| B. subtilis is an important bio-control strain and wide applied in the plant diseases control due to its’well environmental colonization and production of diversity second antimicrobial metabolites. Three families of cyclic lipopeptide-surfactins, iturins and fengycins produced by B. subtilis have well-recognized potential uses in biotechnology and biopharmaceutical applications because of their antagonistic activity in a wide range of phytopathogens such as bacteria, fungi and oomycetes. To further elucidate the detail chemistry and molecular mechanism of bio-control activity of commercial B. subtilis Bs916, we found nine gene clusters directing synthesis of bioactive peptides and polyketides by modularly organized mega-enzymes named non-ribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs) in Bs916 on the basis of its draft genomic sequence. The results also suggested that Bs916 is potential to produce ten kinds of second antimicrobial metabolites. To our interesting, the genome of Bs916 contains four gene clusters for four family lipopeptides production, not only include three well-known gene cluster srf for surfactin synthesis, bmy for bacillomycin L synthesis, fen for fengycin synthesis; but also include a low similarity gene cluster loc presumed for an unknown locillomycin synthesis. Crude lipopeptides extracted from the Bs916 culture broth showed broad antifungal and antibacterial spectrum. The molecular weights determined by High Pressure Liquid Chromatography-Mass Spectrum (HPLC-MS) were attributed to three surfactin isoforms, three bacillomycin L isoforms, five fengycin iso forms and three unkown lipopeptide isoforms (designated New lipopeptides).Bioinformation analysis of the low similarity NRPS gene cluster loc showed that the gene cluster encodes 4 proteins (namely locA, locB, locC and locD), including hexamodular nonribosomal peptide synthetases (NRPS) and a C-terminal thioestrase. Gene deletions confirmed that two NRPS, locD and locC, were required for the biosynthesis of locillomycin. Biochemical analysis of the adenylation domain specificity supported the assignment of this gene cluster as a locillomycin biosynthetic gene cluster. The molecular weights of the locillomycin three homologues were the same as the molecular weights of the new lipopeptides extracted from B. subtilis 916 culture broth by HPLC-MS analysis. The purified locillomycin was prepared by acid precipitation, organic solvent extracts, solid phase extraction and HPLC. The structure of locillomycin was determined by the amino acid analysis, MS and NMR. The locillomycin was a cyclic lipopeptide which a nonapepetide linked to 13-15 C factty acid chain. Comparison of locillomycin with other known lipopeptides, the locillomycin belong to a new family lipopeptide. The locillomycins are also active against bacteria and viruses with a low haemolytic activity. Thus, the compounds of locillomycin have potentially therapeutic applications in natural health products. According to the genome analysis and the chemical structures of the locillomycin final products, we first proposed the biosynthesis pathway of the locillomycin The proposed pathway of nonribosomal peptide biosynthetic exhibited several atypical features, including N-acyl capped cyclic peptide, a nonlinear assembly line and a marocyclizing thioestarase that produced a cyclic lipopeptide including nine amino acid residues, which resulted in distinguished architectures of locillomycins. The structural elucidation and the biosynthetic gene identification of this novel family cyclic lipopeptide provides the basis for future mechanistic study of a nonapeptide assembled by hexamodular NRPS.Recent investigations shed light on that the lipopeptides are able to influence the typical phenotypic features include antimicrobial activity, haemolytic activity, swarming motility, biofilm formation and colony morphology, but it is relatively less known about their exact contribution to these phenotypic features. B. subtilis Bs916 was an effective bio-control strain in control plant disease caused by fungal pathogenic. Interestingly, B. subtilis Bs916 not only co-produce well-known surfactin, bacillomycin L and fengycin, but also produce a novel lipopeptide locillomycin. Accordingly, the genome of B. subtilis Bs916 contained four lipopeptides gene clusters srf, bac,fen and loc which are responsibile for surfactin, bacillomycin L, fengycin and locillomycin biosynthesis respectively. Eight B. subtilis Bs916 derivatives which lost single, double or triple lipopeptides production were constructed and used for the study of five phenotypic features. We found that the bacillomycin L and fengycin contributed mainly to the antifungal activity. While surfactin has weak antifungal activity in vitro, the srf mutant decreased antifungal activity sharply due to impairing the production of fengycin and bacillomycin L. Except to fengycin, the deficiency of other three lipopeptides resulted in the change of colony morphology to variant extent. While the fengycin and locillomycin had little influence on haemolytic activity, swarming motility and biofilm formation, surfactin and bacillomycin L play an important influence on these three phenotypic features in an intricate manner. In a word, the four lipopeptides contributed individually, differently and synergistically to these related phenotypic features of B. subtilis Bs916.Surfactin, bacillomycin L, fengycin and locillomycin have recognized potential uses in biotechnology and biopharmaceutical applications because of their antagonistic activities and haemolytic activities. However, the commercial product of surfactin is expensive and even more the commercial products of bacillomycin L, fengycin and locillomycin are not available at all. In this paper, we reported the methods for rapid isolation and purification of the four lipopeptides from B. subtilis Bs916’s mutants and further analyzed biological function of the four lipopeptides. The purified surfactin, bacillomycin L, fengycin and locillomycin were prepared through fermentation, acid precipitation, organic solvent extracts and solid phase extraction. The purities of the four samples were detected by the HPLC and MS. The results of biological function research showed surfactin and bacillomycin L have strong haemolytic activities, but fengycin and locillomycin only have weak haemolytic activities. While bacillomycin L and fengycin showed strong antifungal activities, surfactin and locillomycin showed antibacterial and antiviral activities. Surfactin and bacillomycin L were able to restore the swarming mobility and biofilm formation of according mutants. In general, the purified surfactin, bacillomycin L, fengycin and locillomycin layed the foundation for future detail elucidation of their biological functions.B. subtilis Bs916 is effective in bio-controlling rice sheath blight by R.solani. While lipopeptides produced by B. subtilis Bs916 was well studied for antagonistic activity, it is relatively less known about their mechanism in environmental colonization. The interaction of B. subilis Bs916-rice sheath-R. solani provides an excellent system to investigate the mechanism of B. subtilis strains colonization on plant leaves. In this paper, we investigated the biofilm formation and colonization on rice sheath of wild type B. subtilis Bs916 and its mutants Asrf, Abmy and Asrf+bmy, which were deficient in surfactin and bacillomycin L production respectively, with the aid of GFP tag. The results showed that B. subtilis Bs916 formed robust biofilm on the rice blight lesion and colonize well on the rice sheath, while its mutants performed very poorly. This is correlated with the pot experiments, in which B. subtilis Bs916 has a better control activity than its mutants. In a word, Surfactin and bacillomycin L contribute differently but synergistically to biocontrol of rice sheath blight in B. subtilis Bs916 through affecting antifungal activity, biofilm formation and colonization. |
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