The isolation efficiency of Salmonella enterica using selective enrichment media varies depending on culture conditions. Tetrathionate broth (TTB) and selenite broth (SB) differ in selectivity and growth support, yet practical guidance on optimizing their use remains limited. To identify appropriate conditions for improving recovery, we first conducted a spike test in which a defined amount of S. enterica was added to Salmonella negative cattle feces. TTB showed improved isolation rates when the inoculum size was reduced and the incubation period was extended, indicating that each factor independently contributed to recovery. In contrast, SB exhibited enhanced recovery when a smaller inoculum was combined with an incubation period appropriate for that inoculum size. We then validated these observations using naturally contaminated chicken liver, pig feces, and crow droppings. After pre‑enrichment, samples were inoculated into TTB, SB, modified semi‑solid Rappaport agar for stab culture (MSRAsc), and Rappaport-Vassiliadis broth under various culture conditions. Although results varied among sample types, TTB and SB generally showed improved isolation rates when cultured under the optimized conditions. MSRAsc achieved the highest overall recovery, but some strains, likely sensitive to dyes, failed to grow. These results indicate that although optimizing TTB and SB enhances isolation efficiency, no single medium is sufficient to recover a broad range of serovars. Therefore, a combined approach using dye‑free TTB alongside MSRAsc is recommended, and SB should also be used when strains exhibiting poor growth in other selective media are suspected.
Bacillus subtilis var. natto is a naturally occurring biotin auxotroph widely used in traditional soybean fermentation. In this study, we evaluated its potential as a microbial biotin biosensor without genetic modification. Using a two-layer agar diffusion assay, growth responses were examined across biotin concentrations ranging from 0.001 to 200 mg/L. Halo diameters increased proportionally with biotin concentrations. The strain exhibited detectable responses over more than five orders of magnitude of biotin concentration without engineering of biotin transport or biosynthetic pathways. These results demonstrate that B. subtilis var. natto can serve as a simple, safe, and cost-effective biotin bioassay platform and highlight the utility of naturally evolved auxotrophic microorganisms as biosensors.
Bacillus subtilis var. natto is distinguished from other B. subtilis strains by the inability to synthesize biotin. To investigate the evolutionary origin and functional consequences of this auxotrophy, we performed a comparative genomic analysis across 35 Bacillus genomes. Loss-of-function mutations in bioW and bioF were restricted to natto and closely related food-associated strains, whereas genes involved in biofilm formation and sporulation remained strongly conserved. To assess the functional effect of biotin availability, we constructed a biotin prototrophic natto strain by restoring the bio operon. Flow cytometric analysis revealed that the auxotrophic strain initiated sporulation earlier, reaching a stable spore ratio of 40% by 24 hours, whereas the prototrophic strain showed delayed but more complete sporulation (>96% by 48 hours). These results suggest that intracellular biotin availability influences sporulation kinetics and that biotin auxotrophy in natto-associated lineages may be a niche adaptation that inadvertently shaped fermentation-associated phenotypes.
To produce new types of sake with a fruity aroma, imparted mainly by ethyl caprylate, a novel cerulenin-resistant strain, NSYC8, was isolated. NSYC8 produces high levels of caprylic acid, the precursor of ethyl caprylate, from the sake yeast, Saccharomyces cerevisiae NSY. An FAS2-1280F mutant was constructed from S. cerevisiae strain, Kyokai S-2, using a self-cloning method. The mutant produced 1.9-fold more caprylic acid than Kyokai S-2. Ethyl caprylate was detected in sake produced by the mutant, but not in sake produced by Kyokai S-2. The mutation enhanced production of caprylic acid and ethyl caprylate, facilitating the improvement of sake yeast strains.
The photosynthetic reaction center (RC) of green sulfur bacteria is divided into two parts: the membrane-embedded RC core (RCC) complex (comprising PscA and PscC) and the water-soluble peripheral subunits (comprising the trimeric Fenna-Matthews-Olson [FMO] protein, PscB, and PscD). The peripheral subunits are bound onto the cytoplasmic surface of the RCC complex, and no method has been identified so far to detach the peripheral subunits to maintain the functionality of the RCC complex. The present study proposes biochemical treatments enabling the reversible dissociation of the peripheral subunits. A pH increased to 11.0 effectively removed almost all the peripheral subunits from the RCC complex. NaCl of more than 1 M destabilized their binding; however, the degrees of impact differed between each subunit. Approximately half of FMO and PscB remained on the RCC complex, whereas PscD demonstrated near-complete dissociation. Continuous washing of the detached subunits led to the near-complete dissociation of all subunits, even when treated with 1 M NaCl. The peripheral subunits under the detaching conditions were in an equilibrated state between the bound and unbound states. Neutralization and desalting after detaching treatments reassembled all the dissociated subunits and recovered the charge-separation activity comparable to that before treatment. Insights from atomic resolution structures indicate that electrostatic interaction with PscA stabilized the binding of the peripheral subunits on the RCC complex.
