Bacillus spp. Isolated from Puba as a Source of Biosurfactants and Antimicrobial Lipopeptides.
ABSTRACT: Several products of industrial interest are produced by Bacillus, including enzymes, antibiotics, amino acids, insecticides, biosurfactants and bacteriocins. This study aimed to investigate the potential of two bacterial isolates (P5 and C3) from puba, a regional fermentation product from cassava, to produce multiple substances with antimicrobial and surface active properties. Phylogenetic analyses showed close relation of isolates P5 and C3 with Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. Notably, Bacillus sp. P5 showed antimicrobial activity against pathogens such as Listeria monocytogenes and Bacillus cereus, in addition to antifungal activity. The presence of genes encoding pre-subtilosin (sboA), malonyl CoA transacylase (ituD), and the putative transcriptional terminator of surfactin (sfp) were detected in Bacillus sp. P5, suggesting the production of the bacteriocin subtilosin A and the lipopeptides iturin A and surfactin by this strain. For Bacillus sp. C3 the presence of sboA and spas (subtilin) genes was observed by the first time in members of B. cereus cluster. Bacillus sp. P5 showed emulsifying capability on mineral oil, soybean biodiesel and toluene, while Bacillus sp. C3 showed emulsifying capability only on mineral oil. The reduction of the surface tension in culture medium was also observed for strain P5, confirming the production of surface-active compounds by this bacterium. Monoprotonated molecular species and adducts of sodium and potassium ions of surfactin, iturin, and fengycin were detected in the P5 culture medium. Comparative MS/MS spectra of the peak m/z 1030 (C14 surfactin A or C15 surfactin B [M+Na]+) and peak m/z 1079 (C15 iturin [M+Na]+) showed the same fragmentation profile of standards, confirming the molecular identification. In conclusion, Bacillus sp. P5 showed the best potential for the production of antifungal, antibacterial, and biosurfactant substances.
Project description:This report demonstrates the usefulness of PCR for the genes spaS and sboA as a means of identifying Bacillus strains with a potential to produce subtilin and subtilosin A. One collection strain and five Bacillus spp. isolated from aquatic environments in the Amazon basin were screened by PCR using primers for sboA and spaS designed specifically for this study. The sequences of the PCR products showed elevated homology with previously described spaS and sboA genes. Antimicrobial peptides were isolated from culture supernatants and analyzed by mass spectrometry. For all samples, the mass spectra revealed clusters with peaks at m/z 3300-3500 Da, corresponding to subtilosin A, subtilin and isoforms of these peptides. These results suggest that the antimicrobial activity of these strains may be associated with the production of subtilosin A and/or subtilin. The PCR used here was efficient in identifying novel Bacillus strains with the essential genes for producing subtilosin A and subtilin.
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:Bacillus subtilis subsp. krictiensis ATCC55079 produces the cyclic lipopeptide antibiotics iturin A-F as well as several surfactins. Here, we analyzed and characterized the biosynthetic genes associated with iturin and surfactin production in this strain. We aligned the sequences of each iturin and surfactin synthetase ORF obtained from a genomic library screen and next generation sequencing. The resulting 37,249-bp and 37,645-bp sequences associated with iturin and surfactin production, respectively, contained several ORFs that are predicted to encode proteins involved in iturin and surfactin biosynthesis. These ORFs showed higher sequence homologies with the respective iturin and surfactin synthetase genes of B. methylotrophicus CAU B946 than with those of B. subtilis RB14 and B. subtilis ATCC6633. Moreover, comparative analysis of the secondary metabolites produced by the wild-type and surfactin-less mutant (with a spectinomycin resistance cassette inserted into the srfAB gene within the putative surfactin gene region) strains demonstrated that the mutant strain showed significantly higher antifungal activity against Fusarium oxysporum than the wild-type strain. In addition, the wild-type strain-specific surfactin high performance liquid chromatography (HPLC) peaks were not observed in the surfactin-less mutant strain. In contrast, the iturin A peak detected by HPLC and liquid chromatography-mass spectrometry (LC/MS) in the surfactin-less mutant strain was 30% greater than that in the wild-type strain. These results suggested that the gene cluster we identified is involved in surfactin biosynthesis, and the biosynthetic pathways for iturin and surfactin in Bacillus strains producing both iturin and surfactin may utilize a common pathway.
Project description:Iturin A and surfactin are antimicrobial lipopeptides produced by antagonistic Bacillus spp. We herein demonstrated that both lipopeptides amended the soil-mediated suppression of the soil-borne disease, Fusarium yellows of tatsoi (Brassica rapa var. rosularis). Significant disease suppression was conferred by the amendments of purified iturin A or surfactin to soil. However, an excess amount of iturin A or surfactin to soil resulted in the loss of disease suppression activity.
Project description:In the present study, a deep-sea bacterial strain designated Bacillus sp. strain wsm-1 was screened and found to exhibit strong antifungal activity against many plant-pathogenic fungi, and corresponding antifungal agents were thereby purified and determined by tandem mass spectrometry to be two cyclic lipopeptide homologs. These homologs, which were different from any previously reported lipopeptides, were identified to possess identical amino acid sequences of ?-amino fatty acid-Asn-Ser-Asn-Pro-Tyr-Asn-Gln and deduced as two novel lipopeptides designated C14 iturin W and C15 iturin W. Electron microscopy observation indicated that both iturin W homologs caused obvious morphological changes and serious disruption of plasma membrane toward fungal cells, while C15 iturin W exhibited more serious cell damages than C14 iturin W did, which was well consistent with the results of the antifungal activity assays. To improve the yield and antifungal activity of iturin W, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W were investigated. The results indicated that supplements of most of the detected carbon and nitrogen sources could increase the yield of C14 iturin W, but inhibit the yield of C15 iturin W, while supplements of tryptone and most of the detected amino acids could increase the yield of both C14 iturin W and C15 iturin W.IMPORTANCE Plant disease caused by pathogenic fungi is one of the most devastating diseases, which affects the food safety of the whole world to a great extent. Biological control of plant diseases by microbial natural products is more desirable than traditional chemical control. In this study, we discovered a novel lipopeptide, iturin W, with promising prospects in biological control of plant diseases. Moreover, the effects of different carbon and nitrogen sources and amino acids on production of C14 iturin W and C15 iturin W would provide a reasonable basis for the optimization of the fermentation process of lipopeptides. Notably, the structure of iturin W was different from that of any previously reported lipopeptide, suggesting that deep-sea microorganisms might produce many novel natural products and have significant potential in the development of biological products in the future.
