Biology subjects

Tokunou, Y.

Publications and source records attributed to Tokunou, Y..

3 recordsLinked to original sources

bioRxiv2025

Challenging the Diffusion Barrier Paradigm: Biofilms Promote Flavin-mediated Electron Shuttling in Shewanella oneidensis

Flavins are ubiquitous diffusible redox mediators that are crucial in enabling extracellular electron transfer (EET) in electroactive bacteria that often form biofilms in both natural and engineered environments. However, the behavior of flavin diffusion within biofilms remains poorly understood. In this study, we developed a colony-based electrochemical platform using interdigitated electrode arrays to quantify the diffusion coefficients of flavins within bacterial biofilms. We found that flavin diffusivity was about 11-fold enhanced in Shewanella oneidensis MR-1 biofilms than in the bulk solution. However, this enhancement was abolished in the gene deletion mutant that lacked the membrane-associated flavin-binding c-type cytochrome OmcA, which suggests that the flavin-OmcA interaction on cell surfaces facilitates flavin diffusion within the biofilm. Notably, the diffusion coefficients of other redox molecules, such as methylene blue and safranin, scarcely improved in biofilms, which validates the inference that flavin-specific interaction-accelerated diffusion occurs in biofilm environments. These findings uncover a phenomenon promoting long-range electron transfer, overturning the prevailing assumption that shuttling-based EET is hindered by slow molecular diffusion in biofilms. Our study highlights the functional importance of cell-surface cytochromes in overcoming the kinetic limitation of diffusion-based electron transfer, thereby shaping the bioenergetics in biofilms.

microbiology↗

bioRxiv2025

Membrane vesicles of Shewanella oneidensis MR-1 enhance denitrification growth in a species-selective manner

Denitrification, a fundamental bacterial respiratory process that occurs in anoxic environments, plays a pivotal role in energy synthesis and the global nitrogen cycle. Although the significance of this process is well-recognized, its regulation within polymicrobial communities remains poorly understood, particularly concerning interspecies interactions. In this study, we investigated the role that bacterial membrane vesicles (MV) play in modulating denitrification across bacterial species. MV is known to carry specific biomolecules such as secondary metabolites, proteins, and nucleic acids, therefore considered to be a secretion pathway. We found that MV produced by Shewanella oneidensis enhanced denitrification in a species-specific manner. Bacteria with highly hydrophobic surfaces tended to respond to denitrification enhancement, suggesting that the MV-bacteria attachment process is the key to generating species specificity. Transcriptome analysis and isotopic metabolite tracking indicated that the MV influenced denitrifying activities, rather than the transcription of denitrification-related genes. We further demonstrated that c-type cytochromes in MV act as key components that enhance denitrification. These insights expand our understanding of bacterial ecology, highlighting the role of membrane vesicles in facilitating respiratory competition and cooperation in polymicrobial communities.

microbiology↗

bioRxiv2019

Basicity of N5 in semiquinone enhances the rate of respiratory electron outflow in Shewanella oneidensis MR-1

Extracellular electron transport (EET) occurs in environmental iron-reducing bacteria and is mediated by an outer membrane multi-heme cytochrome complex (Cyts). It has critical implications for global mineral cycling and electrochemical microbial catalysis. The rate of EET mediated by multiple heme redox centers significantly increases in the presence of flavins and quinones. Their electron free energy does not entirely account for the fact that differential effects on EET rate enhancement vary significantly by factors [≥]100. Here, we report on whole-cell electrochemical analysis of Shewanella oneidensis MR-1 using six flavin analogs and four quinones. We demonstrated that protonation of the nitrogen atom at position 5 (N5) of the isoalloxazine ring is essential for electron outflow acceleration as a bound non-covalent cofactor of Cyts. EET mediated by Cyts was accelerated at a rate dependent on pKa(N5). The EET rate largely decreased in response to the addition of deuterated water (D2O), while low concentration of D2O (4 %) had little impact on electron free energy difference of the heme and non-covalent bound cofactors, strongly suggesting that the protonation of N5 limits the rate of EET. Our findings directly link EET kinetics to proton transport reaction via N5 and provide a basis for the development of novel strategies for controlling EET-associated biological reactions.\n\nSignificance statementThe potential of various small molecules such as flavins and quinones to enhance the rate of extracellular electron transport (EET) has been exploited to develop environmental energy conversion systems. Flavins and quinones have similar molecular structures but their abilities to enhance EET vary by >100x in Shewanella oneidensis MR-1. These large differences are inconsistent with conventional models, which rely on redox potentials or diffusion constant of shuttling electron mediators. In this study, we demonstrated that the basicity of the nitrogen atom of the isoalloxazine ring (N5) enhances the rate of electron outflow when a flavin or quinone is a non-covalent cofactor of S. oneidensis MR-1 outer membrane c-type cytochromes.

biochemistry↗