalysts. A codon-optimized carbonic anhydrase from Sulfurihydrogenibium yellowstonense (SyCA) was produced in the cytoplasm, but the cells showed little activity as a whole-cell biocatalyst. To enhance activity, we fused six signal peptides (SPs) to SyCA for periplasmic expression. The Omp38-SP fusion of SyCA was properly processed to the mature size, yielding higher whole-cell activity. By contrast, the other constructs were either undetectable or remained unprocessed, resulting in lower activities. These results show that periplasmic expression of SyCA is important for efficient CO2 hydration in Tol 5 cells as whole-cell biocatalysts.

bioengineering↗

bioRxiv2025

Grid partitioning image analysis for bacterial cell aggregates

Bacterial cell aggregation plays a fundamental role in surface colonization, stress tolerance, and interspecies metabolite exchange; yet quantitative analysis remains challenging. Here, we introduce grid partitioning image analysis (GPIA), a simple workflow that quantifies the compositional heterogeneity of bacterial aggregates. Confocal laser scanning microscopy (CLSM) images of fluorescently labeled Acinetobacter sp. Tol 5, which exhibits a self-aggregative nature through its cell surface protein AtaA, were partitioned into 2-{micro}m square grids. Grids containing one or no cells were classified as dispersed, whereas those containing multiple cells were classified as aggregates, and the proportion of EGFP-labeled cells within each grid was recorded. Reference images representing dispersed cells, homo-aggregates, and hetero-aggregates produced characteristic EGFP-ratio histograms that matched binomial predictions. When AtaA production in one cell type was decreased, the histogram changed from a symmetric unimodal histogram with the peak at 40-60 % EGFP-ratio to a skewed distribution, indicating that GPIA can detect differences in cell-to-cell affinity. Using the same procedure, we examined six in-frame deletion variants of AtaA. The deletion of the N-terminal head domain alone prevented co-aggregation with full-length AtaA, suggesting that homophilic recognition by this domain mediated self-aggregation, whereas deletions in all other regions had no measurable effect. GPIA, therefore, offers a simple and rapid approach for quantitative studies on bacterial cell aggregation, bridging the gap between qualitative microscopy and quantitative but technically demanding single-cell analysis. GPIA will accelerate research on cell-cell interactions, which are the foundational processes that drive biofilm formation and the assembly of microbial consortia.

microbiology↗

bioRxiv2025

Identification and functional characterization of toluene degradation genes in Acinetobacter sp. Tol 5

Microbial degradation of aromatic compounds provides sustainable solutions for environmental remediation and bioconversion. Acinetobacter sp. Tol 5 is notable for its strong adhesiveness and potential as a biocatalyst for toluene degradation; however, its toluene metabolic pathway has not been fully elucidated. In this study, genomic analysis identified a cluster of genes in Tol 5 highly similar to the well-known tod operon of Pseudomonas putida, encoding enzymes responsible for toluene metabolism. Phylogenetic analyses indicated that these tod genes, unusual among Acinetobacter species, were likely acquired through horizontal gene transfer. Transcriptomic analyses revealed that todF and todC1 are co-transcribed, while the adjacent fadL2 gene, encoding a putative outer membrane transporter corresponding to P. putida todX, is independently transcribed. Functional characterization using gene-knockout mutants revealed that TodC1, the large subunit of dioxygenase, is essential for growth on toluene, whereas FadL2 is not essential. Growth experiments further showed that the todC1 knockout mutant could metabolize benzoate, but not toluene or benzene, confirming that the TOD pathway is the primary route for toluene and benzene degradation in Tol 5. The identification of the functional TOD pathway, which is unique within Acinetobacter, provides genetic and biochemical insights for the development of Tol 5 as an efficient immobilized biocatalyst for the bioremediation and bioconversion of aromatic compounds.

microbiology↗

bioRxiv2024

A new target of multiple lysine methylation in bacteria

The methylation of {varepsilon}-amino groups in protein lysine residues is known to be an important posttranslational modification in eukaryotes. This modification plays a pivotal role in the regulation of diverse biological processes, including epigenetics, transcriptional control, and cellular signaling. Although less studied in prokaryotes, recent research has begun to reveal the potential role of methylation in modulating bacterial immune evasion and adherence to host cells. In this study, we analyzed the cell surface proteins of the toluene-degrading bacterium Acinetobacter sp. Tol 5 by label-free liquid chromatography-mass spectrometry (LC-MS) and found that the lysine residues of its trimeric autotransporter adhesin (TAA), AtaA, are methylated. Over 130 lysine residues of AtaA, consisting of 3,630 amino acids and containing 232 lysine residues, were methylated. We identified the outer membrane protein lysine methyltransferase (OM PKMT) of Tol 5, KmtA, which specifically methylates the lysine residues of AtaA. In the KmtA-deficient mutant, most lysine methylations on AtaA were absent, indicating that KmtA is responsible for the methylation of multiple lysine residues throughout AtaA. Bioinformatic analysis revealed that the OM PKMT genes were widely distributed among gram-negative bacteria, including pathogens with TAAs that promote infectivity, such as Burkholderia mallei and Haemophilus influenzae. Although KmtA has sequence similarities to the OM PKMTs of Rickettsia involved in infectivity, KmtA-like PKMTs formed a distinct cluster from those of the Rickettsia type according to the clustering analysis, suggesting that they are new types of PKMTs. Furthermore, the deletion of Tol 5 KmtA led to an increase in AtaA on the cell surface and enhanced bacterial adhesion, resulting in slower growth. SignificanceMethylation of lysine residues is a posttranslational modification that plays diverse physiological roles in eukaryotes. In prokaryotes however, lysine methylation has been studied only in a limited number of pathogenic bacteria. In this study, we found novel lysine methylation across multiple residues of an outer membrane protein and its methyltransferase, KmtA, in a bacterium from activated sludge. KmtA, along with rickettsial outer membrane protein lysine methyltransferases, which are known to be involved in bacterial pathogenicity, exists in many species of gram-negative bacteria. This finding suggests that methylations are ubiquitous in prokaryotes and are involved in a variety of functions, offering potential strategies for controlling bacterial infections and enhancing the functions of beneficial bacteria for biotechnological applications.

