The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5.

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent capabilities, which include efficiently converting a wide range of substrates into valuable products and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5, an environmentally isolated Gram-negative bacterium, possesses intriguing properties for use in synthetic biology applications. Despite the previous development of genetic tools for the engineering of strain Tol 5, its genetic manipulation has been hindered by low transformation efficiency via electroporation, rendering the process laborious and time-consuming. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding type I and type III restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA without reliance on intermediate Escherichia coli constructs. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our findings provide genetic modification strategies that allow for the domestication of environmentally isolated bacteria, streamlining their utilization in synthetic biology applications.IMPORTANCERecent synthetic biology has sought diverse bacterial chassis from environmental sources to circumvent the limitations of laboratory Escherichia coli strains for industrial and environmental applications. One of the critical barriers in cell engineering of bacterial chassis is their inherent resistance to recombinant DNA, propagated either in vitro or within E. coli cells. Environmental bacteria have evolved defense mechanisms against foreign DNA as a response to the constant threat of phage infection. The ubiquity of phages in natural settings accounts for the genetic intractability of environmental isolates. The significance of our research is in demonstrating genetic modification strategies for the cell engineering of such genetically intractable bacteria. This research marks a pivotal step in the domestication of environmentally isolated bacteria, promising candidates for emerging synthetic biology chassis. Our work thus significantly contributes to advancing their applications across industrial, environmental, and biomedical fields.

Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5.

Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.

Long-term continuous degradation of carbon nanotubes by a bacteria-driven Fenton reaction.

Very few bacteria are known that can degrade carbon nanotubes (CNTs), and the only known degradation mechanism is a Fenton reaction driven by Labrys sp. WJW with siderophores, which only occurs under iron-deficient conditions. No useful information is available on the degradation rates or long-term stability and continuity of the degradation reaction although several months or more are needed for CNT degradation. In this study, we investigated long-term continuous degradation of oxidized (carboxylated) single-walled CNTs (O-SWCNTs) using bacteria of the genus Shewanella. These bacteria are widely present in the environment and can drive the Fenton reaction by alternating anaerobic-aerobic growth conditions under more general environmental conditions. We first examined the effect of O-SWCNTs on the growth of S. oneidensis MR-1, and it was revealed that O-SWCNTs promote growth up to 30 µg/mL but inhibit growth at 40 µg/mL and above. Then, S. oneidensis MR-1 was subjected to incubation cycles consisting of 21-h anaerobic and 3-h aerobic periods in the presence of 30 µg/mL O-SWCNTs and 10 mM Fe(III) citrate. We determined key factors that help prolong the bacteria-driven Fenton reaction and finally achieved long-term continuous degradation of O-SWCNTs over 90 d. By maintaining a near neutral pH and replenishing Fe(III) citrate at 60 d, a degraded fraction of 56.3% was reached. S. oneidensis MR-1 produces Fe(II) from Fe(III) citrate, a final electron acceptor for anaerobic respiration during the anaerobic period. Then, ·OH is generated through the Fenton reaction by Fe(II) and H2O2 produced by MR-1 during the aerobic period. ·OH was responsible for O-SWCNT degradation, which was inhibited by scavengers of H2O2 and ·OH. Raman spectroscopy and X-ray photoelectron spectroscopy showed that the graphitic structure in O-SWCNTs was oxidized, and electron microscopy showed that long CNT fibers initially aggregated and became short and isolated during degradation. Since Shewanella spp. and iron are ubiquitous in the environment, this study suggests that a Fenton reaction driven by this genus is applicable to the degradation of CNTs under a wide range of conditions and will help researchers develop novel methods for waste treatment and environmental bioremediation against CNTs.

Simple Method for the Creation of a Bacteria-Sized Unilamellar Liposome with Different Proteins Localized to the Respective Sides of the Membrane.

