Biology subjects

Sakata, N.

Publications and source records attributed to Sakata, N..

11 recordsLinked to original sources

bioRxiv2026

Rapid enhancement of antibiotic resistance and divergent remodeling of gene expression in single cells of bacteria

Exposing bacterial cell populations to antibiotics often results in the emergence of descendants with enhanced resistance. Phenotypic heterogeneity and global physiological responses are thought to contribute to resistance evolution. However, identifying the specific stages of drug exposure where phenotypic heterogeneity and physiological responses play critical roles remains elusive. Using long-term single-cell lineage tracking of an Escherichia coli resistance model, here we reveal that resistance-enhanced cells emerge rapidly within a few generations in a clonal bacterial cell population under chloramphenicol (Cp) exposure and stably transmit the enhanced resistance for hundreds of generations in the presence of Cp. High expression of a resistance protein was advantageous during the initial period of drug exposure, but conferred only a marginal fitness advantage during the period of resistance enhancement. Transcriptomic profiles of resistance-enhanced cell populations were lineage-specific, suggesting that multiple gene expression states can support enhanced resistance. Altogether, these results suggest that an interplay between initial phenotypic heterogeneity and divergent remodeling of gene expression drives the rapid acquisition of enhanced antibiotic resistance.

microbiology↗

bioRxiv2025

Context-dependent activation and evolutionary buffering of a mating pheromone in fission yeast

The evolution of mating signals drives reproductive isolation and speciation across diverse lineages. However, how short peptide pheromones, typically subject to strong structural constraints, achieve functional diversification remain unclear. In the fission yeast Schizosaccharomyces pombe, a previously established library of 153 single-amino acid variants of the mating pheromone M-factor was applied to large-scale competition assays under varied mating conditions. Mutations deleterious under standard conditions became advantageous at specific environmental pH levels, demonstrating context-dependent pheromone function activation. Synthetic peptide assays confirmed that certain substitutions act as environmental molecular switches. Comparative analysis with the closely related Schizosaccharomyces octosporus species identified a permissive mutation that mitigates the effects of otherwise inactivating changes, enabling an evolutionary route without intermediate fitness loss. Our findings reveal how short peptide signals evolve via environmentally contingent activation and compensatory interactions, offering a mechanistic framework for understanding the ecological and evolutionary dynamics of mating communication.

evolutionary biology↗

bioRxiv2022

A Drosophila model of diabetic neuropathy reveals a crucial role of proteasome activity in the glia

Diabetic peripheral neuropathy (DPN) is the most common chronic, progressive complication of diabetes mellitus. The main symptom is sensory loss; the molecular mechanisms are not fully understood. We found that Drosophila fed a high-sugar diet, which induces diabetes-like phenotypes, exhibit impairment of noxious heat avoidance. The impairment of heat avoidance was associated with shrinkage of the leg neurons expressing the Drosophila transient receptor potential channel Painless. Using a candidate genetic screening approach, we identified proteasome modulator 9 as one of the modulators of impairment of heat avoidance. We further showed that proteasome inhibition in the glia reversed the impairment of noxious heat avoidance, and heat-shock proteins and exosome secretion in the glia mediated the effect of proteasome inhibition. Our results establish Drosophila as a useful system for exploring molecular mechanisms of diet-induced peripheral neuropathy and propose that the glial proteasome is one of the candidate therapeutic targets for DPN.

neuroscience↗

bioRxiv2022

Controlling stomatal aperture, a potential strategy for managing plant bacterial disease

Bacterial blight of crucifers caused by Pseudomonas cannabina pv. alisalensis (Pcal) inflicts great damage on crucifer production. To explore efficient and sustainable strategies for Pcal disease control, we here investigated and screened for amino acids with reduced disease development. We found that exogenous foliar application with multiple amino acids reduced disease symptoms and bacterial populations in cabbage after spray-inoculation, but not syringe-inoculation. These results indicate that these amino acids showed a protective effect before Pcal entered plants. Therefore, we observed stomatal responses, which is a main gateway for Pcal entry into the apoplast, after amino acid treatments, and found several amino acids induce stomatal closure. Moreover, our findings demonstrated that reducing stomatal aperture width can limit bacterial entry into plants, leading to reduced disease symptoms. Therefore, managing stomatal aperture can be a new powerful strategy for controlling bacterial disease.

