Nonribosomal peptide synthase gene clusters for lipopeptide biosynthesis in Bacillus subtilis 916 and their phenotypic functions.

Bacillus cyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features of Bacillus strains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology. Bacillus subtilis 916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome of B. subtilis 916 contains four nonribosomal peptide synthase (NRPS) gene clusters, srf, bmy, fen, and loc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studying B. subtilis 916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activity in vitro, the strain mutated in srfAA had significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other than fen resulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion, B. subtilis 916 coproduces four families of LPs which contribute to the phenotypic features of B. subtilis 916 in an intricate way.

Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916.

Three families of Bacillus cyclic lipopeptides--surfactins, iturins, and fengycins--have well-recognized potential uses in biotechnology and biopharmaceutical applications. This study outlines the isolation and characterization of locillomycins, a novel family of cyclic lipopeptides produced by Bacillus subtilis 916. Elucidation of the locillomycin structure revealed several molecular features not observed in other Bacillus lipopeptides, including a unique nonapeptide sequence and macrocyclization. Locillomycins are active against bacteria and viruses. Biochemical analysis and gene deletion studies have supported the assignment of a 38-kb gene cluster as the locillomycin biosynthetic gene cluster. Interestingly, this gene cluster encodes 4 proteins (LocA, LocB, LocC, and LocD) that form a hexamodular nonribosomal peptide synthetase to biosynthesize cyclic nonapeptides. Genome analysis and the chemical structures of the end products indicated that the biosynthetic pathway exhibits two distinct features: (i) a nonlinear hexamodular assembly line, with three modules in the middle utilized twice and the first and last two modules used only once and (ii) several domains that are skipped or optionally selected.

Bacillomycin L and surfactin contribute synergistically to the phenotypic features of Bacillus subtilis 916 and the biocontrol of rice sheath blight induced by Rhizoctonia solani.

The antagonistic activity of lipopeptides in Bacillus subtilis 916 has been well documented, yet relatively little is known about their mechanism in biofilm formation and environmental colonization. This study sought to examine the interaction of B. subtilis 916 on Rhizoctonia solani-infected rice sheath to elucidate the mechanism of colonization on plant leaves. Results showed that the mutants Δbac, Δsrf, and Δsrf + bac of B. subtilis 916, deficient in bacillomycin L and surfactin production, respectively, not only altered colony morphology but also changed swarming motility, reduced antagonistic activity, and decreased biofilm formation. In particular, biofilm formation in mutant Δbac, not Δsrf or Δsrf + bac, were restored with addition of surfactin and bacillomycin L at 10 and 50 µg/mL, respectively. Moreover, surfactin and bacillomycin L were able to restore or enhance swarming motility in the corresponding mutants at 10 µg/mL, respectively. With the aid of green fluorescent protein tagging, it was demonstrated that B. subtilis 916 formed a robust biofilm on the rice sheath blight lesion and colonized well on R. solani-infected rice sheath, while its corresponding mutants performed poorly. These observations also correlated with the rice cultivar pot experiments, in which B. subtilis 916 exhibited greater biocontrol than its mutants. Our results suggest that surfactin and bacillomycin L contribute differently but synergistically to the biocontrol of rice sheath blight in B. subtilis 916 through its antifungal activity, biofilm formation, and colonization.

Improved YOLOv5-based for small traffic sign detection under complex weather.

Traffic sign detection is a challenging task for unmanned driving systems. In the traffic sign detection process, the object size and weather conditions vary widely, which will have a certain impact on the detection accuracy. In order to solve the problem of balanced detecting precision of traffic sign recognition model in different weather conditions, and it is difficult to detect occluded objects and small objects, this paper proposes a small object detection algorithm based on improved YOLOv5s in complex weather. First, we add the coordinate attention(CA) mechanism in the backbone, a light-weight yet effective module, embedding the location information of traffic signs into the channel attention to improve the feature extraction ability of the network. Second, we exploit effectively fine-grained features about small traffic signs from the shallower layers by adding one prediction head to YOLOv5s. Finally, we use Alpha-IoU to improve the original positioning loss CIoU, improving the accuracy of bbox regression. Applying this model to the recently proposed CCTSDB 2021 dataset, for small objects, the precision is 88.1%, and the recall rate is 79.8%, compared with the original YOLOv5s model, it is improved by 12.5% and 23.9% respectively, and small traffic signs can be effectively detected under different weather conditions, with low miss rate and high detection accuracy. The source code will be made publicly available at https://github.com/yang-0706/ImprovedYOLOv5s .