Artificial cells are membrane vesicles mimicking cellular functions. To date, giant unilamellar vesicles made from a single lipid membrane with a diameter of 10 µm or more have been used to create artificial cells. However, the creation of artificial cells that mimic the membrane structure and size of bacteria has been limited due to technical restrictions of conventional liposome preparation methods. Here, we created bacteria-sized large unilamellar vesicles (LUVs) with proteins localized asymmetrically to the lipid bilayer. Liposomes containing benzylguanine-modified phospholipids were prepared by combining the conventional water-in-oil emulsion method and the extruder method, and green fluorescent protein fused with SNAP-tag was localized to the inner leaflet of the lipid bilayer. Biotinylated lipid molecules were then inserted externally, and the outer leaflet was modified with streptavidin. The resulting liposomes had a size distribution in the range of 500-2000 nm with a peak at 841 nm (the coefficient of variation was 10.3%), which was similar to that of spherical bacterial cells. Fluorescence microscopy, quantitative evaluation using flow cytometry, and western blotting proved the intended localization of different proteins on the lipid membrane. Cryogenic electron microscopy and quantitative evaluation by α-hemolysin insertion revealed that most of the created liposomes were unilamellar. Our simple method for the preparation of bacteria-sized LUVs with asymmetrically localized proteins will contribute to the creation of artificial bacterial cells for investigating functions and the significance of their surface structure and size.

A novel inverse membrane bioreactor for efficient bioconversion from methane gas to liquid methanol using a microbial gas-phase reaction.

BACKGROUND: Methane (CH4), as one of the major energy sources, easily escapes from the supply chain into the atmosphere, because it exists in a gaseous state under ambient conditions. Compared to carbon dioxide (CO2), CH4 is 25 times more potent at trapping radiation; thus, the emission of CH4 to the atmosphere causes severe global warming and climate change. To mitigate CH4 emissions and utilize them effectively, the direct biological conversion of CH4 into liquid fuels, such as methanol (CH3OH), using methanotrophs is a promising strategy. However, supplying biocatalysts in an aqueous medium with CH4 involves high energy consumption due to vigorous agitation and/or bubbling, which is a serious concern in methanotrophic processes, because the aqueous phase causes a very large barrier to the delivery of slightly soluble gases. RESULTS: An inverse membrane bioreactor (IMBR), which combines the advantages of gas-phase bioreactors and membrane bioreactors, was designed and constructed for the bioconversion of CH4 into CH3OH in this study. In contrast to the conventional membrane bioreactor with bacterial cells that are immersed in an aqueous phase, the filtered cells were placed to face a gas phase in the IMBR to supply CH4 directly from the gas phase to bacterial cells. Methylococcus capsulatus (Bath), a representative methanotroph, was used to demonstrate the bioconversion of CH4 to CH3OH in the IMBR. Cyclopropanol was supplied from the aqueous phase as a selective inhibitor of methanol dehydrogenase, preventing further CH3OH oxidation. Sodium formate was added as an electron donor to generate NADH, which is necessary for CH3OH production. After optimizing the inlet concentration of CH4, the mass of cells, the cyclopropanol concentration, and the gas flow rate, continuous CH3OH production can be achieved over 72 h with productivity at 0.88 mmol L-1 h-1 in the IMBR, achieving a longer operation period and higher productivity than those using other types of membrane bioreactors reported in the literature. CONCLUSIONS: The IMBR can facilitate the development of gas-to-liquid (GTL) technologies via microbial processes, allowing highly efficient mass transfer of substrates from the gas phase to microbial cells in the gas phase and having the supplement of soluble chemicals convenient.

Growth phase-dependent production of the adhesive nanofiber protein AtaA in Acinetobacter sp. Tol 5.

AtaA, the sticky, long, and peritrichate nanofiber protein from Acinetobacter sp. Tol 5, mediates autoagglutination and is highly adhesive to various material surfaces, resulting in a biofilm. Although the production of the adhesive nanofiber protein is likely to require a large amount of energy and material sources, the relationship between AtaA fiber production and cell growth remains unknown. Here, we report the growth phase-dependent AtaA fiber production in Tol 5. We examined the ataA gene expression in different growth phases using a reporter gene assay with an originally developed reporter plasmid and using reverse transcription-quantitative polymerase chain reaction. Bacterial cells with surface-displayed AtaA at different growth phases were immunostained and analyzed using fluorescence flow cytometry and confocal laser scanning microscopy. The results indicate that Tol 5 modulated the amount of surface-displayed AtaA at the transcriptional level. AtaA production was low in the early growth phase but remarkably increased in the late growth phase, covering the whole bacterial cell with AtaA fibers in the stationary phase. Tol 5 displayed AtaA fibers poorly in the early growth phase and showed less autoagglutination and adhesiveness than those in the stationary phase. Although Tol 5 grew as fast as its ataA-deficient mutant in the early growth phase, the optical density of Tol 5 culture was slightly lower than that of the ataA-deficient mutant in the late growth phase. Based on these experimental results, we propose the growth-phase-dependent production of AtaA fiber for efficient and fast cell growth.

