Strong inhibitory activities and action modes of lipopeptides on lipase.
ABSTRACT: Lipopeptides have been reported to exhibit anti-obesity effects. In this study, we obtained a Bacillus velezensis strain FJAT-52631 that could coproduce iturins, fengycins, and surfactins. Results showed that the FJAT-52631 crude lipopeptide, purified fengycin, iturin, and surfactin standards exhibited strong inhibition activities against lipase with dose-dependence manners (half maximal inhibitory concentration (IC50)?=?0.011, 0.005, 0.056, and 0.005?mg/mL, respectively). Moreover, fengycin and surfactin had the comparable activities with orlistat, but iturin not. It was revealed that the inhibition mechanism and type of the lipopeptides were reversible and competitive. The quenching mechanism of lipase was static and only one binding site between lipase and lipopoeptide was inferred from the fluorescence analysis. The docking analysis displayed that fengycin and surfactin could directly interact with the active amino acid residues (Ser or Asp) of lipase, but not with iturin. Our work suggests that the B. velezensis lipopeptides would have great potential to act as lipase inhibitors.
Project description:BACKGROUND:There is an urgent need to discover biocontrol agents to control bacterial wilt. This study reports on a new lipopeptide-producing biocontrol strain FJAT-46737 and explores its lipopeptidic compounds, and this study investigates the antagonistic effects of these compounds. RESULTS:Based on a whole genome sequence analysis, the new strain FJAT-46737 was identified as Bacillus velezensis, and seven gene clusters responsible for the synthesis of bioactive secondary metabolites in FJAT-46737 were predicted. The antimicrobial results demonstrated that FJAT-46737 exhibited broad-spectrum antimicrobial activities in vitro against three bacteria and three fungi. Pot experiments showed that the control efficiencies for tomato bacterial wilt of the whole cultures, the 2-fold diluted supernatants and the crude lipopeptide of FJAT-46737 were 66.2%, 82.0%, and 96.2%, respectively. The above results suggested that one of the antagonistic mechanisms of FJAT-46737 was the secretion of lipopeptides consisting of iturins, fengycins and surfactins. The crude lipopeptides had significant antagonistic activities against several pathogens (including Ralstonia solanacearum, Escherichia coli and Fusarium oxysporum) and fengycins were the major antibacterial components of the lipopeptides against R. solanacearum in vitro. Furthermore, the rich organic nitrogen sources (especially yeast extracts) in the media promoted the production of fengycin and surfactin by FJAT-46737. The secretion of these two lipopeptides was related to temperature fluctuations, with the fengycin content decreasing by 96.6% and the surfactins content increasing by 59.9% from 20?°C to 40?°C. The optimal temperature for lipopeptide production by FJAT-46737 varied between 20?°C and 25?°C. CONCLUSIONS:The B. velezensis strain FJAT-46737 and its secreted lipopeptides could be used as new sources of potential biocontrol agents against several plant pathogens, and especially the bacterial wilt pathogen R. solanacearum.
Project description:Probiotics may offer an attractive alternative for standard antibiotic therapy to treat Clostridium difficile infections (CDI). In this study, the antibacterial mechanism in vitro of newly isolated B. amyloliquefaciens C-1 against C. difficile was investigated. The lipopeptides surfactin, iturin, and fengycin produced by C-1 strongly inhibited C. difficile growth and viability. Systematic research of the bacteriostatic mechanism showed that the C-1 lipopeptides damage the integrity of the C. difficile cell wall and cell membrane. In addition, the lipopeptide binds to C. difficile genomic DNA, leading to cell death. Genome resequencing revealed many important antimicrobial compound-encoding clusters, including six nonribosomal peptides (surfactins (srfABCD), iturins (ituABCD), fengycins (fenABCDE), bacillibactin (bmyABC), teichuronic, and bacilysin) and three polyketides (bacillaene (baeEDLMNJRS), difficidin (difABCDEFGHIJ), and macrolactin (mlnABCDEFGHI)). In addition, there were other beneficial genes, such as phospholipase and seven siderophore biosynthesis gene clusters, which may contribute synergistically to the antibacterial activity of B. amyloliquefaciens C-1. We suggest that proper application of antimicrobial peptides may be effective in C. difficile control.
