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 ity 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, upregulated. These results indicate that AvaKa coninduced oxidative stress likely underlies the dehydration sensitivity of DRavaKa.
Homologous recombination-based genetic engineering, or recombineering, has greatly improved the precision and flexibility of bacterial genome manipulation. However, most existing approaches still require in vitro handling of target DNA, including PCR amplification, restriction digestion, or genomic DNA extraction prior to recombination. Here, an in vivo capture system enabling rapid cloning of bacterial chromosomal segments was developed, allowing direct recovery of genomic loci into plasmid vectors without prior amplification or in vitro DNA assembly. The system is based on λ Red-mediated homologous recombination between a linear PCR-amplified plasmid backbone and chromosomal loci in Gram-negative bacteria. Evaluation using the rcsA locus in Salmonella enterica revealed a minimal homology requirement for productive recombination. Homology arms of 10-20 bp supported plasmid recovery but failed to yield detectable on-target recombinants, whereas 30-40 bp arms reproducibly enabled accurate chromosomal segment capture. Under optimized conditions, recovered clones showed high on-target fidelity as confirmed by colony PCR and phenotypic analysis. Multiple independent recombinant plasmids were further validated by whole-plasmid sequencing, confirming precise junction formation and faithful recovery of the targeted chromosomal regions. Collectively, this study establishes a practical framework for an in vivo capture system for bacterial chromosomal segments and defines key parameters required for reliable recovery of accurate recombinant plasmids.
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.