Perturbation by Antimicrobial Bacteria of the Epidermal Bacterial Flora of Rainbow Trout in Flow-Through Aquaculture.

The bacterial flora of the epidermal mucus of fish is closely associated with the host's health and susceptibility to pathogenic infections. In this study, we analyzed the epidermal mucus bacteria of rainbow trout (Oncorhynchus mykiss) reared in flow-through aquaculture under environmental perturbations. Over ~2 years, the bacteria present in the skin mucus and water were analyzed based on the 16S rDNA sequences. The composition of the mucus bacterial community showed significant monthly fluctuations, with frequent changes in the dominant bacterial species. Analysis of the beta- and alpha-diversity of the mucus bacterial flora showed the fluctuations of the composition of the flora were caused by the genera Pseudomonas, Yersinia, and Flavobacterium, and some species of Pseudomonas and Yersinia in the mucus were identified as antimicrobial bacteria. Examination of the antimicrobial bacteria in the lab aquarium showed that the natural presence of antimicrobial bacteria in the mucus and water, or the purposeful addition of them to the rearing water, caused a transition in the mucus bacteria community composition. These results demonstrate that specific antimicrobial bacteria in the water or in epidermal mucus comprise one of the causes of changes in fish epidermal mucus microflora.

Fabrication of a highly protective 3D-printed mask and evaluation of its viral filtration efficiency using a human head mannequin.

Facemasks are one of the most effective and low-cost prophylactics for COVID-19. In the spring 2020, when a severe shortage of facemasks occurred worldwide, various types of 3D-printed masks were designed and proposed. However, the protective effects conferred by most of these masks were not experimentally evaluated. Here, we provide a new simple design of 3D-printed mask and evaluate its protective effect in a viral filtration test using a human head mannequin. The developed mask can be constructed with a low-cost 3D printer, with an approximate production cost of US $4. This mask has three parts: the main part, wearing parts, and a piece of non-woven fabric filter. The volume of the filter, which needs to be changed daily, was reduced to approximately 1/10 of that of commercially available surgical masks used in this study. The developed mask is fabricated from polylactic acid, a biodegradable plastic, and its surface contour contacting the face may be adjusted after softening the material with hot water at 60-80°C. The viral filtration efficiency of the developed mask was found to be over 80%. This performance is better than that of commercially available facemasks, such as surgical masks and cloth masks, and equal to those of KN95 and KF94.

Recent advances in research on biointerfaces: From cell surfaces to artificial interfaces.

Biointerfaces are regions where biomolecules, cells, and organic materials are exposed to environmental media or come in contact with other biomaterials, cells, and inorganic/organic materials. In this review article, six research topics on biointerfaces are described to show examples of state-of-art research approaches. First, biointerface design of nanoparticles for molecular detection is described. Functionalized gold nanoparticles can be used for sensitive detection of various target molecules, including chemical compounds and biomolecules, such as DNA, proteins, cells, and viruses. Second, the interaction between bacterial cell surfaces and material surfaces, including the introduction of advances in analytical methods and theoretical calculations, are explained as well as their applications to bioprocesses. Third, bioconjugation technologies for localizing functional proteins at biointerfaces are introduced, in particular, by focusing the potential of enzymes as a catalytic tool for designing different types of bioconjugates that function at biointerfaces. Forth topics is focusing on lipid-protein interaction in cell membranes as natural biointerfaces. Examples of membrane lipid engineering are introduced, and it is mentioned how their compositional profiles affect membrane protein functions. Fifth topic is the physical method for molecular delivery across the biointerface being developed currently, such as highly efficient nanoinjection, electroporation, and nanoneedle devices, in which the key is how to perforate the cell membrane. Final topic is the chemical design of lipid- or polymer-based RNA delivery carriers and their behavior on the cell interface, which are currently attracting attention as RNA vaccine technologies targeting COVID-19. Finally, future directions of biointerface studies are presented.

Single-cell adhesion force mapping of a highly sticky bacterium in liquid.