Project description:The banyan endophyte, Bacillus subtilis K1, produces a complex mixture of lipopeptides exhibiting potent antifungal activity. These lipopeptides were purified by high-performance liquid chromatography and analyzed using MALDI-TOF-MS as well as liquid chromatography coupled with ESI-MS. A heterogenous mixture of lipopeptides belonging to three different families of cyclic lipopeptides, viz., fengycins, iturins and surfactins, was detected in the cell-free extracellular extract of B. subtilis K1. The detailed mass spectrometric characterization revealed the presence of four variants of iturin A and three variants of iturin C varying in the ?-amino fatty acid chain length from C<sub>13</sub> to C<sub>17</sub>. The MS/MS of monovalent alkali metal ion adducts (Na and K) of iturin suggested the Glu<sup>4</sup> as a binding site for metal ion. The LC-ESI-MS/MS analysis of surfactins enabled the identification of seven surfactin variants with the variations in Val/Ile/Leu at position 4 and C<sub>13</sub>-C<sub>17</sub> ?-hydroxy fatty acids. This study demonstrates the application of tandem mass spectrometry in identification of closely related lipopeptides from a heterogenous mixture obtained from a natural source. Furthermore, this is the first report of an endophytic bacillus strain co-producing so many variants of surfactins and iturins.
Project description:To isolate Bacillus velezensis mutants with improved antifungal activity for use in the biological control of phytopathogenic fungi, wild-type Bacillus velezensis KRF-001 producing iturin, surfactin, and fengycin was irradiated by ultraviolet (UV) rays. The in vitro and in vivo antifungal activities of UV mutants and characterization of the cyclic lipopeptides produced by a selected mutant were examined. A mutant strain yielding high levels of iturin showed over 2-fold higher antifungal activity than the wild-type against Fusarium oxysporum. A potent suppressive effect of the mutant was also observed on spore germination of Botrytis cinerea, the causative agent of cucumber gray mold, at different butanol extract concentrations. Further analysis of the mutant by real-time PCR and high-performance liquid chromatography revealed increased expression of iturin and surfactin biosynthesis genes as well as enhanced production of iturin and surfactin metabolites. However, the amounts of fengycin obtained from the mutant strain BSM54 were significantly lesser than those of iturin and surfactin. Particularly, iturin A production by the mutant was 3.5-fold higher than that of the wild-type, suggesting that the higher antifungal activity of the mutant against F. oxysporum resulted from the increased expression of biosynthesis genes associated with iturin production. The commercial greenhouse experiment using soil naturally infested with Sclerotinia sclerotiorum (sclerotinia rot) and F. oxysporum (fusarium wilt) showed that the mutant strain reduced sclerotinia rot and fusarium wilt diseases (P = 0.05) more effectively than the wild-type and commercially available product Cillus® in Korea. These results suggest that the mutant with high iturin yield is a potential candidate for the development of a biological control agent in agriculture.
Project description:Non-self-recognition of microorganisms partly relies on the perception of microbe-associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA-regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long-lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP-non-producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.
Project description:A potential antagonist, designated strain Bacillus subtilis MBCU5 was previously isolated from vermicompost-amended soils of Gandhinagar, Gujarat, India. Crude allelochemicals from strain MBCU5 displayed strong antifungal activity against Macrophomina phaseolina as well as Rhizoctonia solani. These crude allelochemicals were tentatively identified as iturin, fengycin and surfactin through TLC and HPTLC analysis. Lipopeptides produced by MBCU5 were identified by MALDI-TOF-MS and LC-ESI-MS/MS analysis showed that iturin homologues (m/z 1020-1120), surfactin (m/z 1008.7 and m/z 1022.7), fengycin A and fengycin B (m/z 1400-1550) types of allelochemicals which are responsible for antifungal activity against pathogens. PCR analysis showed presence of genes (i.e. Iturin A synthetase KJ531680 and Surfactin synthetase KJ601726) involved in the biosynthesis of allelochemicals. Many reports showed lipopeptides from Bacillus species; this is the first report executed of multifarious allelochemicals from vermicompost-amended soil due to the presence of predominant Bacillus species.