plant biology↗

bioRxiv2022

Purifying selection enduringly acts on the sequence evolution of highly expressed proteins in Escherichia coli

The evolutionary speed of a protein sequence is constrained by its expression level, with highly expressed proteins evolving relatively slowly. This negative correlation between expression levels and evolutionary rates (known as the E-R anticorrelation) has already been widely observed in past macroevolution between species from bacteria to animals. However, it remains unclear whether this seemingly general law also governs recent evolution, including past and de novo, within a species. However, the advent of genomic sequencing and high-throughput phenotyping, particularly for bacteria, has revealed fundamental gaps between the two evolutionary processes and has provided empirical data opposing the possible underlying mechanisms which are widely believed. These conflicts raise questions about the generalization of the E-R anticorrelation and the relevance of plausible mechanisms. To explore the ubiquitous impact of expression level on molecular evolution, and to test the relevance of the possible underlying mechanisms, we analyzed the genome sequences of 99 strains of Escherichia coli for microevolution in nature. We also analyzed genomic mutations accumulated under laboratory conditions as a model of de novo microevolution. Here, we show that the E-R anticorrelation is significant in both past and de novo microevolution in E. coli. Our data also confirmed ongoing purifying selection acting on highly expressed genes. Ongoing selection included codon-level purifying selection, supporting the relevance of the underlying mechanisms. However, their contributions to the constraints in recent evolution might be smaller than previously expected from past macroevolution.

evolutionary biology↗

bioRxiv2020

Covering soybean leaves with cellulose nanofiber changes leaf surface hydrophobicity and confers resistance against Phakopsora pachyrhizi

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi, an obligate biotrophic fungal pathogen, is the most devastating soybean production disease worldwide. Currently, timely fungicide application is the only means to control ASR in the field. We investigated cellulose nanofiber (CNF) application on ASR disease management. CNF-treated leaves showed reduced lesion number after P. pachyrhizi inoculation compared to control leaves, indicating that covering soybean leaves with CNF confers P. pachyrhizi resistance. We also demonstrated that formation of P. pachyrhizi pre-infection structures including germ-tubes and appressoria, and also gene expression related to these formations, such as chitin synthases (CHSs), were significantly suppressed in CNF-treated soybean leaves compared to control leaves. Moreover, contact angle measurement revealed that CNF converts soybean leaf surface properties from hydrophobic to hydrophilic. These results suggest that CNF can change soybean leaf surface hydrophobicity, conferring resistance against P. pachyrhizi, based on the reduced expression of CHSs, as well as reduced formation of pre-infection structures. This is the first study to investigate CNF application to control field disease.

plant biology↗

bioRxiv2020

Coronatine contributes Pseudomonas cannabina pv. alisalensis virulence by overcoming both stomatal and apoplastic defenses in dicot and monocot plants