The sticky bacterium Acinetobacter sp. Tol 5 adheres to various material surfaces via its cell surface nanofiber protein, AtaA. This adhesiveness has only been evaluated based on the amount of cells adhering to a surface. In this study, the adhesion force mapping of a single Tol 5 cell in liquid using the quantitative imaging mode of atomic force microscopy (AFM) revealed that the adhesion of Tol 5 was near 2 nN, which was 1-2 orders of magnitude higher than that of other adhesive bacteria. The adhesion force of a cell became stronger with the increase in AtaA molecules present on the cell surface. Many fibers of peritrichate AtaA molecules simultaneously interact with a surface, strongly attaching the cell to the surface. The adhesion force of a Tol 5 cell was drastically reduced in the presence of 1% casamino acids but not in deionized water (DW), although both liquids decrease the adhesiveness of Tol 5 cells, suggesting that DW and casamino acids inhibit the cell approaching step and the subsequent direct interaction step of AtaA with surfaces, respectively. Heterologous production of AtaA provided non-adhesive Acinetobacter baylyi ADP1 cells with a strong adhesion force to AFM tip surfaces of silicon and gold.

Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli.

Cell immobilization is an important technique for efficiently utilizing whole-cell biocatalysts. We previously invented a method for bacterial cell immobilization using AtaA, a trimeric autotransporter adhesin from the highly sticky bacterium Acinetobacter sp. Tol 5. However, except for Acinetobacter species, only one bacterium has been successfully immobilized using AtaA. This is probably because the heterologous expression of large AtaA (1 MDa), that is a homotrimer of polypeptide chains composed of 3,630 amino acids, is difficult. In this study, we identified the adhesive domain of AtaA and constructed a miniaturized AtaA (mini-AtaA) to improve the heterologous expression of ataA. In-frame deletion mutants were used to perform functional mapping, revealing that the N-terminal head domain is essential for the adhesive feature of AtaA. The mini-AtaA, which contains a homotrimer of polypeptide chains from 775 amino acids and lacks the unnecessary part for its adhesion, was properly expressed in E. coli, and a larger amount of molecules was displayed on the cell surface than that of full-length AtaA (FL-AtaA). The immobilization ratio of E. coli cells expressing mini-AtaA on a polyurethane foam support was significantly higher compared to the cells with or without FL-AtaA expression, respectively. The expression of mini-AtaA in E. coli had little effect on the cell growth and the activity of another enzyme reflecting the production level, and the immobilized E. coli cells could be used for repetitive enzymatic reactions as a whole-cell catalyst.

Complete Genome Sequence of the Highly Adhesive Bacterium Acinetobacter sp. Strain Tol 5.

Acinetobacter sp. strain Tol 5 is a nonpathogenic Gram-negative bacterium with biotechnological and environmental applications. Here, we report the complete genome sequence of Acinetobacter sp. strain Tol 5, which has a genome size of 4,799,506 bp and a G+C content of 38.1%.

Development of a Biocontained Toluene-Degrading Bacterium for Environmental Protection.

Biocontainment is a safeguard strategy for preventing uncontrolled proliferation of genetically engineered microorganisms (GEMs) in the environment. Biocontained GEMs are designed to survive only in the presence of a specific molecule. The design of a pollutant-degrading and pollutant-dependent GEM prevents its proliferation after cleaning the environment. In this study, we present a biocontained toluene-degrading bacterium based on Acinetobacter sp. Tol 5. The bamA gene, which encodes an essential outer membrane protein, was deleted from the chromosome of Tol 5 but complemented with a plasmid carrying a bamA gene regulated by the Pu promoter and the regulatory protein XylR. The resultant strain (PuBamA) degraded toluene, similarly to the wild-type Tol 5. Although the cell growth of the PuBamA strain was remarkably inhibited after toluene depletion, escape mutants emerged at a frequency of 1 per 5.3 × 10-7 cells. Analyses of escape mutants revealed that insertion sequences (ISs) carrying promoters were inserted between the Pu promoter and the bamA gene on the complemented plasmid. MinION deep sequencing of the plasmids extracted from the escape mutants enabled the identification of three types of ISs involved in the emergence of escape mutants, suggesting a strategy for reducing it. IMPORTANCE GEMs are beneficial for various applications, including environmental protection. However, the risks of GEM release into the environment have been debated for a long time. If a pollutant is employed as a specific molecule for a biocontainment system, GEMs capable of degrading pollutants are available for environmental protection. Nevertheless, to our knowledge, biocontained degraders for real pollutants have not been reported in academic journals so far. This is possibly due to the difficulty in the expression of enzymes for degrading pollutants in a tractable bacterium such as Escherichia coli. On the other hand, bacteria with enzymes for degrading pollutants are often intractable as a host of GEMs due to the shortage of tools for genetic manipulation. This study reports the feasibility of a biocontainment strategy for a toluene degrader. Our results provide useful insights into the construction of a GEM biocontainment system for environmental protection.

