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 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.

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.

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.

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.