P. cannabina pv. alisalensis (Pcal) is a causative agent of bacterial blight of crucifer including cabbage, radish, and broccoli. Importantly, Pcal can infect not only a wide range of Brassicaceae, but also green manure crops such as oat. However, Pcal virulence mechanisms have not been investigated and are not fully understood. We focused on coronatine (COR) function, which is one of the well-known P. syringae pv. tomato DC3000 virulence factors, in Pcal infection processes on both dicot and monocot plants. Cabbage and oat plants dip-inoculated with a Pcal KB211 COR mutant ({Delta}cmaA) exhibited reduced virulence compared to Pcal WT. Moreover, {Delta}cmaA failed to reopen stomata on both cabbage and oat, suggesting that COR facilitates Pcal entry through stomata into both plants. Furthermore, cabbage and oat plants syringe-infiltrated with {Delta}cmaA also showed reduced virulence, suggesting that COR is involved in overcoming not only stomatal-based defense, but also apoplastic defense. Indeed, defense related genes, including PR1 and PR2, were highly expressed in plants inoculated with {Delta}cmaA compared to Pcal WT, indicating that COR suppresses defense-related genes of both cabbage and oat. Additionally, SA accumulation increases after {Delta}cmaA inoculation compared to Pcal WT. Taken together, COR contributes to cause disease by suppressing stomatal-based defense and apoplastic defense in both dicot and monocot plants. This is the first study to investigate COR functions in the interaction of Pcal and different host plants (dicot and monocot plants) using genetically and biochemically defined COR deletion mutants. Author summaryDisease outbreaks caused by new Pseudomonas syringae isolates are problems worldwide. P. cannabina pv. alisalensis (Pcal) causes bacterial blight on a wide range of cruciferous plants and bacterial brown spot on oat plants. Although P. syringae deploys a variety of virulence factors, Pcal virulence factors have not been investigated. We focused on coronatine (COR) function, which is one of the well-known P. syringae virulence factors. COR is a non-host-specific phytotoxin and contributes to P. syringae growth and lesion formation or expansion in several host plants. COR function has been mainly studied in the model pathogen P. syringae pv. tomato DC3000 and model the plant Arabidopsis thaliana. Thus, COR roles in Pcal infection especially on monocot plants have not been well studied. Therefore, we investigated COR role in Pcal interaction with both dicot and monocot plants. Here, we revealed that COR functions as a multifunctional suppressor to manage Pcal virulence on both plants.

plant biology↗

bioRxiv2020

Distinct specificity of two pheromone G-protein coupled receptors, Map3 and Mam2, in fission yeast species.

Most sexually reproducing organisms have the ability to recognize individuals of the same species. In ascomycetes including yeasts, potential mating between cells of opposite mating-type depends on the molecular recognition of two peptidyl mating pheromones by their corresponding G-protein coupled receptors (GPCRs). Although such pheromone/receptor systems are likely to function in both mate choice and prezygotic isolation, very few studies have focus on the differences in pheromone/receptor system between mating types. The fission yeast Schizosaccharomyces pombe has two mating types (sexes), Plus (P) and Minus (M). Here we investigated the specificity of the two GPCRs, Mam2 and Map3, for their respective pheromones, P-factor and M-factor, in fission yeast. First, we switched GPCRs between S. pombe and the closely related species Schizosaccharomyces octosporus, which showed that SoMam2 (Mam2 of S. octosporus) is partially functional in S. pombe, whereas SoMap3 (Map3 of S. octosporus) is not interchangeable. Next, we swapped individual domains of Mam2 and Map3 with the respective domains in SoMam2 and SoMap3, which revealed differences between the receptors both in the intracellular regions that regulate the downstream signaling of pheromones and in the molecular recognition for pheromone binding. In particular, we demonstrated that two amino acid residues of Map3, F214 and F215, are essential for the specificity of M-factor recognition. Thus, the differences in these two GPCRs are likely to reflect the significantly distinct specificities of their respective pheromone/receptor systems; that is, the specificity of Map3 is more stringent than that of Mam2. We speculate that this sexual asymmetry might allow ascomycete fungi to generate novel prezygotic barriers within a population, while maintaining strong mate choice. Our genetic analyses also contribute to our understanding of the receptors that comprise the Class D GPCRs belonging to the fungal pheromone receptor family. Author summaryCourtship signals play a key role in the fertilization processes of living beings from animals to microorganisms and, in particular, sex pheromones are involved in differentiating among species. On the other hand, changes in a pheromone/receptor system might also alter species-specificity during the selection of a mating partner, which is likely to facilitate prezygotic isolation. Here we demonstrate a distinct difference in the specificity of GPCRs for pheromones between cells of opposite mating-type in the fission yeast Schizosaccharomyces pombe, by conducting a comprehensive genetic analysis and comparison of these proteins between closely related fission yeast species. Our finding of sexual asymmetry due to the relative strictness of receptors in S. pombe suggests that it might be one of the driving forces behind the formation of new species. Such differences in pheromone/receptor interactions between the sexes may occur in a variety of life-forms such as insects and amphibians; hence, the findings of this study might be extended to other organisms. In addition, this study of S. pombe pheromone receptors sheds light on the functions of the unique Class D GPCRs.