Switching Between Methanol Accumulation and Cell Growth by Expression Control of Methanol Dehydrogenase in Methylosinus trichosporium OB3b Mutant.

Methanotrophs have been used to convert methane to methanol at ambient temperature and pressure. In order to accumulate methanol using methanotrophs, methanol dehydrogenase (MDH) must be downregulated as it consumes methanol. Here, we describe a methanol production system wherein MDH expression is controlled by using methanotroph mutants. We used the MxaF knockout mutant of Methylosinus trichosporium OB3b. It could only grow with MDH (XoxF) which has a cerium ion in its active site and is only expressed by bacteria in media containing cerium ions. In the presence of 0 µM copper ion and 25 µM cerium ion, the mutant grew normally. Under conditions conducive to methanol production (10 µM copper ion and 0 µM cerium ion), cell growth was inhibited and methanol accumulated (2.6 µmol·mg-1 dry cell weight·h-1). The conversion efficiency of the accumulated methanol to the total amount of methane added to the reaction system was ~0.3%. The aforementioned conditions were repeatedly alternated by modulating the metal ion composition of the bacterial growth medium.

Metabolic alteration of Methylococcus capsulatus str. Bath during a microbial gas-phase reaction.

This study demonstrates the metabolic alteration of Methylococcus capsulatus (Bath), a representative bacterium among methanotrophs, in microbial gas-phase reactions. For comparative metabolome analysis, a bioreactor was designed to be capable of supplying gaseous substrates and liquid nutrients continuously. Methane degradation by M. capsulatus (Bath) was more efficient in a gas-phase reaction operated in the bioreactor than in an aqueous phase reaction operated in a batch reactor. Metabolome analysis revealed remarkable alterations in the metabolism of cells in the gas-phase reaction; in particular, pyruvate, 2-ketoglutarate, some amino acids, xanthine, and hypoxanthine were accumulated, whereas 2,6-diaminopimelate was decreased. Based on the results of metabolome analysis, cells in the gas-phase reaction seemed to alter their metabolism to reduce the excess ATP and NADH generated upon increased availability of methane and oxygen. Our findings will facilitate the development of efficient processes for methane-based bioproduction with low energy consumption.

Establishing a Percutaneous Infection Model Using Zebrafish and a Salmon Pathogen.

To uncover the relationship between skin bacterial flora and pathogen infection, we developed a percutaneous infection model using zebrafish and Yersinia ruckeri, a pathogen causing enteric redmouth disease in salmon and in trout. Pathogen challenge, either alone or together with pricking by a small needle, did not cause infection of the fish. However, cold stress given by water temperature shift from the optimum 28 °C for zebrafish to 20 °C caused fatal infection of injured fish following pathogen challenge. We investigated the effects of cold stress, injury, and pathogen challenge, alone and in combination, on fish skin bacterial flora using 16S rDNA metagenomics. We found that cold stress drastically altered the skin bacterial flora, which was dominated by Y. ruckeri on infected fish. In addition, fish whose intrinsic skin bacterial flora was disrupted by antibiotics had their skin occupied by Y. ruckeri following a challenge with this pathogen, although the fish survived without injury to create a route for invasion into the fish body. Our results suggest that the intrinsic skin bacterial flora of fish protects them from pathogen colonization, and that its disruption by stress allows pathogens to colonize and dominate their skin.

The extracellular juncture domains in the intimin passenger adopt a constitutively extended conformation inducing restraints to its sphere of action.

Enterohemorrhagic and enteropathogenic Escherichia coli are among the most important food-borne pathogens, posing a global health threat. The virulence factor intimin is essential for the attachment of pathogenic E. coli to the intestinal host cell. Intimin consists of four extracellular bacterial immunoglobulin-like (Big) domains, D00-D2, extending into the fifth lectin subdomain (D3) that binds to the Tir-receptor on the host cell. Here, we present the crystal structures of the elusive D00-D0 domains at 1.5 Å and D0-D1 at 1.8 Å resolution, which confirms that the passenger of intimin has five distinct domains. We describe that D00-D0 exhibits a higher degree of rigidity and D00 likely functions as a juncture domain at the outer membrane-extracellular medium interface. We conclude that D00 is a unique Big domain with a specific topology likely found in a broad range of other inverse autotransporters. The accumulated data allows us to model the complete passenger of intimin and propose functionality to the Big domains, D00-D0-D1, extending directly from the membrane.