Osmotolerant rhizobacteria, Bacillus altitudinis TR-19, has been subjected to osmotic stress by cultivating the cells in: (1) Luria Bertani (LB) broth medium, (2) LB broth supplemented with 1.8M NaCl, and (3) LB broth supplemented with 20% glucose incubated at 370C with shaking. Samples were drawn periodically for growth analysis and metabolome profile. The results demonstrated that cultivation of the osmotolerant rhizobacteria in 1.8M NaCl-supplemented LB medium and 20% glucose-supplemented LB medium prolonged the adaptation growth phase, suggesting that NaCl at 1.8 M and 20% glucose reduced cell growth rate, although the cells were still capable of growing at such high osmotic stress. Metabolites synthesised under different growth conditions, in rich media (LB broth) and under osmotic stress (1.8M NaCl-supplemented LB medium and 20% glucose-supplemented LB medium) also demonstrated striking differences in their profile. Under osmotic conditions in NaCl, much less metabolites were found than under non-stress condition, suggesting the changes in metabolites synthesis pathway. Under 20% glucose stress conditions, by using 20% glucose-supplemented LB medium, the number of metabolites were also found reduced but more metabolites were observed than under NaCl stress. Different metabolome profile under non-stress conditions, NaCl-stress, and glucose stress were also observed, suggesting the changes in the metabolic pathway. One of the metabolite synthesised under osmotic condition was betaine which is known as the derivative of amino acid glycine. Betaine was synthesised under NaCl and glucose stress. Under non-stress condition, choline was not detected, suggesting that betaine and choline are important compatible solutes under NaCl stress.
Acetylcholine (ACh) is widely recognized as a neurotransmitter in animals; however, its potential relevance to microbial physiology remains largely unexplored. Here, we report that the BetI regulatory system of Escherichia coli, previously characterized as a choline-responsive osmoprotective pathway, exhibits a measurable transcriptional response to extracellular ACh. Using a recombinant reporter strain, we demonstrate that ACh induces BetI-dependent gene expression and that the shape and sensitivity of the response curve are markedly altered by genetic perturbations of pathway components, including BetT overexpression and BetA deletion. While the underlying molecular mechanism remains unresolved, these results establish that E. coli can functionally respond to ACh at the whole-cell level. Given that ACh is readily hydrolyzed into metabolites relevant to microbial metabolism and is supplied in animal tissues from sources beyond neurons, our findings suggest that acetylcholine responsiveness may be more widespread among host-associated microorganisms than previously appreciated.
Plastids retain their own genomic DNA (plastid DNA, ptDNA) which must be faithfully maintained for proper organelle function. However, the molecular mechanisms responsible for ptDNA maintenance remain poorly understood, particularly in red algae. Here, we identified a plastid-targeted RecQ-like helicase (ptRECQ) in the unicellular red alga Cyanidioschyzon merolae. Biochemical assays demonstrated that ptRECQ possesses ATP-dependent helicase activity and preferentially unwinds splayed DNA substrates that mimic replication fork structures. ptrecQ mutant cells were hypersensitive to nalidixic acid, a ptDNA gyrase inhibitor that induces replication stress, indicating a role for ptRECQ in plastid genome maintenance under replication stress. Phylogenetic analyses showed that ptRECQ is broadly conserved in red algae, absent from green algae and land plants, and phylogenetically distinct from cyanobacterial RecQ proteins. These results suggest that red algae employ a ptDNA maintenance system that differs from that of the green lineage.
Narezushi is a traditional Japanese fermented food made from fish and rice. Nezushi is a regional variety of izushi-style narezushi produced in the Hida (Gero) and Oku-Mino (Gujo) regions of Gifu Prefecture. In this study, nezushi was analyzed to clarify its bacterial communities using both culture-dependent and culture-independent approaches. Nezushi (Gero) was dominated almost exclusively by Latilactobacillus sakei, whereas nezushi (Gujo) showed greater diversity with Latilactobacillus sakei and Leuconostoc lactis. Izushi (Tokachi) closely resembled nezushi (Gero), while izushi (Ishikari) resembled nezushi (Gujo). In contrast, kabura‑zushi (Kanazawa) exhibited a distinct community dominated by Leuconostoc spp. These results indicate that psychrotrophic lactic acid bacteria, primarily Latilactobacillus sakei and Leuconostoc spp., drive fermentation in izushi‑style narezushi, while regional processing conditions shape microbial composition. This study provides the first microbiological characterization of nezushi, revealing previously unrecognized features of its fermentation ecology.
