Visualizing extracellular vesicle biogenesis in gram-positive bacteria using super-resolution microscopy.

BACKGROUND: Recently, bacterial extracellular vesicles (EVs) have been considered to play crucial roles in various biological processes and have great potential for developing cancer therapeutics and biomedicine. However, studies on bacterial EVs have mainly focused on outer membrane vesicles released from gram-negative bacteria since the outermost peptidoglycan layer in gram-positive bacteria is thought to preclude the release of EVs as a physical barrier. RESULTS: Here, we examined the ultrastructural organization of the EV produced by gram-positive bacteria using super-resolution stochastic optical reconstruction microscopy (STORM) at the nanoscale, which has not been resolved using conventional microscopy. Based on the super-resolution images of EVs, we propose three major mechanisms of EV biogenesis, i.e., membrane blebbing (mechanisms 1 and 2) or explosive cell lysis (mechanism 3), which are different from the mechanisms in gram-negative bacteria, despite some similarities. CONCLUSIONS: These findings highlight the significant role of cell wall degradation in regulating various mechanisms of EV biogenesis and call for a reassessment of previously unresolved EV biogenesis in gram-positive bacteria.

Preparation of eutectic substrate mixtures for enzymatic conversion of ATC to L-cysteine at high concentration levels.

High concentration eutectic substrate solutions for the enzymatic production of L-cysteine were prepared. Eutectic melting of binary mixtures consisting of D,L-2-amino-Δ(2)-thiazoline-4-carboxylic acid (ATC) as a substrate and malonic acid occurred at 39 °C with an ATC mole fraction of 0.5. Formation of eutectic mixtures was confirmed using SEM, SEM-EDS, and XPS surface analyses. Sorbitol, MnSO4, and NaOH were used as supplements for the enzymatic reactions. Strategies for sequential addition of five compounds, including a binary ATC mixture and supplements, during preparation of eutectic substrate solutions were established. Eutectic substrate solutions were stable for 24 h. After 6 h of enzymatic reactions, a 550 mM L-cysteine yield was obtained from a 670 mM eutectic ATC solution.

Genome-scale metabolic modeling and in silico analysis of opportunistic skin pathogen Cutibacterium acnes.

Cutibacterium acnes, one of the most abundant skin microbes found in the sebaceous gland, is known to contribute to the development of acne vulgaris when its strains become imbalanced. The current limitations of acne treatment using antibiotics have caused an urgent need to develop a systematic strategy for selectively targeting C. acnes, which can be achieved by characterizing their cellular behaviors under various skin environments. To this end, we developed a genome-scale metabolic model (GEM) of virulent C. acnes, iCA843, based on the genome information of a relevant strain from ribotype 5 to comprehensively understand the pathogenic traits of C. acnes in the skin environment. We validated the model qualitatively by demonstrating its accuracy prediction of propionate and acetate production patterns, which were consistent with experimental observations. Additionally, we identified unique biosynthetic pathways for short-chain fatty acids in C. acnes compared to other GEMs of acne-inducing skin pathogens. By conducting constraint-based flux analysis under endogenous carbon sources in human skin, we discovered that the Wood-Werkman cycle is highly activated under acnes-associated skin condition for the regeneration of NAD, resulting in enhanced propionate production. Finally, we proposed potential anti-C. acnes targets by using the model-guided systematic framework based on gene essentiality analysis and protein sequence similarity search with abundant skin microbiome taxa.

Bacteria detection and species identification at the single-cell level using super-resolution fluorescence imaging and AI analysis.

The skin microbiome is thought to play a critical role in maintaining skin health and protecting against infection. While most microorganisms that live on the skin are harmless or even beneficial, some can cause skin infections or other health problems, emphasizing the importance of diagnosis of the composition and diversity of the skin flora. However, conventional diagnostic methods for evaluation of the skin microbiome are not sensitive enough to detect bacteria at low concentrations and suffer from poor specificity, thus limiting early diagnosis of bacterial infections. In this study, we developed novel approaches for bacterial species detection and identification methods with single-cell sensitivity using super-resolution microscopy and AI-based image analysis: a protein quantification-based method and an AI-based bacterial image analysis method. We demonstrate that these methods can differentiate between common bacterial members of the skin flora, including Staphylococcus aureus and Staphylococcus epidermidis, and different ribotypes of Cutibacterium acnes, both in purified bacterial samples and in scaling skin samples. The advantages of these methods, including the lack of time-consuming amplification or purification steps and single-cell level detection sensitivity, allow early diagnosis of bacterial infections, even from bacterial samples at extremely low concentrations, thus showing promise as a next-generation platform for microbiome detection as single-cell diagnostics.

Effects of associated bacteria on the pathogenicity and reproduction of the insect-parasitic nematode Rhabditis blumi (Nematoda: Rhabditida).

