Biology subjects

Yokoyama, F.

Publications and source records attributed to Yokoyama, F..

4 recordsLinked to original sources

bioRxiv2026

Biofilm Initiation via Extracellular Matrix Production Driven by Cell Orientation Patterning in Growing Escherichia coli Populations

Unicellular microorganisms can transition to multicellular states that enhance survival under environmental fluctuations. In bacteria, one such state is the biofilm, defined by the production of an extracellular matrix. Although many aspects of biofilm formation have been extensively studied, how matrix production is spatially initiated within a growing population remains largely unknown. Here we show that cell orientation patterning is associated with the spatial initiation of Rcs stress-response activation, which is known to be involved in extracellular matrix production. Using an Escherichia coli strain reporting mechanically induced stress responses, we observed that Rcs activation was accompanied by mechanically regulated out-of-plane growth under agar-pad confinement. Notably, this activation preferentially emerged near dense topological defects, where cell orientation mismatches and growth-induced pressure builds up. By controlling confinement geometry with microfluidic devices, we regulated cell orientation patterns and defect distributions, thereby biasing the spatial pattern of Rcs activation. These findings indicate an association between cell orientation patterning and the spatial activation of a stress-response pathway involved in extracellular matrix production, suggesting a possible physical link to biofilm initiation.

microbiology↗

bioRxiv2024

Development of a new genotype-phenotype linked antibody screening system

Antibodies are powerful tools for the therapy and diagnosis of various diseases. In addition to conventional hybridoma-based screening, recombinant antibody-based screening has become a common choice; however, its application is hampered by two factors: 1) screening starts after Ig gene cloning and recombinant antibody production only, and 2) the antibody is composed of paired chains, heavy and light, commonly expressed by two independent expression vectors. Here, we introduce a method for the rapid screening of recombinant monoclonal antibodies by establishing a Golden Gate-based dual-expression vector and in-vivo expression of membrane-bound antibodies. Using this system, we demonstrate the rapid isolation of influenza cross-reactive antibodies with high affinity from immunized mice within 7 days. This system is particularly useful for isolating therapeutic or diagnostic antibodies, e.g., during foreseen pandemics. Impact StatementA Golden Gate-based dual-expression vector enables rapid screening, which facilitates efficient isolation of high-affinity cross-reactive antibodies for therapeutic or diagnostic use and provides a crucial advance for pandemic preparedness.

bioengineering↗

bioRxiv2023

Capturing and Detecting of Extracellular Vesicles Derived from Single Escherichia coli Mother Cells

Cells have a phenotypic heterogeneity even in isogeneic populations. Differences in secretion of substances have been well-investigated with single mammalian cells. However, studies on the heterogeneity of secreted substances at the single-bacterial-cell level are challenging due to the small size, motility, and rapid proliferation of bacterial cells such as Escherichia coli. Here, we propose a microfluidic device to achieve an isolated culture of single bacterial cells and capture of extracellular vesicles (EVs) secreted from individuals. The device has winding channels to trap single rod-shaped E. coli cells at their entrances. Isolated single mother cells grew constantly up to 24 h, while their daughter cells were removed by flow. The flow carried EVs of the trapped cells along the channel, whose surface was rendered positively charged to electrostatically capture negatively charged EVs, followed by staining with a lipophilic dye to detect EVs by microscopy. Our results underline that the amounts of segregated EVs vary among cells. Moreover, individual responses to perturbation using a membrane-perturbing antibiotic were observed in growth dynamics and EV secretion of living-alone bacteria. The proposed method can be applied to detect other secreted substances of interest, possibly paving the way for elucidating unknown heterogeneities in bacteria.

microbiology↗

bioRxiv2020

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium, Shewanella vesiculosa HM13

Bacteria secrete and utilize nanoparticles, called extracellular membrane vesicles (EMVs), for survival in their growing environments. Therefore, the amount and components of EMVs should be tuned in response to the environment. However, how bacteria regulate vesiculation in response to the extracellular environment remains largely unknown. In this study, we identified a putative sensor protein, HM1275, involved in the induction of vesicle production in a hypervesiculating Gram-negative bacterium, Shewanella vesiculosa HM13. This protein was predicted to possess typical sensing and signaling domains of sensor proteins, such as methyl-accepting chemotaxis proteins. Comparison of vesicle production between the hm1275-disrupted mutant and the parent strain revealed that HM1275 is involved in lysine-induced hypervesiculation. Moreover, HM1275 has sequence similarity to a biofilm dispersion protein, BdlA, of Pseudomonas aeruginosa PAO1, and hm1275 disruption increased the amount of biofilm. Thus, this study showed that the induction of vesicle production and suppression of biofilm formation in response to lysine concentration are under the control of the same putative sensor protein.

microbiology↗