This study systematically elucidated the microbial community succession and functional gene dynamics during the postharvest spoilage process of Stropharia rugosoannulata by integrating physiological and biochemical indicators with metagenomic analysis. The experimental results demonstrated that as storage time extended, the activities of antioxidant enzymes (superoxide dismutase, peroxidase) in S. rugosoannulata significantly declined, while the content of membrane lipid peroxidation product malondialdehyde increased, leading to compromised cell membrane integrity and creating favorable conditions for microbial colonization. Metagenomic analysis revealed that during the spoilage phase (post-harvest day 14), the relative abundance of Pseudomonadota increased to 85.7%, with Pseudomonas replacing Ewingella as the absolutely dominant microbial population. Further functional gene analysis showed that the post-harvest day 14 exhibited significant enrichment of glycosyltransferases (GT0, GT1, GT2, GT4) and carbohydrate-binding modules (CBM10, CBM16, CBM50), along with pectinase (GH78), chitinase (GH19), and polysaccharide-modifying enzymes (CE4, CE11). This indicated a metabolic shift towards cell wall synthesis and substrate recognition. In contrast, the post-harvest day 7, prior to fruiting body softening, demonstrated high expression of glycoside hydrolases (GH1, GH2, GH4, GH94) and carbohydrate esterase CE8, focusing on the degradation of cellulose and starch. These findings, for the first time from a molecular ecology perspective, clarify that the essence of postharvest spoilage in S. rugosoannulata is a quality deterioration process driven by a Pseudomonas-dominated microbial community. The study provided a basis for the development of targeted antibacterial preservation strategies.
Lactic acid bacteria (LAB) are widely recognized for their health benefits and are commonly incorporated into functional foods. However, their survival and metabolic performance depend on the availability of appropriate substrates. This study evaluated the ability of LAB isolated from Dioscorea species to utilize starch from Dioscorea luzonensis, an endemic plant in the Philippines. Among the isolates tested, Limosilactobacillus fermentum PJG11 demonstrated the highest efficiency in converting D. luzonensis starch into glucose. This enhanced starch utilization can be explained by the upregulation of the α-amylase gene when the strain was cultured with its natural substrate, resulting in accelerated starch granule degradation. These findings underscore the importance of using natural, plant-derived substrates in the development of LAB-based functional foods to support optimal bacterial activity and improve carbohydrate breakdown for enhanced nutritional value.
Sanghuangporus baumii polysaccharides (SBP) are recognized for their valuable pharmacological activities, driving increasing interest in their medicinal potential. However, the biosynthetic pathway of SBP remains incompletely characterized. Phosphoglucose isomerase (PGI), a key enzyme in carbohydrate metabolism, catalyzes the reversible isomerization between glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P) and is hypothesized to regulate polysaccharide biosynthesis in this fungus. In this study, the pgi gene from S. baumii (sbpgi) was cloned and created sbpgi-silenced mutants using RNA interference (RNAi) to investigate its function. Silencing sbpgi resulted in an approximately 20% reduction in mycelial biomass but concurrently enhanced the production of exopolysaccharide (EPS) and intracellular polysaccharide (IPS) by approximately 2.0-fold and 1.9-fold after 9 days, respectively. Furthermore, suppression of sbpgi expression markedly decreased the content of cell wall β-1,3-glucan (by ~23%) while increasing chitin deposition by about 1.7-fold, leading to alterations in cell wall architecture, including thickness, and changes in stress tolerance. Transcriptional analysis revealed that sbpgi silencing significantly upregulated the expression of key genes in the polysaccharide biosynthetic pathway, including ugpg and pmm, highlighting the critical regulatory role of sbpgi in polysaccharide production. Our findings provide a foundation for metabolic engineering strategies to develop high-yielding strains for the industrial production of SBP.
