Kenji Yokota

Ligilactobacillus ruminis is a flagellated lactic acid bacterium found in the intestines of various mammals, including humans. Although this species harbors a complete flagellar gene cluster, flagella formation has not been observed in human-derived strains, and the underlying regulatory mechanisms remain unknown. Here, we isolated a motility-acquired mutant of L. ruminis ATCC 25644 that exhibited full flagellation and a measurable chemotactic response under acidic conditions (pH 3.0). Whole-genome sequencing revealed a ~35 kb deletion encompassing multiple regulatory genes. Functional complementation identified a single response regulator, designated FpsR (flagellation-piliation switchover regulator), as a central switch that suppresses flagella formation while promoting pilus expression. The motility-acquired mutant displayed reduced pilus production, diminished adhesion to murine intestinal mucus and fibronectin, and increased susceptibility to acid (pH 3.0) and bile (0.25-0.5%), resulting in a complete loss of intestinal colonization in a murine model. Furthermore, while flagellin from the motile strain activated TLR5 and induced proinflammatory responses comparable to those of pathogenic bacteria, no such inflammation was observed in vivo, likely due to the strain's colonization failure. These findings reveal FpsR as a previously unrecognized genetic mechanism that coordinates motility and mucosal colonization in a human commensal bacterium and provide insight into how flagella are regulated and silenced in the gut environment to support host-microbe symbiosis.

Bacillus velezensis TCG15 is a bacterial isolate from soil that shows antifungal activity against Aspergillus spp. and Candida spp. This study aimed to characterize its antifungal activity. TCG15 produces cyclic lipopeptides (cLPs), including surfactin A, plipastatin, and bacillomycin L. We obtained the complete sequence of the operon encoding nonribosomal peptide synthetase for bacillomycin L biosynthesis, bmyL operon (bmyLDABC), using genome analysis of TCG15. The bmyLD gene-disrupted mutant did not produce bacillomycin L, plipastatin, or surfactin A. The mutant showed no antifungal activity, indicating an essential role of cLPs. Among the cLPs, bacillomycin L was identified as the key antifungal compound. This activity was enhanced by surfactin A, which showed no antifungal activity alone. These findings suggested that the synergistic interaction between bacillomycin L and surfactin A contributed to the antifungal activity of TCG15.

Surfactin is a cyclic lipopeptide produced by Bacillus spp., consisting of a β-hydroxy fatty acid and a heptapeptide synthesized by non-ribosomal peptide synthetases. Surfactin congeners (A, B, and C) differ in amino acid substitutions, with Leu7 in surfactin A replaced by Val in B and Ile in C. Our LC–MS analysis revealed that the elution profiles of surfactin-producing strains could be classified into two distinct patterns under identical culture conditions, corresponding to surfactin A and C production. This suggests that endogenous factors influence surfactin production. Therefore, we aimed to identify the genetic factor that regulates surfactin congener production. The srfA operon for surfactin A biosynthesis in B. subtilis, composed of four open reading frames (ORFs), is srfAABCD. Comparative genomic analysis between the B. subtilis JCM 1465 srfA operon and the TUA12 surfactin biosynthesis genes examined in this study revealed that the operon responsible for surfactin A biosynthesis is distinct, exhibiting 68.7%, 69.2%, 84.7%, and 67.4% homology with the four ORFs, respectively. Similarly, the operon for Ptrs2 surfactin C biosynthesis showed 68.7%, 69.2%, 64.4%, and 67.1% homology. These differences indicate that the identified surfactin A and C biosynthetic operons are novel genetic variants. Further analysis identified the adenylation domain responsible for selecting Ile7 in surfactin C via domain substitution in a surfactin A-producing strain. Average nucleotide identity analysis showed that the surfactin A and C operons were found in B. velezensis and B. amyloliquefaciens, respectively. Our findings suggest that surfactin congener production is species-dependent, with the srf operon specifically distributed in Bacillus spp.

Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of brown blotch disease of mushrooms, and are the toxins that cause the brown spots. We previously reported that M...

Many flagellated bacteria possess multiple flagellins, but the roles and the compositions of each flagellin are diverse and poorly understood. In Ligilactobacillus agilis BKN88, there are two active flagellin gene paralogues but their function and composition in its flagellar filaments have not been described. The aim of this study is to find the function and composition of the flagellins by employing mutant strains each of which expresses a single flagellin or a modified flagellin. Two single flagellin-expressing strains were both flagellated while the number of flagella per cell in the single flagellin-expressing derivatives was lower than that in the wild type. Nonetheless, these derivative strains were apparently equally motile as the wild type. This indicates that either flagellin is sufficient for cell motility. The immunological activity via Toll-like receptor 5 of the single flagellin-expressing strains or purified single flagellins was readily detectable but mostly variably weaker than that of the wild type. The flagellar filaments of wild type L. agilis BKN88 were more acid-/thermo-stable than those of single flagellin-expressing derivatives. Using a combination of immunoprecipitation and flagellin-specific staining, wild type BKN88 appeared to possess heteropolymeric flagellar filaments consisting of both flagellins and each flagellin appeared to be equally distributed throughout the filaments. The results of this study suggest that the two flagellins together form a more robust filament than either alone and are thus functionally complementary.

Tolaasins are lipodepsipeptides secreted by Pseudomonas tolaasii, the causal agent of bacterial blotch on several kinds of cultivated mushrooms. Our previous study reported on tolaasin detoxification by Microbacterium sp. K3-5 as a potential biocontrol of the disease. In this study, the tolaasin-detoxifying activities of various type strains of Microbacterium spp. were evaluated through chemical and biological assays. The bacterial cells of all tested strains of Microbacterium spp. showed tolaasin I-elimination from liquid phase. However, the toxin activities of tolaasins were still retained on the tolaasin-treated bacterial cells of all Microbacterium strains except M. foliorum NBRC 103072^T. Furthermore, intact tolaasin I was recovered from the tolaasin-treated bacterial cells of all tested strains except M. foliorum NBRC 103072^T. Our data reveal that Microbacterium spp. can be characterized as effective tolaasin I-eliminating bacteria through cell adsorption, but that this adsorption alone is insufficient for actual tolaasin detoxification. The biological degradation process must be needed to carry out the detoxification.

Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.

Iturin A and surfactin are antimicrobial lipopeptides produced by antagonistic Bacillus spp. We herein demonstrated that both lipopeptides amended the soil-mediated suppression of the soil-borne disease, Fusarium yellows of tatsoi (Brassica rapa var. rosularis). Significant disease suppression was conferred by the amendments of purified iturin A or surfactin to soil. However, an excess amount of iturin A or surfactin to soil resulted in the loss of disease suppression activity.