Search

Cyclic lipopeptides (CLPs) are potent secondary metabolites with diverse biological functions. Bacillus strains primarily produce CLPs of three key families, namely, iturins, fengycins, and surfactins, each comprising structural variants characterized by a cyclic peptide linked to a fatty acid chain. Despite extensive research on CLPs, the individual roles of these analogues and their proportion in driving biological activity have remained largely overlooked. In this study, we purified and chemically characterized CLP variants from Bacillus velezensis UMAF6639 and tested them individually for their antifungal and plant growth-promoting effects. We isolated 5 fractions containing iturin A analogues (from C 13 to C 17 ), 5 fengycin fractions (containing C 16 , C 17 , and C 18 fengycin A and C 14 , C 15 , C 16 , and C 17 fengycin B), and 5 surfactin fractions (from C 12 to C 16 ). We show how antifungal activity and seed radicle growth promotion relied on the lipopeptide structural variant and concentration based on the physiological ratio calculated for each lipopeptide variant. Notably, we found that the most toxic variants were the least abundant, which likely minimized autotoxicity while preserving bioactivity. This balance is achieved through synergistic interactions with more abundant, less aggressive analogues. Furthermore, certain fengycin and surfactin variants were shown to increase bacterial population density and exopolysaccharide production, crucial strategies for microbial competition with significant ecological impacts. In addition to advancing basic knowledge, our findings will support the development of precision biotechnological innovations, offering targeted solutions to drive sustainable food production and preservation strategies.

Bacillus spp. produce numerous antimicrobial metabolites. Among these metabolites, cyclic lipopeptides (CLP) including fengycins, iturins, and surfactins are known to have varying antifungal activity against phytopathogenic fungi. The differential activities of CLP have been attributed to diverse mechanisms of action on fungal membranes. However, the precise biochemical determinants driving their antifungal modes of action have not been conclusively identified. In this study, three plant pathogenic fungi of varying lipopeptide sensitivities, Alternaria solani, Cladosporium cucumerinum, and Fusarium sambucinum, were studied to determine how their cell membrane lipid compositions may confer sensitivity and/or tolerance to fengycin, iturin, and surfactin. Results indicated that sensitivity to all three lipopeptides correlated with lower ergosterol content and elevated phospholipid fatty acid unsaturation. Fungal sensitivity to surfactin was also notably different than fengycin and iturin, as surfactin was influenced more by lower phosphatidylethanolamine amounts, higher levels of phosphatidylinositol, and less by phospholipid fatty acyl chain length. Results from this study provide insight into the fungal membrane composition of A. solani, F. sambucinum, and C. cucumerinum and the specific membrane characteristics influencing the antifungal effectiveness of fengycin, iturin, and surfactin. Understanding of these determinants should enable more accurate prediction of sensitivity-tolerance outcomes for other fungal species exposed to these important CLP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Marine microbes secrete exopolymeric substances (EPS), which surrounds the biofilm and inhibits the fungal growth. Elucidation of the structure and function of the extracellular exopolymeric substances is of vital relevance therapeutically. The active compound responsible for bioactivity was purified and characterized using TLC, LC/MS/MS, GC/MS and FT-IR. Bioactivity of the characterized cyclic peptides (CLPs) against azole resistant and susceptible Candida strains were examined for growth and biofilm formation using scanning electron microscopy, flow cytometry, confocal microscopy. In the present study we identified bioactive cyclic peptides from marine isolated Neobacillus drentensis that exhibited promising tensio-active properties and antifungal efficacy against azole resistant and susceptible Candida albicans. The cluster is composed of five CLP isoforms which were sequenced and identified as new peptides with compositional and structural variations in the amino acid sequence and fatty acid chain. In vitro cytotoxic activity of CLPs was tested in human fibroblast normal cells. We have observed that the CLPs repressed the Candida albicans growth and multiplication by inhibiting the biofilm formation and disruption of branching filamentous hyphae. CLPs have been found to arrest the C. albicans cell cycle by a block at G1-S transition followed by apoptotic cell death. The current studies suggest these natural marine derived CLPs function as potential anti-biofilm agents against azole C. albicans resistant strains., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fengycins are cyclic lipo-depsipeptides produced by Bacillus spp. that display potent antifungal properties but are chemically unstable. This instability has meant that no total synthesis of any fengycin has been published. Here we report the synthesis of fengycin A analogues that display enhanced antifungal properties and chemical stability under both basic and acidic conditions. The analogues prepared also demonstrate that the fengycin core structure can be modified and simplified without the loss of antifungal activity.