genetics↗

bioRxiv2020

Acibenzolar-S-methyl activates stomatal-based defense systemically in Japanese radish by inducing peroxidase-dependent reactive oxygen species production

Acibenzolar-S-methyl (ASM) is a well-known plant activator, which is a synthetic analog of salicylic acid (SA). Recently, copper fungicides and antibiotics are major strategies for controlling bacterial diseases. However, resistant strains have already been found. Therefore, there is an increasing demand for sustainable new disease control strategies. We investigated the ASM disease control effect against Pseudomonas cannabina pv. alisalensis (Pcal), which causes bacterial blight on Japanese radish. In this study, we demonstrated that ASM effectively suppressed Pcal disease symptom development associated with reduced bacterial populations on Japanese radish leaves. Interestingly, we also demonstrated that ASM activated systemic acquired resistance (SAR), including stomatal-based defense, not only on ASM treated leaves, but also on untreated upper and lower leaves. Reactive oxidative species (ROS) are essential second messengers in stomatal-based defense. We found that ASM induced stomatal closure by inducing ROS production through peroxidase. These results indicate that stomatal closure induced by ASM treatment is effective for preventing Pcal pathogen invasion into plants, and in turn reduction of disease development.

plant biology↗

bioRxiv2020

High-throughput laboratory evolution and evolutionary constraints in Escherichia coli

Understanding the constraints that shape the evolution of antibiotic resistance is critical for predicting and controlling drug resistance. Despite its importance, however, a systematic investigation for evolutionary constraints is lacking. Here, we performed a high-throughput laboratory evolution of Escherichia coli under the addition of 95 antibacterial chemicals and quantified the transcriptome, resistance, and genomic profiles for the evolved strains. Using interpretable machine learning techniques, we analyzed the phenotype-genotype data and identified low dimensional phenotypic states among the evolved strains. Further analysis revealed the underlying biological processes responsible for these distinct states, leading to the identification of novel trade-off relationships associated with drug resistance. We also report a novel constraint that leads to decelerated evolution. These findings bridge the genotypic, gene expression, and drug resistance space and lead to a better understanding of evolutionary constraints for antibiotic resistance.

evolutionary biology↗

bioRxiv2017

High-Throughput Laboratory Evolution Of Escherichia coli Under Multiple Stress Environments

Bacterial cells have a remarkable capacity to adapt and to evolve to environmental changes. Although many mutations contributing to adaptive evolution have been identified, the relationship between the mutations and the phenotypic changes responsible for fitness gain has yet to be fully elucidated. For a better understanding of phenotype-genotype relationship in evolutionary dynamics, we performed high-throughput laboratory evolution of Escherichia coli under various stress conditions using an automated culture system. One measure of phenotype, transcriptome analysis, revealed that the expression changes which occurred during the evolution were generally similar among the strains evolved in the same stress environment. We also found several genes and gene functions for which mutations were commonly fixed in the strains resistant to the same stress, and whose effects on resistance were verified experimentally. We demonstrated that the integration of transcriptome and genome data enables us to extract the mechanisms for stress resistance.\n\nAuthor summaryUnderstanding the relationship between phenotypic and genetic changes is a fundamental goal in evolutionary biology, which can provide insights into the past and future evolutionary trajectories. Evolution of microorganisms in a laboratory has been the primary approach to clarify the mappings of phenotypic and genotypic changes. Here, we performed high-throughput laboratory evolution with bacteria using an automated culture system, to quantify phenotypic and genotypic changes occurred under various stress conditions. We identified various stress-specific gene expression changes and mutations, and contributions of them to fitness gain were validated. These results demonstrated that the integration of phenotypic and genotypic changes makes it possible to extract the mechanisms for stress resistance evolution, which will contribute to bioengineering applications.

evolutionary biology↗