The filamentous sulfur-oxidizing bacterium Thiothrix nivea produces glucosaminoglucan (GG), which is β-1,4-linked copolymer of glucose and glucosamine. GG is a major component of the sheath which covers the bacterial filament, and the bacterium uses CO2 and organic acids as carbon sources in the presence of H2S. Similar to chitin, N-acetylated GG (NGG) can be degraded by lysozyme. However, it has not yet been confirmed whether NGG can be degraded by chitinase. Glycoside hydrolase 18 (GH18) chitinase is present in mammals and is produced by various microorganisms. In this study, the degradation of NGG by GH18 chitinase was investigated. When GH18 chitinase was added to the NGG suspension, a decrease in turbidity and an increase in reducing power were observed, indicating that it can degrade NGG. HPLC analysis and Nuclear magnetic resonance spectroscopy revealed that the enzyme degrades NGG into a specific disaccharide β-Glc-(1→4)-GlcNAc. Accordingly, it was found that GH18 chitinase hydrolyzes the GlcNAc→Glc linkage but not the Glc→GlcNAc linkage. The NGG degradation rate was much lower than that of chitin. Hence, NGG was recognized as a material with better biological durability than that of chitin. NGG-chitin composites with different NGG contents were prepared, and as the NGG content increased, the susceptibility of the hydrogel to chitinase decreased. Importantly, this study proposes a novel method for controlling the stability of chitin hydrogels using GH18 chitinase and enhances current understanding of the enzyme.

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.

Hfq is an RNA chaperone conserved across diverse bacterial lineages that regulates gene expression by facilitating the pairing of small RNAs with their target mRNAs. It influences both the translation and stability of specific transcripts, thereby contributing to complex post-transcriptional regulatory networks. We have previously shown that overproduction of Hfq inhibits cell division by suppressing the expression of the essential cell division protein FtsZ. Based on this observation, we developed a novel antibiotic screening system using an Escherichia coli strain harboring an IPTG-inducible hfq gene to identify compounds that interfere with Hfq-mediated RNA metabolism. We tested representative inhibitors of DNA synthesis, protein synthesis, and cell wall synthesis; however, none restored colony formation. In contrast, rifampicin, an inhibitor of RNA polymerase, restored colony formation, as expected for a compound interfering with Hfq function. Sublethal concentrations of rifampicin restored colony formation of the Hfq-overproducing cells. Western blot analysis revealed that Hfq expression levels decreased upon rifampicin treatment, indicating that rifampicin was a false-positive hit. To eliminate such false-positive hits, a rifampicin-resistant rpoB mutation was introduced into the indicator strain. As expected, rifampicin did not restore growth of the rifampicin-resistant rpoB strain. This assay provides a useful platform for identifying compounds that interfere with Hfq-mediated RNA metabolism.

Biological pre-treatment of lignocellulosic waste is crucial for efficient fermentation in second-generation biofuel production. Halophilic bacteria produce glycosyl hydrolases with enhanced stability for industrial application. This study aimed to characterise putative glycosyl hydrolases (cellulases and hemicellulases) encoded in the genome of the halophilic bacterium Meridianimaribacter sp. CL38 using bioinformatic approaches, in order to address the scarcity of structural information for this genus. Four putative glycosyl hydrolases (GH9, GH3, GH144 and GH16) were analysed, focusing on their functional domains and structural properties. Phylogenetic analysis indicated a close relationship with other members of the Flavobacteriaceae family. Structural analyses revealed significant predicted halophilic and thermostable properties, suggesting these putative enzymes may endure industrial conditions. This study provides insights into putative glycosyl hydrolase candidates for efficient lignocellulosic waste pre-treatment.

Although Bacillus subtilis natto is a highly beneficial probiotic bacterium, its genetic engineering is heavily constrained by low natural transformability. To provide a competence-independent route for DNA delivery, we developed a minimized mobilizable plasmid system. We demonstrated that the pCJ plasmid was successfully transferred into B. subtilis natto via conjugation, achieving knockout of the BSNT10618 gene, selected for its predicted role in restricting incoming DNA. Furthermore, systematic replicon evaluation revealed that a pE194-derived rolling-circle replicon maintained high transfer efficiency, enabling successful marker-free genome editing at the malS locus in the laboratory strain B. subtilis 168 via both transformation and conjugation. While demonstrated primarily in strain 168, this system provides a reliable foundation to expand future genome editing and genetic engineering applications in the industrial natto strain.

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.

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.

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.

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.

Microorganisms on the human skin and host form a close and complex community. Abnormal proliferation of certain commensal microorganisms can disrupt this balanced interaction, resulting in the development of skin diseases. Cosmetics is a significant factor affecting the balance of the skin microbiome. We focused a major commensal coagulase-negative Staphylococcus and evaluated the growth effect by five cosmetic ingredients including polyamines. Two polyamines showed notable effects, although others showed no impact effects. They significantly reduced the turbidity during the active growth phase and recovered in the stationary phase. During the death phase, they prolonged the stationary phase turbidity in a concentration-dependent manner, continued by the end of experiments. Our findings could be a previously uncharacterized effect under these experimental conditions.

Band-pass filters, which selectively transmit signals within a defined range of input magnitudes, are fundamental components of signal-processing systems. In cellular gene circuits, band-pass behavior has likewise been pursued as a mean to implement complex signal-processing functions. However, previously reported genetic band-pass circuits have typically relied not only on a large number of regulatory components but also on transcriptional cascades involving multiple transcription factors, resulting in long DNA sequences and increased circuit complexity. Here, we first propose a band-pass gene circuit that operates without transcriptional cascades. By co-expressing two variants of the transcription factor BetI that exhibit opposite input-response behaviors-one acting as an inducer-dependent activator and the other as an inducer-dependent repressor-band-pass filtering is achieved solely through differential tuning of their inducer sensitivities. This minimal architecture enables gene expression only within a specific range of intracellular choline concentrations. Furthermore, we demonstrate that this cascade-free band-pass circuit can be exploited to generate spatial expression patterns in Escherichia coli populations in response to a choline diffusion gradient, illustrating its utility for pattern formation in multicellular contexts.