Three bacteria, Alcaligenes faecalis , Flavobacterium sp., and Providencia vermicola , were isolated from dauer juveniles of Rhabditis blumi . The pathogenic effects of the bacteria against 4th instar larvae of Galleria mellonella were investigated. Providencia vermicola and Flavobacterium sp. showed 100% mortality at 48 h after haemocoelic injection, whereas A. faecalis showed less than 30% mortality. Dauer juveniles showed 100% mortality against G. mellonella larvae, whereas axenic juveniles, which do not harbor associated bacteria, exhibited little mortality. All of the associated bacteria were used as a food source for nematode growth, and nematode yield differed with bacterial species. Among the bacterial species, P. vermicola was most valued for nematode yield, showing the highest yield of 5.2 × 10(4) nematodes/mL in the plate. In bacterial cocultures using two of the three associated bacteria, one kind stimulated the other. The highest total bacterial yield of 12.6 g/L was obtained when the inoculum ratio of P. vermicola to A. faecalis was 10:1. In air-lift bioreactors, the nematode growth rate increased with an increasing level of dissolved oxygen. The maximum nematode yield of 1.75 × 10(5) nematodes/mL was obtained at 192 h with an aeration rate of 6 vvm.

Effect of the skincare product on facial skin microbial structure and biophysical parameters: A pilot study.

Daily use of cosmetics is known to affect the skin microbiome. This study aimed to determine the bacterial community structure and skin biophysical parameters following the daily application of a skincare product on the face. Twenty-five Korean women, who used the same skincare product for four weeks participated in the study. During this period, skin hydration, texture, sebum content, and pH were measured, and skin swab samples were collected on the cheeks. The microbiota was analyzed using the MiSeq system. Through these experiments, bacterial diversity in facial skin increased and the microbial community changed after four weeks of skincare product application. The relative abundance of Cutibacterium and Staphylococcus increased, significant changes in specific bacterial modules of the skin microbial network were observed, and skin hydration and texture improved. It was suggested that daily use of skincare products could affect the microbial structure of facial skin as well as the biophysical properties of the facial skin. These findings expand our understanding of the role of skincare products on the skin environment.

Development of inhibitors against lipase and alpha-glucosidase from derivatives of monascus pigment.

New derivatives of monascus pigment were produced during Monascus fermentation by the addition of unnatural amino acids, and the inhibitory activities of the derivatives against diet-related lipase and alpha-glucosidase were tested. Derivatives with penicillamine (H-Pen), cyclohexylalanine (H-Cha), butylglycine (L-t-Bg), and norleucine (H-Nle) showed relatively high inhibitory activities against lipase. The H-Pen derivative exhibited the highest inhibitory activity, with an IC(50) (50% inhibition) value of 24.0 microM. The four derivatives all showed noncompetitive inhibition patterns against lipase. The inhibition constant (K(i)) of the H-Pen derivative was estimated to be 20.7 microM. The H-Pen derivative also exhibited a relatively high inhibitory activity against alpha-glucosidase, with an IC(50) value of 50.9 microM. The inhibition pattern of the H-Pen derivative against alpha-glucosidase appeared to be of a mixed type. The inhibition constants K(i) and were estimated to be 25.9, and 98.9 microM, respectively.

Lipase-catalyzed synthesis of sorbitol-fatty acid esters at extremely high substrate concentrations.

Lipase-catalyzed synthesis of sorbitol-fatty acid esters was performed in eutectic media with extremely high substrate concentrations. Homogeneous eutectic melts of sorbitol and fatty acids of C6-C16 were prepared using an adjuvant mixture. Enhanced homogeneity of mixtures was confirmed by X-ray diffraction analysis. The substrate concentration was 3.63-6.67 M in the eutectic media, whereas in organic media the concentration was below 0.10 M. Esters were synthesized with an immobilized Candida antarctica lipase, and optimum conditions were analyzed. Compared to reactions in organic media, the initial reaction rate of ester synthesis and the overall productivity were significantly enhanced in eutectic media while the conversion yields were similar. Based on the kinetic analysis, highly viscous eutectic media were shown to influence the initial reaction rate and the apparent activation energy resulting in diffusion limitations.

Effective isopropanol-butanol (IB) fermentation with high butanol content using a newly isolated Clostridium sp. A1424.