Water is indispensable to life, yet some cyanobacteria inhabit hyper-arid deserts and withstand complete desiccation. However, the molecular mechanisms enabling such acclimation remain incompletely understood. In Anabaena sp. strain PCC 7120, the gene avaKa, which encodes a protein of unknown function, has been shown to be required for desiccation tolerance. Here, we characterized the avaKa disruptant DRavaKa under dehydration-related stress conditions. DRavaKa displayed hypersensitivity to EDTA, and transcripts of iron-deficiency-inducible genes (isiA1, isiB, furA, and sufB) were elevated even in the absence of EDTA. Whole-cell absorption spectra of DRavaKa revealed a blue shift of the chlorophyll absorption peak, a characteristic feature of iron-deficient cyanobacteria. In addition, the oxidative-stress-inducible gene trxA2 was likewise upregulated. These results indicate that AvaKa contributes to iron homeostasis and that iron deficiency-induced oxidative stress likely underlies the dehydration sensitivity of DRavaKa.
Bacillus thuringiensis (Bt) has broad spectrum multipotent functionalities for pest and disease suppression, and growth promotion (PGP) of plants. Therefore, potency of 27 rice rhizospheric and 2 commercial Bt isolates was assessed for biocidal and PGP traits. Functionally rhizospheric Bts were broadly superior than commercial Bts. Virulence of the Bts varied against rice leaf folder (LF, Cnaphalocrocis medinalis) and stripe stem borer (SSB, Chilo suppresalis) larvae in laboratory, net house and field tests. Drosophila diet (DD) incorporation, cut leaf and field assays proved virulence of 5-9 Bt isolates against LF larvae with LC50s 1.99 - 6.31 x 108, 2.18 x 106 - 2.25 x 109 and 3.16 x 106 - 1.25 x 109 bacteria-spore-crystal (BSC)/ml, respectively, and TB261 was most (LC50s 2.18 x 106 - 3.98 x 108 BSC/ml) infective. DD and cut stem assays for SSB proved virulence of 5 and 6 Bts with LC50s 9.20 x 106 - 3.62 x 108 and 9.21 x 106- 3.24 x 108 BSC/ml, respectively, and maximum (LC50s 9.20 - 9.21 x 108 BSC/ml) infectivity of TB263. Eight Bts inhibited 1-4 out of 7 rice pathogens and 16 Bts antagonized 1-4 out of 9 entomopathogenic fungi. Biocidal principles of the Bts were cell wall/membrane hydrolyzing exoenzymes, toxin/inhibitors and crystal toxins. Furthermore, the Bts were also inhibited by 3 insecticides and 2 fungicides. The Bts possessed 1-4 PGP and phytostimulation traits also. The potent rhizospheric Bt can be prospected for overall improvement/sustenance of rice.
Plastics are indispensable in modern society, but their increasing production and disposal pose serious environmental challenges, including pollution and the depletion of non-renewable resources. Poly(ethylene furanoate) (PEF), a bio-based polyester composed of ethylene glycol and 2,5-furandicarboxylic acid (FDCA), is attracting attention as a sustainable alternative to poly(ethylene terephthalate) (PET). In this study, we developed a fully biotechnological upcycling system for PEF. Our approach involved enzymatic depolymerization of PEF to release FDCA, followed by microbial conversion of FDCA into polyhydroxyalkanoate (PHA), a biodegradable polyester. From soil samples enriched with FDCA as the sole carbon source, we isolated two bacterial strains: Pseudomonas sp. S8-1 and Caballeronia sp. S8-5. These strains produced medium-chain-length and short-chain-length PHAs, respectively, in defined medium containing FDCA. For enzymatic depolymerization, we employed the thermostable ICCG variant (F243I/D238C/S283C/Y127G) of leaf-branch compost cutinase, known for its high PET-degrading activity. The depolymerization of PET by this enzyme was enhanced by the addition of calcium carbonate (CaCO3) powder to suppress acidification. Furthermore, the enzyme retained high activity even after partial purification by heat treatment at 60°C and efficiently depolymerized PEF as well. Finally, the PEF degradation solution was successfully utilized as a carbon source for PHA production by strain S8-5. These results demonstrate a proof-of-concept biorecycling system for PEF and represent a first step toward sustainable plastic management.
An efficient bacterial consortium (designated BPA-1), comprising Bacillus subtilis SX-6, Pseudomonas sp. SX-10, and Georgenia sp. SY-1, was successfully constructed for the decolorization of the azo dye Congo Red (CR). BPA-1 exhibited significant thermotolerance and heavy metal resistance, achieving over 90% CR decolorization within 60 h at 47°C under co-stress conditions with Zn²⁺, Mn²⁺, and Pb²⁺ (50 mg/L each). The consortium demonstrated broad substrate specificity, effectively decolorizing 12 structurally diverse azo dyes. Enzymatic assays revealed the involvement of laccase, manganese peroxidase, lignin peroxidase, and azoreductase in CR biodegradation. Metabolic pathway analysis indicated a three-stage degradation mechanism: (1) Asymmetric cleavage of azo bonds (-N=N-) generated 4,4'-diazaldenylbiphenyl and 4-amino-1-naphthalenesulfonic acid (Intermediate II); (2) Deamination converted Intermediate II to 3,4-dihydroxy-1-naphthalenesulfonic acid, followed by desulfurization to form naphthalene-1,2,3,4-tetraol; (3) Complete mineralization of intermediates occurred through subsequent oxidative steps. Notably, 4,4'-diazaldenylbiphenyl was further transformed into 4,4'-diaminobiphenyl, confirming the consortium's capacity for multi-step detoxification.
