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.

This study examined the contribution of GltB on biofilm formation and biocontrol efficiency of B. subtilis Bs916. 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. 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. 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.

Herein, a bactericide-secreting Bacillus strain, potentially useful as a biocontrol agent, was isolated from the commercial Yanjing Natto food. Following the biochemical and physiological evaluation, the molecular identification was performed using 16S rDNA sequencing of polymerase chain reaction-amplified DNA that confirmed the natto isolate as Bacillus subtilis natto (B. subtilis natto). The biocontrol (microbial inhibitory) capability of B. subtilis natto was investigated against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and yeast (Yarrowia lipolytica) and recorded. The antimicrobial activity of B. subtilis natto was further enhanced by optimizing the growth medium for optimal bactericides secretion. Under optimized conditions, B. subtilis natto exhibited much higher inhibitory activity against S. aureus with a zone of inhibition diameter up to 27 mm. After 48 h incubation, the optimally yielded B. subtilis natto broth was used to extract and purify the responsible bactericides by silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography. Structural identification of purified bactericides (designated as NT-5, NT-6, and NT-7) from B. subtilis natto was performed by ¹³C-nuclear magnetic resonance (NMR) and mass spectral analyses. The NMR comparison also revealed that NT-5, NT-6, and NT-7 had identical structures, except for the fatty chain. In summary, the present study suggests the improved biocontrol and/or microbial inhibitory potential of newly isolated bactericides secreting B. subtilis natto.