Single-Cell Mass Spectroscopic Analysis for Quantifying Active Metabolic Pathway Heterogeneity in a Bacterial Population on an Electrode.

Microbial electrochemical catalysis based on respiratory reactions coupled with extracellular electron transport (EET), which is critical for bioenergy applications, strongly depends on the biocompatibility of the electrode material. However, the comparison of materials for such physiological responses has been difficult because of the lack of a quantitative assay for characterizing cellular metabolism at the electrode surface. Here, we developed a single-cell analysis method specific for the cells attached to the electrode to quantify active metabolic pathway heterogeneity as an index of physiological cell/electrode interaction, which generally increases with metabolic robustness in the microbial population. Nanoscale secondary ion mass spectrometry followed by microbial current production with model EET-capable bacteria, Shewanella oneidensis MR-1 and its mutant strains lacking carbon assimilation pathways, showed that different active metabolic pathways resulted in nearly identical 13C/15N assimilation ratios for individual cells in the presence of isotopically labeled nutrients, demonstrating a correlation between the 13C/15N ratio and the active metabolic pathway. Compared to the nonelectrode conditions, the heterogeneity of the assimilated 13C/15N ratio was highly enhanced on the electrode surface, suggesting that the metabolic robustness of the microbial population increased through the electrochemical interaction with the electrode. The present methodology enables us to quantitatively compare and screen electrode materials that increase the robustness of microbial electrocatalysis.

Impact of tightening environmental regulations against long-chain perfluoroalkyl acids on composition of durable water repellents containing side-chain fluorinated polymers.

Tightening of environmental regulations against long-chain perfluoroalkyl acids (PFAAs) since the 2000s may have led to significant increases in the occurrence of short-chain PFAAs in the environment. Understanding the impact of the regulations on composition of durable water repellents (DWRs) is imperative to guide implementation of pragmatic actions during their use and end-of-life treatment. Substantial decreases in the frequencies of detection and concentrations of long-chain PFAAs and long-chain PFAA-precursors, and substantial increases in those of short-chain PFAAs and short-chain PFAA-precursors, have been observed in the impurities and hydrolysis products of side-chain fluorinated polymers (SCFPs). Comparison of profiles among the DWRs containing fluorinated ingredients in 2011 indicated that DWRs containing C8F17- and C10F21-SCFPs were the dominant products and accounted for 90 % of the samples, whereas DWRs containing C4F9- and C6F13-SCFPs were the dominant products and accounted for 70 % of the samples collected in 2021. Tightening of the regulations have caused decreasing applications of long-chain SCFPs and increasing use of short-chain SCFPs in DWRs containing fluorinated ingredients. The ingredients of one DWR were changed from PFAS-free alternatives to short-chain SCFPs, whereas those of another DWR were changed from short-chain SCFPs to PFAS-free alternatives. The presence of unexplained extractable organic fluorine has been observed in DWRs containing fluorinated ingredients, which may be difficult to be hydrolyzed and form known compounds. A historical series of DWRs available from before and after the tightening of regulations and a multifaceted analytical technique consisting of combustion ion chromatographic and mass spectrometric approaches combined with two extraction techniques involving ultrasonic treatment and alkaline hydrolysis revealed the impact of tightening regulations on composition of DWRs.

Enhancement of cell growth by uncoupling extracellular electron uptake and oxidative stress production in sediment sulfate-reducing bacteria.

Microbial extracellular electron uptake (EEU) from solid electron donors has critical implications for microbial energy acquisition in energy-limited environments as well as electrochemical microbial technologies. Although EEU supplies sufficient energy to support cellular growth, additional soluble electron donors are required for most microbes to grow on electrode surfaces. Here, we demonstrated that the minimization of exogenous and endogenous oxidative stress greatly enhanced the growth rate of the sediment EEU-capable sulfate-reducing bacterium Desulfovibrio ferrophilus IS5 on an electrode without the addition of a soluble electron donor. Single-cell activity analysis by nanoscale secondary ion mass spectrometry showed that the metabolic activity of IS5 cells on the electrode was significantly enhanced following incubation in an H-type reactor, which was configured to reduce the exposure of cells to the potential oxidative stress source of the Pt counter electrode (CE). Additionally, the highest metabolic activity was observed at an electrode potential of -0.4 V (versus the standard hydrogen electrode), where electron uptake rate was not at peak. Compared to a single-chamber reactor, incubation in an H-type reactor at -0.4 V shortened the cell doubling time by 50-fold, which resulted in sufficient anabolism for cell replication (15N/Ntotal > 50%). The production of strongly oxidizing species at the CE was confirmed by X-ray photoelectron spectroscopy and inductively coupled plasma mass spectrometry analyses. Transcriptome analysis revealed overexpression of antioxidative genes in cells incubated at a potential with higher current production. These results suggested that higher levels of endogenous oxidative species were produced by a more reduced electron-transport chain from trace amounts of oxygen in the reactor, thereby lowering cell activity. In conclusion, EEU may enable sediment microbes to undergo enhanced cell growth and to find niches on minerals under anaerobic energy-limited conditions, where oxidative stress is much less likely to be present.