Process Description of an Unconventional Biofilm Formation by Bacterial Cells Autoagglutinating through Sticky, Long, and Peritrichate Nanofibers.

In this study, we elucidated the formation process of an unconventional biofilm formed by a bacterium autoagglutinating through sticky, long, and peritrichate nanofibers. Understanding the mechanisms of biofilm formation is essential to control microbial behavior and improve environmental biotechnologies. Acinetobacter sp. Tol 5 autoagglutinate through the interaction of the long, peritrichate nanofiber protein AtaA, a trimeric autotransporter adhesin. Using AtaA, without cell growth or extracellular polymeric substances production, Tol 5 cells quickly form an unconventional biofilm. The process forming this unconventional biofilm started with cell-cell interactions, proceeded to cell clumping, and led to the formation of large cell aggregates. The cell-cell interaction was described by Derjaguin-Landau-Verwey-Overbeek (DLVO) theory based on a new concept, which considers two independent interactions between two cell bodies and between two AtaA fiber tips forming a discontinuous surface. If cell bodies cannot collide owing to an energy barrier at low ionic strengths but approach within the interactive distance of AtaA fibers, cells can agglutinate through their contact. Cell clumping proceeds following the cluster-cluster aggregation model, and an unconventional biofilm containing void spaces and a fractal nature develops. Understanding its formation process would extend the utilization of various types of biofilms, enhancing environmental biotechnologies.

Extracellular electron transfer mediated by a cytocompatible redox polymer to study the crosstalk among the mammalian circadian clock, cellular metabolism, and cellular redox state.

The circadian clock is an endogenous biological timekeeping system that controls various physiological and cellular processes with a 24 h rhythm. The crosstalk among the circadian clock, cellular metabolism, and cellular redox state has attracted much attention. To elucidate this crosstalk, chemical compounds have been used to perturb cellular metabolism and the redox state. However, an electron mediator that facilitates extracellular electron transfer (EET) has not been used to study the mammalian circadian clock due to potential cytotoxic effects of the mediator. Here, we report evidence that a cytocompatible redox polymer pMFc (2-methacryloyloxyethyl phosphorylcholine-co-vinyl ferrocene) can be used as the mediator to study the mammalian circadian clock. EET mediated by oxidized pMFc (ox-pMFc) extracted intracellular electrons from human U2OS cells, resulting in a longer circadian period. Analyses of the metabolome and intracellular redox species imply that ox-pMFc receives an electron from glutathione, thereby inducing pentose phosphate pathway activation. These results suggest novel crosstalk among the circadian clock, metabolism, and redox state. We anticipate that EET mediated by a redox cytocompatible polymer will provide new insights into the mammalian circadian clock system, which may lead to the development of new treatments for circadian clock disorders.

Native display of a huge homotrimeric protein fiber on the cell surface after precise domain deletion.

AtaA, a trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, exhibits nonspecific, high adhesiveness to abiotic surfaces. For identification of the functional domains of AtaA, precise design of domain-deletion mutants is necessary so as not to cause undesirable structural distortion. Here, we designed and constructed three types of AtaA mutants from which the same domain, FGG1, was deleted. The first mutant was designed to preserve the periodicity of hydrophobic residues in the coiled-coil segments sandwiching the deleted region. After the deletion, the protein was properly displayed on the cell surface and had the same adhesive function as the wild type. Transmission electron microscopy (TEM) imaging and circular dichroism (CD) spectroscopy showed that its isolated passenger domain had the same fiber structure as in the AtaA wild type. In contrast, a mutant designed to disturb the coiled-coil periodicity at the deletion site failed to reach the cell surface. Although secretion occurred for the mutant designed with a flexible connector between the coiled coils, the cells exhibited a decrease in adhesiveness. Furthermore, TEM imaging of the mutant fibers showed bending at the fiber tip and changes in their CD spectrum indicated a decrease in secondary structure content. Thus, we succeeded to natively display the huge homotrimeric fiber structure of AtaA on the cell surface after precise deletion of a domain, maintaining the proper folding state and adhesive function by preserving its coiled-coil periodicity. This strategy enables us to construct various domain-deletion mutants of AtaA without structural distortion for complete functional mapping.