BACKGROUND: Acetone-butanol-ethanol fermentation has been studied for butanol production. Alternatively, to achieve acetone-free butanol production, use of clostridium strains producing butanol and 1,3-propanediol (1,3-PDO) from glycerol, natural and engineered isopropanol-butanol-ethanol (IBE) producers has been attempted; however, residual 1,3-PDO and acetone, low IBE production by natural IBE producers, and complicated gene modification are limitations. RESULTS: Here, we report an effective isopropanol and butanol (IB) fermentation using a newly isolated Clostridium sp. A1424 capable of producing IB from various substrates with a small residual acetone. Notably, this strain also utilized glycerol and produced butanol and 1,3-PDO. After 46.35 g/L of glucose consumption at pH 5.5-controlled batch fermentation, Clostridium sp. A1424 produced 9.43 g/L of butanol and 13.92 g/L of IB at the productivity of 0.29 and 0.44 g/L/h, respectively, which are the highest values in glucose-based batch fermentations using natural IB producers. More interestingly, using glucose-glycerol mixtures at ratios ranging from 20:2 to 14:8 led to not only acetone-free and 1,3-PDO-free IB fermentation but also enhanced IB production along with a much higher butanol content (butanol/isopropanol ratio of 1.81 with glucose vs. 2.07-6.14 with glucose-glycerol mixture). Furthermore, when the mixture of glucose and crude glycerol at the ratio of 14:8 (total concentration of 35.68 g/L) was used, high butanol/isopropanol ratio (3.44) and butanol titer (9.86 g/L) were achieved with 1.4-fold enhanced butanol yield (0.28 g/g) and productivity (0.41 g/L/h) compared to those with glucose only at pH 5.5. CONCLUSIONS: A newly isolated Clostridium sp. A1424 was able to produce butanol and isopropanol from various carbon sources. The productivity and titer of butanol and total alcohol obtained in this study were higher than the previously reported results obtained using other natural IB producers. Use of the mixture of glucose and glycerol was successful to achieve acetone-free, 1,3-PDO-free, and enhanced IB production with higher yield, productivity, and selectivity of butanol compared to those with glucose only, providing great advantages from the perspective of carbon recovery to alcohols. This notable result could be accomplished by isolating an effective IB producer Clostridium sp. A1424 as well as by utilizing glucose-glycerol mixtures.

Butyric acid production from softwood hydrolysate by acetate-consuming Clostridium sp. S1 with high butyric acid yield and selectivity.

The aim of this work was to study the butyric acid production from softwood hydrolysate by acetate-consuming Clostridium sp. S1. Results showed that Clostridium sp. S1 produced butyric acid by simultaneously utilizing glucose and mannose in softwood hydrolysate and, more remarkably, it consumed acetic acid in hydrolysate. Clostridium sp. S1 utilized each of glucose, mannose, and xylose as well as mixed sugars simultaneously with partially repressed xylose utilization. When softwood (Japanese larch) hydrolysate containing glucose and mannose as the main sugars was used, Clostridium sp. S1 produced 21.17g/L butyric acid with the yield of 0.47g/g sugar and the selectivity of 1 (g butyric acid/g total acids) owing to the consumption of acetic acid in hydrolysate. The results demonstrate potential of Clostridium sp. S1 to produce butyric acid selectively and effectively from hydrolysate not only by utilizing mixed sugars simultaneously but also by converting acetic acid to butyric acid.

Eutectic formation analysis of amino acid mixtures using molecular dynamics simulations.

The mechanism of eutectic formation was investigated via computer-aided molecular dynamics techniques based on experimental results. The CBZ group mixtures CBZ-l-Asp/d-AlaNH2 x HCl/methanol, CBZ-l-Asp/l-PheOMe x HCl/methanol, and CBZ-l-Tyr/l-ArgNH2 x 2HCl/methanol formed transparent eutectic melts. The non-CBZ group mixtures l-Asp/d-AlaNH2 x HCl/methanol, l-Asp/l-PheOMe x HCl/methanol, and l-Tyr/l-ArgNH2 x 2HCl/methanol did not form eutectic melts. According to molecular dynamics simulation results, increase in the kinetic energy values of eutectic forming mixtures was much larger than the increase in potential energy over a temperature shift from 298 to 333 K. However, the results for non-eutectic forming mixtures were reversed. The Coulomb interaction energies of eutectic forming mixtures significantly decreased, because eutectic melting can increase the mobility of molecules in the mixtures. The enhancement of molecular mobility was confirmed by increased self-diffusion constant values, and the change of solid-to-liquid phase was detected by radial distribution function results. The periodic boundary conditions for calculation of molecular dynamics were found to be reliable.

Preparation of a nanoporous CaCO3-coated TiO2 electrode and its application to a dye-sensitized solar cell.

A nanoporous CaCO3 overlayer-coated TiO2 thick film was prepared by the topotactic thermal decomposition of Ca(OH)2, and its performance as an electrode of a dye-sensitized solar cell was investigated. As compared to bare TiO2, nanoporous CaCO3-coated TiO2 provided higher specific surface area and, subsequently, a larger amount of dye adsorption; this in turn increased short-circuit current (Jsc). Furthermore, the CaCO3 coating demonstrated increased impedance at the TiO2/dye/electrolyte interface and increased the lifetime of the photoelectrons, indicating the improved retardation of the back electron transfer, which increases Jsc, open-circuit voltage (Voc), and fill factor (ff). Thereby, the energy conversion efficiency (eta) of the solar cell improved from 7.8 to 9.7% (an improvement of 24.4%) as the nanoporous CaCO3 layer was coated onto TiO2 thick films.