The hydrophobicity in a chemically modified side-chain of cysteine residues of thanatin is related to antimicrobial activity against Micrococcus luteus.

The chemically modified thanatins with the methyl group (CH(3)), ethyl group (C(2)H(5)), and normal-octyl group (C(8)H(17)) at the side-chain of cysteine residues were synthesized. The octyl group modified form exhibited 8-fold higher antimicrobial activity against Micrococcus luteus than wild type thanatin. It was found that there was an equilateral correlation between antimicrobial activity and side-chain hydrophobicity at the cysteine residues in thanatin.

Cation-limited kinetic model for microbial extracellular electron transport via an outer membrane cytochrome C complex.

Outer-membrane c-type cytochrome (OM c-Cyt) complexes in several genera of iron-reducing bacteria, such as Shewanella and Geobacter, are capable of transporting electrons from the cell interior to extracellular solids as a terminal step of anaerobic respiration. The kinetics of this electron transport has implications for controlling the rate of microbial electron transport during bioenergy or biochemical production, iron corrosion, and natural mineral cycling. Herein, we review the findings from in-vivo and in-vitro studies examining electron transport kinetics through single OM c-Cyt complexes in Shewanella oneidensis MR-1. In-vitro electron flux via a purified OM c-Cyt complex, comprised of MtrA, B, and C proteins from S. oneidensis MR-1, embedded in a proteoliposome system is reported to be 10- to 100-fold faster compared with in-vivo estimates based on measurements of electron flux per cell and OM c-Cyts density. As the proteoliposome system is estimated to have 10-fold higher cation flux via potassium channels than electrons, we speculate that the slower rate of electron-coupled cation transport across the OM is responsible for the significantly lower electron transport rate that is observed in-vivo. As most studies to date have primarily focused on the energetics or kinetics of interheme electron hopping in OM c-Cyts in this microbial electron transport mechanism, the proposed model involving cation transport provides new insight into the rate detemining step of EET, as well as the role of self-secreted flavin molecules bound to OM c-Cyt and proton management for energy conservation and production in S. oneidensis MR-1.

Characterization of candidate intermediates in the Black Box of the ecdysone biosynthetic pathway in Drosophila melanogaster: Evaluation of molting activities on ecdysteroid-defective larvae.

Early steps of the biosynthetic pathway of the insect steroid hormone ecdysone remains the "Black Box" wherein the characteristic ecdysteroid skeleton is built. 7-Dehydrocholesterol (7dC) is the precursor of uncharacterized intermediates in the Black Box. The oxidation step at C-3 has been hypothesized during conversion from 7dC to 3-oxo-2,22,25-trideoxyecdysone, yet 3-dehydroecdysone is undetectable in some insect species. Therefore, we first confirmed that the oxidation at C-3 occurs in the fruitfly, Drosophila melanogaster using deuterium-labeled cholesterol. We next investigated the molting activities of candidate intermediates, including oxidative products of 7dC, by feeding-rescue experiments for Drosophila larvae in which an expression level of a biosynthetic enzyme was knocked down by the RNAi technique. We found that the administration of cholesta-4,7-dien-3-one (3-oxo-Δ4,7C) could overcome the molting arrest of ecdysteroid-defective larvae in which the expression level of neverland was reduced. However, feeding 3-oxo-Δ4,7C to larvae in which the expression levels of shroud and Cyp6t3 were reduced inhibited molting at the first instar stage, suggesting that this steroid could be converted into an ecdysteroid-antagonist in loss of function studies of these biosynthetic enzymes. Administration of the highly conjugated cholesta-4,6,8(14)-trien-3-one, oxidized from 3-oxo-Δ4,7C, did not trigger molting of ecdysteroid-defective larvae. These results suggest that an oxidative product derived from 7dC is converted into ecdysteroids without the formation of this stable conjugated compound. We further found that the 14α-hydroxyl moiety of Δ4-steroids is required to overcome the molting arrest of larvae in loss of function studies of Neverland, Shroud, CYP6T3 or Spookier, suggesting that oxidation at C-14 is indispensable for conversion of these Δ4-steroids into ecdysteroids via 5ß-reduction.

Actinopyrone D, a new downregulator of the molecular chaperone GRP78 from Streptomyces sp.

A new downregulator of the molecular chaperone GRP78, actinopyrone D, was isolated together with a known related compound, PM050463, from Streptomyces sp. RAG92. The molecular formula of actinopyrone D was established as C25H36O4 by high-resolution FAB-MS. NMR spectroscopic analysis revealed the structure of actinopyrone D, which consists of an α-methoxy-γ-pyrone ring and a C17 side chain containing a cis olefin moiety. Actinopyrone D and PM050463 dose-dependently inhibited 2-deoxyglucose-induced luciferase expression in HT1080 human fibrosarcoma cells transfected with a luciferase reporter plasmid containing the GRP78 promoter. Actinopyrone D inhibited GRP78 protein expression and induced cell death under endoplasmic reticulum stress.