Nanoscale Secondary Ion Mass Spectrometry Analysis of Individual Bacterial Cells Reveals Feedback from Extracellular Electron Transport to Upstream Reactions

Saito, Junki; Hashimoto, Kazuhito; Okamoto, Akimitsu

doi:10.5796/electrochemistry.85.444

Cited by 10 publications

(15 citation statements)

References 14 publications

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“…The ITO electrode samples were prepared based on methodology as previously described ( Saito et al, 2017 ). ITO electrodes with cell attachments were cut into 7 mm × 7 mm pieces to fit the NanoSIMS sample holder.…”

Section: Methodsmentioning

confidence: 99%

Electrode Potential Dependency of Single-Cell Activity Identifies the Energetics of Slow Microbial Electron Uptake Process

Deng

Okamoto

2018

Front. Microbiol.

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Electrochemical measurements have been widely applied to study microbial extracellular electron transport processes. However, because electrochemistry detects not only microbial electron transport but also other reactions, background signals comparable to or larger than microbial ones hamper the identification of microbial electrochemical properties. This problem is crucial especially for the detection of electron uptake processes by slow-growing microbes in low-energy subsurface sediments, as the environmental samples contain electrochemically active humus and mineral particles. In this study, we report a cell-specific stable isotope analysis to quantify the electrode potential dependency of anabolic activity in individual cells for identifying the electron uptake energetics of slow-growing bacteria. Followed by the incubation of Desulfovibrio ferrophilus IS5 cells with isotopic 15N-ammonium as the sole N source on electrodes poised at potentials of -0.2, -0.3, -0.4, and -0.5 V [vs. standard hydrogen electrode (SHE)], we conducted nanoscale secondary ion mass spectroscopy (NanoSIMS) to quantify 15N assimilation in more than 100 individual cells on the electrodes. We observed significant 15N assimilation at potentials of -0.4 and more 15N assimilation at -0.5 V, which is consistent with the onset potential for electron uptake via outer-membrane cytochromes (OMCs). The activation of cell energy metabolism was further examined by transcriptome analysis. Our results showed a novel methodology to study microbial electron uptake energetics. The results also serve as the first direct evidence that energy acquisition is coupled to the electron uptake process in sulfate-reducing bacteria that are ubiquitous in the subsurface environments, with implications on the electron-fueled subsurface biosphere hypothesis and other microbial processes, such as anaerobic iron corrosion and anaerobic methane oxidation.

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Section: Methodsmentioning

confidence: 99%

Electrode Potential Dependency of Single-Cell Activity Identifies the Energetics of Slow Microbial Electron Uptake Process

Deng

Okamoto

2018

Front. Microbiol.

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“…Single-cell metabolic assay was conducted for checking the C. matruchotii activity on the ITO electrode. C. matruchotii cells incubated on the ITO surface at +0.4 V (vs SHE) in DM containing 10 mM 13 C-glucose as a carbon source and 18.7 mM 15 NH 4 Cl as a nitrogen source for 24 h were used for quantifying the assimilation, as they can be employed for assessing the cellular metabolic activity owing to their coupling with the extent of anabolism [ 18 , 43 ]. Before carrying out the NanoSIMS, cells attached to the ITO surface were washed thrice, and subsequently fixed in 2.5% ( v / v ) glutaraldehyde.…”

Section: Methodsmentioning

confidence: 99%

Metabolic Current Production by an Oral Biofilm Pathogen Corynebacterium matruchotii

2020

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The development of a simple and direct assay for quantifying microbial metabolic activity is important for identifying antibiotic drugs. Current production capabilities of environmental bacteria via the process called extracellular electron transport (EET) from the cell interior to the exterior is well investigated in mineral-reducing bacteria and have been used for various energy and environmental applications. Recently, the capability of human pathogens for producing current has been identified in different human niches, which was suggested to be applicable for drug assessment, because the current production of a few strains correlated with metabolic activity. Herein, we report another strain, a highly abundant pathogen in human oral polymicrobial biofilm, Corynebacterium matruchotii, to have the current production capability associated with its metabolic activity. It showed the current production of 50 nA/cm2 at OD600 of 0.1 with the working electrode poised at +0.4 V vs. a standard hydrogen electrode in a three-electrode system. The addition of antibiotics that suppress the microbial metabolic activity showed a significant current decrease (>90%), establishing that current production reflected the cellular activity in this pathogen. Further, the metabolic fixation of atomically labeled 13C (31.68% ± 2.26%) and 15N (19.69% ± 1.41%) confirmed by high-resolution mass spectrometry indicated that C. matruchotii cells were metabolically active on the electrode surface. The identified electrochemical activity of C. matruchotii shows that this can be a simple and effective test for evaluating the impact of antibacterial compounds, and such a method might be applicable to the polymicrobial oral biofilm on electrode surfaces, given four other oral pathogens have already been shown the current production capability.

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“…Particularly for biofilm on the electrode, NanoSIMS specifically analyzes cells that strongly attach to the surface of the electrode as planktonic, and loosely attached cells are removed in the sample preparation process. Moreover, this method for single-cell analysis of metabolic pathways can be applied to any EET-capable bacteria, as it does not require gene engineering (Saito et al, 2017(Saito et al, , 2020 Confocal laser scanning microscopy (CLSM) is a specialized form of microscopy that is used to produce high-resolution and sharp images of cellular and polymeric biofilm components in three dimensions (Bjarnsholt et al, 2013;Nwaneshiudu et al, 2012). 3D imaging is achieved because the confocal optics can be focused on a very small volume in the biofilm.…”

Section: Nano-scale Secondary Ion Mass Spectroscopymentioning

confidence: 99%

Pathogens electrogenicity as a tool for in-situ metabolic activity monitoring and drug assessment in biofilms

2021

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Concerns regarding increased antibiotic resistance arising from the emergent properties of biofilms have spurred interest in the discovery of novel antibiotic agents and techniques to directly estimate metabolic activity in biofilms. Although a number of methods have been developed to quantify biofilm formation, real-time quantitative assessment of metabolic activity in label-free biofilms remains a challenge. Production of electrical current via extracellular electron transport (EET) has recently been found in pathogens and appears to correlate with their metabolic activity. Accordingly, monitoring the production of electrical currents as an indicator of cellular metabolic activity in biofilms represents a new direction for research aiming to assess and screen the effects of antimicrobials on biofilm activity. In this article, we reviewed EET-capable pathogens and the methods to monitor biofilm activity to discuss advantages of using the capability of pathogens to produce electrical currents and effective combination of these methods. Moreover, we discussed EET mechanisms by pathogenic and environmental bacteria and open questions for the physiological roles of EET in pathogen's biofilm. The present limitations and possible future directions of in situ biofilm metabolic activity assessment for large-scale screening of antimicrobials are also discussed.

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Nanoscale Secondary Ion Mass Spectrometry Analysis of Individual Bacterial Cells Reveals Feedback from Extracellular Electron Transport to Upstream Reactions

Cited by 10 publications

References 14 publications

Electrode Potential Dependency of Single-Cell Activity Identifies the Energetics of Slow Microbial Electron Uptake Process

Electrode Potential Dependency of Single-Cell Activity Identifies the Energetics of Slow Microbial Electron Uptake Process

Metabolic Current Production by an Oral Biofilm Pathogen Corynebacterium matruchotii

Pathogens electrogenicity as a tool for in-situ metabolic activity monitoring and drug assessment in biofilms

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