microbiology↗

bioRxiv2023

Structural and functional insights into the complex formation of a trimeric autotransporter adhesin with a peptidoglycan-binding periplasmic protein

Trimeric autotransporter adhesins (TAAs) are outer membrane (OM) proteins that are widely distributed in gram-negative bacteria and are involved primarily in adhesion to biotic and abiotic surfaces, cell agglutination, and biofilm formation. TAAs consist of a passenger domain, which is secreted onto the cell surface, and a transmembrane domain, which forms a pore in the OM to secrete and anchor the passenger domain. Because the interactions between TAAs and chaperones or dedicated auxiliary proteins during secretion are short-lived, TAAs are thought to reside on the OM without forming complexes with other proteins after secretion. In this study, we aimed to clarify the interactions between an Acinetobacter TAA, AtaA, and a peptidoglycan (PG)-binding periplasmic protein, TpgA. Pull-down assays using recombinant proteins identified the interacting domains. X-ray crystallography at 2.6 [A] resolution revealed an A3B3 heterohexameric complex structure composed of the N-terminal domain of TpgA and the transmembrane domain of AtaA. TpgA-N consists of two short helices and three antiparallel {beta} strands, yielding an {beta}{beta}{beta} topology similar to BamE. However, the regions corresponding to BamE interfaces with BamA and BamD differ in TpgA-N. All-atom molecular dynamics simulations and mutational assays revealed that both electrostatic and hydrophobic interactions contribute to stable complex formation. Bioinformatic analyses indicate that the TAA-TpgA complex occurs in a wide range of species. These findings will contribute to a better understanding of TAAs and the cell envelope. ImportanceGram-negative bacteria have specialized secretion systems (SSs) that translocate molecules from the cytoplasm to the extracellular space. Type V SSs have a simpler structure consisting of a functional passenger domain and a transmembrane domain involved in the secretion and anchoring of the passenger domain. Here, we provide the first direct evidence that a trimeric autotransporter adhesin (TAA) exported by a type Vc system forms a stable complex with a peptidoglycan-binding periplasmic protein. The 2.6 [A] structure of the A3B3 heterohexamer, together with simulation and mutational data, reveals complementary electrostatic and hydrophobic contacts that stabilize flexible loops on the periplasmic face of the TAA transmembrane barrel. Conservation of the taa-tpgA gene cassette and of key interface residues across diverse genera suggests that this coupling is a common strategy for tuning TAA stability and indicates that the envelope architecture of many TAAs, including those related to pathogenicity, is more elaborate than previously appreciated.

microbiology↗

bioRxiv2023

Development of a fast feature extraction method for SARS-CoV-2 spike sequences using amino acid physicochemical properties

COVID-19 continues to spread today, leading to an accumulation of SARS-CoV-2 virus mutations in databases, and large amounts of genomic datasets are currently available. However, due to these large datasets, utilizing this amount of sequence data without random sampling is challenging. Major difficulties for downstream analyses include the increase in the dimension size along with the conversion of sequences into numerical values when using conventional amino acid representation methods, such as one-hot encoding and k-mer-based approaches that directly reflect sequences. Moreover, these sequences are deficient in physicochemical characteristics, such as structural information and hydrophilicity; hence, they fail to accurately represent the inherent function of the given sequences. In this study, we utilized the physicochemical properties of amino acids to develop a rapid and efficient approach for extracting feature parameters that are suitable for downstream processes of machine learning, such as clustering. A fixed-length feature vector representation of a spike sequence with reduced dimensionality was obtained by converting amino acid residues into physicochemical parameters. Next, t-distributed stochastic neighbor embedding (t- SNE), a method for dimensionality reduction and visualization of high-dimensional data, was performed, followed by density-based spatial clustering of applications with noise (DBSCAN). The results show that by using the physicochemical properties of amino acids rather than conventional methods that directly represent sequences into numerical values, SARS-CoV-2 spike sequences can be clustered with sufficient accuracy and a shorter runtime. Interestingly, the clusters obtained by using amino acid properties include subclusters that are distinct from those produced utilizing the method for the direct representation of amino acid sequences. A more detailed analysis indicated that the contributing parameters of this novel cluster identified exclusively when utilizing the physicochemical properties of amino acids significantly differ from one another. This suggests that representing amino acid sequences by physicochemical properties might enable the identification of clusters with enhanced sensitivity compared to conventional methods. Author summaryOne of the major causes of the global threat of SARS-CoV-2 is the rapid emergence of its variants. While analyzing these variants is crucial for understanding the mechanism of outbreaks, the expansion of database size is becoming a barrier for effective analysis. In this study, we provide an approach that allows researchers without vast computational resources to comprehensively analyze the variants of SARS-CoV-2 spike by representing the sequences using the physicochemical properties of amino acids. The result of clusters derived using this method demonstrates not only an accuracy comparable to the conventional approaches of directly converting sequences into numerical values but also indicates the potential for more detailed clustering outcomes. The results suggest that our approach is valuable for the rapid identification of characteristic residues in new variants of SARS-CoV-2 and other viruses that may arise in the future.

bioinformatics↗