Loss of GltB Inhibits Biofilm Formation and Biocontrol Efficiency of Bacillus subtilis Bs916 by Altering the Production of γ-Polyglutamate and Three Lipopeptides.

AIMS: This study examined the contribution of GltB on biofilm formation and biocontrol efficiency of B. subtilis Bs916. METHODS AND RESULTS: The gltB gene was identified through a biofilm phenotype screen and a bioinformatics analysis of serious biofilm formation defects, and then a gltB single knockout mutant was constructed using homologous recombination. This mutant demonstrated severe deficits in biofilm formation and colonisation along with significantly altered production ofγ-polyglutamate (γ-PGA) and three lipopeptide antibiotics (LPs) as measured by a transcriptional analysis of both the wild type B. subtilis Bs916 and the gltB mutant. Consequently, the mutant strain retained almost no antifungal activity against Rhizoctonia solani and exhibited decreased biocontrol efficiency against rice sheath blight. Very few gltB mutant cells colonised the rice stem, and they exhibited no significant nutrient chemotaxis compared to the wild type B. subtilis Bs916. The mechanism underlying these deficits in the gltB mutant appears to be decreased significantly in production of γ-PGA and a reduction in the production of both bacillomycin L and fengycin. Biofilm restoration of gltB mutant by additionγ-PGA in the EM medium demonstrated that biofilm formation was able to restore significantly at 20 g/L. CONCLUSIONS: GltB regulates biofilm formation by altering the production ofγ-PGA, the LPs bacillomycin L and fengcin and influences bacterial colonisation on the rice stem, which consequently leads to poor biocontrol efficiency against rice sheath blight. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of a key regulatory protein (GltB) that is involved in biofilm regulation and its regulation mechanism and biocontrol efficiency by B. subtilis.

Relationships between 25-hydroxyvitamin D and nocturnal enuresis in five- to seven-year-old children.

BACKGROUND: Vitamin D has been recognized to contribute to various physiological processes. However, no study has investigated serum 25-hydroxyvitamin D [25(OH)D] concentrations in children with nocturnal enuresis (NE) in the English literature. OBJECTIVE: In the present study, serum 25(OH)D concentrations were measured in five- to seven-year-old children with NE and compared with those in non-enuretic children to investigate whether there was any relationship between 25(OH)D and NE as the first time in the literature. DESIGN: Two hundred forty-seven five- to seven-year-old children were recruited from Taizhou, Zhejiang Province, China. Serum 25(OH)D concentrations were measured, and the structured questionnaire was administered to the parents of all children. Low 25(OH)D was defined as serum 25(OH)D concentrations below 20 ng/ml. RESULTS: The prevalence of NE was 7.3% in the group of children with 25(OH)D concentrations that exceeded 20 ng/ml; this prevalence was much lower than the 17.5% observed in the group of children with 25(OH)D concentrations below 20 ng/ml (p<0.05). After adjusting for potential confounders, serum 25(OH)D (≥20 ng/ml) was significantly associated with NE and represented a protective factor against NE (OR = 0.31, 95%CI = 0.092, 1.0, P<0.05). A nonlinear relationship between 25(OH)D and NE was observed. The prevalence of NE decreased with increasing 25(OH)D concentrations above 19 ng/ml. Additionally, children exhibiting higher frequencies of bedwetting had lower 25(OH)D concentrations [5-7 times/week: 18.3±4.8; 2-4 times/week: 20.9±4.1; 0-1 times/week: 23.6±6.4 (ng/ml), P<0.05)]. CONCLUSIONS: Low 25(OH)D was associated with an increased risk of NE in children aged five to seven years.