Project description:Oomycete Phytophthora infestans [(Mont.) de Bary] is the cause of potato late blight, a plant disease which poses a serious threat to our global food security and is responsible for huge economic losses worldwide. Lipopeptides produced by Bacillus species are known to be potent antibacterial compounds against many plant pathogens. In this study, we show that Bacillus megaterium WL-3 has an antagonistic effect against potato late blight. Electrospray ionization mass spectrometry (ESI-MS) revealed that lipopeptides derived from the WL-3 strain contained three subfamilies, surfactin (C13 - C15), Iturin A (C14 - C16), and Fengycin A (C15 - C19). The Iturin A and Fengycin A lipopeptide families were each confirmed to have anti-oomycete effects against P. infestans mycelium growth as well as obvious controlling effects against potato late blight in greenhouse experiments and field assays. Furthermore, Iturin A and Fengycin A were able to promote plant photosynthetic efficiency, plant growth, and potato yield. Most importantly, the combination of Iturin A and Fengycin A (I + F) was superior to individual lipopeptides in controlling potato late blight and in the promotion of plant growth. The results of this study indicate that B. megaterium WL-3 and its lipopeptides are potential candidates for the control of late blight and the promotion of potato plant growth.
Project description:Plant growth promoting rhizobacteria (PGPR) provide an effective and environmentally sustainable method to protect crops against pathogens. The spore-forming Bacilli are attractive PGPR due to their ease of storage and application. Here, we characterized two rhizosphere-associated Bacillus velezensis isolates (Y6 and F7) that possess strong antagonistic activity against Ralstonia solanacearum and Fusarium oxysporum under both laboratory and greenhouse conditions. We identified three lipopeptide (LP) compounds (surfactin, iturin and fengycin) as responsible for the antimicrobial activity of these two strains. We further dissected the contribution of LPs to various biological processes important for rhizosphere colonization. Although either iturin or fengycin is sufficient for antibacterial activity, cell motility and biofilm formation, only iturin plays a primary role in defense against the fungal pathogen F. oxysporum. Additionally, we found that LP production is significantly stimulated during interaction with R. solanacearum. These results demonstrate the different roles of LPs in the biology of B. velezensis and highlight the potential of these two isolates as biocontrol agents against phytopathogens.
Project description:Bacillus amyloliquefaciens strain RHNK22 isolated from groundnut rhizosphere showed direct and indirect plant growth-promoting traits along with biosurfactant activity and reduction in surface tension of water. Biosurfactants were identified as lipopeptides (surfactin, iturin, and fengycin) by molecular and biochemical analysis in our studies.
Project description:In this research, a strain with broad-spectrum antimicrobial activities was isolated from the gastrointestinal tract of hairtail (Trichiurus haumela) and identified as Bacillus siamensis JFL15 through morphological, 16S rRNA, and average nucleotide identity analyses. The genome of B. siamensis JFL15 was sequenced, and three gene clusters involved in the biosynthesis of surfactin (srf), bacillibactin (dhb), and fengycin (fen) were predicted through antiSMASH analysis. The combined genomics-metabolics profiling of the strain revealed 20 active compounds, which belong to four main types of cyclic lipopeptides produced by Bacillus species: bacillibactin, iturin, fengycin, and surfactin. Among these lipopeptides, two high-purity antifungal components, namely, components b and c, were successfully identified as iturin A and bacillomycin F. The minimum inhibitory concentrations (MICs) of iturin A for Magnapothe grisea, Rhizoctorzia solani, and Colletotrichum gloeosporioides were 125.00, 62.50, and 125.00 ?g/ml, respectively, whereas the MICs of bacillomycin F for these three organisms were 62.50, 31.25, and 62.50 ?g/ml, respectively. The mechanism of bacillomycin F and iturin A against M. grisea was also investigated. Scanning electron microscopy (SEM) indicated that the surface of the hypha treated with iturin A or bacillomycin F became sunk, lumpy, and wrinkled. The diversity of the identified and predicted compounds from B. siamensis JFL15 suggested that this strain might be a promising biocontrol agent for an effective and environmentally friendly control of pathogenic microorganisms. To the best of our knowledge, this study is the first to describe cyclic lipopeptides purified and identified from B. siamensis.