Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

Abstract

cBacillus 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.

Safety Properties and Probiotic Potential of <i>Bacillus subtilis</i> KATMIRA1933 and <i>Bacillus amyloliquefaciens</i> B-1895

“…The hemolytic mechanism in Bacillus is not fully understood. However, recent studies have been performed to identify the gene(s) responsible for hemolytic activity [23] [24]. Even though the hemolytic activity of some Bacillus spp., such as Bacillus subtilis, is less than in pathogens [25], these isolates may be considered unsafe for food or personal health care applications until the effect of this virulence factor is either eliminated, modified, or confirmed as causing no harm to the eukaryotic host.…”

Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916

“…In addition to the three conventional gene clusters, srf (for surfactins), bmy (for bacillomycin Ls), and fen (for fengycins), B. subtilis 916 also contains a fourth NRPS gene cluster, loc (GenBank accession no. KF866134) (34). Further analysis of the loc open reading frames led to the conclusion that the loc gene cluster was a potential candidate for the biosynthesis of an unknown family of lipopeptides called locillomycins.…”

“…Identification and bioinformatic analyses of the loc gene cluster were employed as described in our previous work (34). The draft genome and complete genome sequences of B. subtilis 916 were recently analyzed (GenBank accession no.…”

“…While NRPSs for lipopeptide production in B. subtilis 916 were reported recently, and the locillomycin also described briefly (34), in the present study we describe in detail the novel structure and unique biosynthesis of the new locillomycin family of nonribosomal lipopeptides and its biological function. In this study, our results strongly suggest that locillomycins have molecular features not observed in the three families of surfactins, iturins, and fengycins, including a unique nonapeptide sequence and macrocyclization.…”

Screening of a <i>Bacillus subtilis</i> Strain Producing Multiple Types of Cyclic Lipopeptides and Evaluation of Their Surface-tension-lowering Activities

“…Using total DNA from the strain as a template, the non-ribosomal peptide synthase-encoding genes were amplified by PCR with the forward and reverse primer sets SrfA-AF 5 -ACACAGATATCAGGCAAGC-3 and SrfA-AR 5 -GTCCCATCGTTCCTTCACA-3 for srfAA surfactin, 908 bp PCR product 23 -ACTGAAGCGATTCGCAAGTT-3 and LchAAR 5 -TC-GCTTCATATTGTGCGTTC-3 for lchAA lichenysin, 472 bp PCR product 25 . PCR reactions were performed using Premix Taq TM Takara Shuzo, Kyoto, Japan as recommended by the manufacturer, and the PCR conditions were as described previously 26 .…”