Project description:BACKGROUND:Biosurfactants, being highly biodegradable, ecofriendly and multifunctional compounds have wide applications in various industrial sectors including environmental bioremediation. Surfactin, a member of lipopeptide family, which is considered as one of the most powerful biosurfactants due to its excellent emulsifying activities as well as environmental and therapeutic applications. Therefore, the aim of this study was to investigate the newly isolated bacterial strain S2MT for production of surfactin-like biosurfactants and their potential applications for oil-contaminated soil remediation. RESULTS:In this study, the strain S2MT was isolated from lake sediment and was identified as Bacillus nealsonii based on transmitted electron microscopy (TEM) and 16S rRNA ribo-typing. The strain S2MT produced biosurfactant that reduced the surface tension (34.15?±?0.6 mN/m) and displayed excellent emulsifying potential for kerosene (55?±?0.3%). Additionally, the maximum biosurfactant product yield of 1300 mg/L was achieved when the composition of the culture medium was optimized through response surface methodology (RSM). Results showed that 2% glycerol and 0.1% NH4NO3 were the best carbon/nitrogen substrates for biosurfactant production. The parameters such as temperature (30 °C), pH (8), agitation (100 rpm), NH4NO3 (0.1%) and NaCl (0.5%) displayed most significant contribution towards surface tension reduction that resulted in enhanced biosurfactant yield. Moreover, the extracted biosurfactants were found to be highly stable at environmental factors such as salinity, pH and temperature variations. The biosurfactants were characterized as cyclic lipopeptides relating to surfactin-like isoforms (C13-C15) using thin-layer chromatography (TLC), Ultra high performance liquid chromatography and mass spectrometry (UHPLC-MS). The crude biosurfactant product displayed up to 43.6?±?0.08% and 46.7?±?0.01% remediation of heavy engine-oil contaminated soil at 10 and 40 mg/L concentrations, respectively. CONCLUSION:Present study expands the paradigm of surfactin-like biosurfactants produced by novel isolate Bacillus nealsonii S2MT for achieving efficient and environmentally acceptable soil remediation as compared to synthetic surfactants.
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:Bacillus spp. produce a broad spectrum of lipopeptide biosurfactants, among which surfactin, iturin and fengycin are widely studied families. The goals of this study were to characterize the biosurfactant activity of Bacillus spp. and to investigate their motility and biofilm formation capabilities. In addition, we extracted lipopeptides from these bacteria to assess their antifungal activities and analyzed these products by mass spectrometry (MS). B. amyloliquefaciens FZB42, Bacillus sp. NH 217 and B. subtilis NH-100 exhibited excellent biosurfactant and surface spreading activities, whereas B. atrophaeus 176s and Paenibacillus polymyxa C1225 showed moderate activity, and B. subtilis 168 showed no activity. Strains FZB42, NH-100, NH-217, 176s and CC125 exhibited excellent biofilm formation capabilities. Lipopeptide extracts displayed good antifungal activity against various phytopathogens and their associated diseases, such as Fusarium moniliforme (rice bakanae disease), Fusarium oxysporum (root rot), Fusarium solani (root rot) and Trichoderma atroviride (ear rot and root rot). Lipopeptide extracts of these strains also showed hemolytic activity, demonstrating their strong potential to produce surfactants. LCMS-ESI analyses identified the presence of surfactin, iturin and fengycin in the extracts of Bacillus strains. Thus, the strains assayed in this study show potential as biocontrol agents against various Fusarium and Trichoderma species.
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:Surfactin, a lipopeptide produced by Bacillus species, has been used for the oral delivery of insulin. In this study, another lipopeptide of iturin was tested for its ability to orally delivery insulin alone or plus surfactin. Iturin could form co-precipitate with insulin at acidic pH values. After treatment by ultrasonification, the structure of coprecipitate was destroyed that led to a significant decrease in hypoglycemic effect after oral administration. Iturin weakly binds to (Kd?=?257??M) and induce insulin structure more compact that is favorable for insulin uptake by the intestine. After being coated with Acryl-Eze by lyophilization, the coprecipitate formed the spherical enteric-coated insulin microparticles delivered by iturin with a relative oral bioavailability of 6.84% in diabetic mice. For further improving oral hypoglycemic effect, surfactin was added to form the spherical enteric-coated insulin microparticles in a formulation containing insulin, Acryl-Eze, iturin and surfactin at a ratio of 1:1:0.5: 0.5 (w/w), with an insulin encapsulation efficiency of 66.22%. The enteric-coated insulin microparticles delivered by iturin plus surfactin showed a classical profile for controlled release in the intestine with a relative bioavailability of 7.67% after oral administration, which could effectively control the postprandial blood glucose at a level about 50% of the initial one just like the subcutaneous injection. Collectively, iturin plus surfactin is more efficient for oral delivering insulin than the sole one, and the resultant enteric-coated insulin microparticles are potential for the development of oral insulin to control postprandial blood glucose in diabetic patients.