Superoxide dismutases (SODs) play crucial roles in protecting cells against oxidative stress by catalyzing the dismutation of superoxide radicals. In Aspergillus nidulans, five putative SOD genes have been predicted in the genome; however, their comparative expression profiles and physiological functions remain largely uncharacterized. In this study, we analyzed the expression levels of all five SOD genes at different growth stages and examined the oxidative stress sensitivity of corresponding gene-disrupted strains. We found that sodA exhibited high and constitutive expression across all growth stages, while sodB was predominantly expressed in conidia (asexual spores). Disruption mutants of sodA and sodB showed increased sensitivity to oxidative agents, confirming their functional importance. Subcellular fractionation and SOD activity assays revealed that SodA was localized in the cytoplasm, whereas SodB was primarily localized in mitochondria. These results highlight the growth stage-specific expression and distinct cellular roles of SodA and SodB in A. nidulans, providing novel insights into the oxidative stress defense system in filamentous fungi.
Superoxide dismutases (SODs) play crucial roles in cellular oxidative stress defense. In Aspergillus nidulans, SodB is a mitochondria-localized SOD whose physiological function remains poorly understood. Here, we show that a ΔsodB mutant displays impaired growth on non-fermentable carbon sources including acetate, ethanol, threonine, and Tween 20/80, suggesting compromised mitochondrial function. Oxygen consumption assays using an extracellular oxygen consumption reagent revealed a ~50% reduction in respiratory activity in the ΔsodB strain compared to the wild type. When mitochondrial respiration was inhibited by Antimycin A or salicylhydroxamic acid, giant colony growth was equally suppressed across wild-type, ΔsodA, ΔsodB, and complemented strains. However, conidial production was significantly reduced in ΔsodB under Antimycin A treatment, and morphological abnormalities in conidiophore heads were observed under this condition. These results indicate that SodB is not only involved in mitochondrial respiration but also required for maintaining normal sporulation under mitochondrial stress conditions. This study provides new insights into the role of mitochondrial ROS defense systems in filamentous fungal development.
At the 2025 Osaka/Kansai Expo, a bacterial-bioluminescence-based lighting system, called BIOLIGHT, was exhibited. It consists of 80 liters of liquid culture medium and produces enough brightness to illuminate a room. In this study, to make clear the relationship between the liquid culture thickness and the brightness using BIOLIGHT, the world's largest liquid culture aquarium of bioluminescent bacteria, we investigated the brightness of the bacterial liquid culture in relation to optical density (OD). The theoretical brightness of BIOLIGHT was calculated using the transmittance of the liquid culture at 475 nm (the peak luminescence wavelength) derived from the measured OD and was then compared with the brightness actually measured. The calculated (theoretical) brightness was lower than the measured one, suggesting that the light output of BIOLIGHT is influenced not only by cell-induced light shielding but also by another factor, presumably forward scattering. Additionally, depth-dependent brightness measurements showed that brightness became saturated at a liquid culture thickness greater than 7 cm. These findings will contribute to the design of future lighting solutions using bacterial bioluminescence.
Methionine gamma-lyase enzyme was isolated and purified from Mucor irregularis PQ344458 fungal isolates, that obtained from plant root, the isolates were identified through observation of their colony morphological features, implementation of PCR and DNA sequencing via sanger-chain termination approach, then data of DNA sequence alignment, phylogenetic tree, percent identity was generated. Through implementation of several stages that involved using of ion-exchange chromatography, gel-filtration chromatography, ammonium sulphate, enzyme isolation and purification stages were accomplished. The enzyme extract then, was analyzed for its protein content, specific activity and Impact of pH, temperature, inhibitors and activators on its kinetics. Additionally, MTT and DPPH radical scavenging assays were carried-out to reveal information about anti-cancer and anti-oxidant activities of methionine gamma-lyase enzyme. MTT assay results of %viable cells were 15% for HeLa cells and 6.6% for U937 cells at maximum concentration of the enzyme extract. Moreover, DPPH scavenging activity results were 82% at maximum concentration.