Background: Microorganisms, including Bacillus species are used to help control plant pathogens, thereby reducing reliance on synthetic pesticides in agriculture. Bacillus velezensis strain 1B-23 has been shown to reduce symptoms of bacterial disease caused by Clavibacter michiganensis subsp. michiganensis in greenhouse-grown tomatoes, with in vitro studies implicating the lipopeptide surfactin as a key antimicrobial. While surfactin is known to be effective against many bacterial pathogens, it is inhibitory to a smaller proportion of fungi which nonetheless cause the majority of crop diseases. In addition, knowledge of optimal conditions for surfactin production in B. velezensis is lacking., Results: Here, B. velezensis 1B-23 was shown to inhibit in vitro growth of 10 fungal strains including Candida albicans, Cochliobolus carbonum, Cryptococcus neoformans, Cylindrocarpon destructans Fusarium oxysporum, Fusarium solani, Monilinia fructicola, and Rhizoctonia solani, as well as two strains of C. michiganensis michiganensis. Three of the fungal strains (C. carbonum, C. neoformans, and M. fructicola) and the bacterial strains were also inhibited by purified surfactin (surfactin C, or [Leu7] surfactin C15) from B. velezensis 1B-23. Optimal surfactin production occurred in vitro at a relatively low temperature (16 °C) and a slightly acidic pH of 6.0. In addition to surfactin, B. velenzensis also produced macrolactins, cyclic dipeptides and minor amounts of iturins which could be responsible for the bioactivity against fungal strains which were not inhibited by purified surfactin C., Conclusions: Our study indicates that B. velezensis 1B-23 has potential as a biocontrol agent against both bacterial and fungal pathogens, and may be particularly useful in slightly acidic soils of cooler climates.

A marine-sediment-derived bacterium Bacillus velezensis SH-B74 can produce cyclic lipopeptides (CLPs). This study presented the isolation, characterization, and activity evaluation of a new CLP from the bacterial cultures of the strain SH-B74. Multiple chromatographic methods (solid-phase extraction and reversed-phase high-performance liquid chromatography) were applied to the purifying procedure of CLP, and the structural characterization of the new CLP was conducted by various spectroscopy (1D and 2D nuclear magnetic resonance together with Fourier transform infrared spectroscopy) and spectrometry (liquid chromatography-mass spectrometry, high-resolution mass spectrometry and tandem mass spectrometry) techniques as well as Marfey's method. The results displayed that the new CLP (anteiso-C 15 Ile 2,7 surfactin, 1) consists of a peptidic backbone of L-Glu 1 , L-Ile 2 , D-Leu 3 , L-Val 4 , L-Asp 5 , D-Leu 6 , L-Ile 7 , and an anteiso-C 15 type saturated fatty acid chain. Further activity assay showed that the new CLP displays activity on the inhibition of the appressoria formation of rice blast causal pathogen Magnaporthe oryzae. To sum up, the results presented the perspective of potential application of the new CLP as a green agrichemical to control M. oryzae.

A strain of Bacillus methylotrophicus was isolated from a soil sample collected in Aswan eastern desert, which is known for its extremely arid climate. After fermentation of the strain in liquid culture and subsequent extraction, a bioassay-guided isolation procedure yielded five compounds: 2-benzyl-4H-pyran-4-one, named bacillopyrone (1), pyrophen (2), macrolactin A (3) and the cyclopeptides malformin A1 (4), and bacillopeptin A (5). The structures were determined by interpretation of nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS) data. This is the first report on the isolation of compounds 1 and 2 from Bacillus species; compound 1 was reported previously as synthetic product. Bacillopyrone (1) exhibited moderate activity against the Gram-negative Chromobacterium violaceum with minimum inhibitory concentration 266.6 μg/mL, while macrolactin A (3) and malformin A1 (4) inhibited Staphylococcus aureus (minimum inhibitory concentrations 13.3 and 133.3 μg/mL, respectively).

Six seongsanamides were isolated from the culture broth of Bacillus safensis KCTC 12796BP, and their structures were elucidated by spectroscopic data analysis combined with Marfey's method, electronic circular dichroism calculations, and biosynthetic gene cluster analysis. Compounds 1-4 were bicyclic peptides with isodityrosine residues; 5 and 6 were monocyclic peptides. Only the bicyclic seongsanamides inhibited degranulation and LTC 4 /PGD 2 generation in IgE/Ag-stimulated bone marrow-derived mast cells. Oral administration of 1 suppressed mast cell-dependent passive cutaneous anaphylaxis reaction.

Aims: Biofilms are composed of micro-organisms within a matrix of chemically complex polymer compounds and from these structures many unknown competitive factors are suggested that many considered are important consequences for biological control. This research was undertaken to study further the endophyte, Bacillus mojavensis and its relationships to biofilm and two classes of lipopeptides considered relevant for biocontrol of plant pathogens., Methods and Results: Laser ablation electrospray ionization mass spectrometry and conventional MS/MS were used to study in situ biofilm production and the production of lipopeptides fengycin and surfactin in different strains of B. mojavensis in plate and test tube culture on two media. All strains were capable of producing biofilm in vitro along with the accumulation of surfactin and fengycin although no concentration-dependent relationship between lipopeptide accumulation and biofilm was observed., Conclusion: All strains studied produce biofilms in culture with the accumulated surfactin and fengycin, demonstrating that endophytic bacteria also produced biofilms., Significance and Impact of the Study: This study demonstrates that this endophytic species produced biofilms along with two biocontrol compounds of which one, surfactin, considered by others as a quorum sensor, highlighting its ecological role as a signalling mechanism in planta., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

The potential of the Bacillus genus to antagonize phytopathogens is associated with the production of cyclic lipopeptides. Depending upon the type of lipopeptide, they may serve as biocontrol agents that are eco-friendly alternatives to chemical fertilizers. This study evaluates the biocontrol activity of surfactin-producing Bacillus (SPB) strains NH-100 and NH-217 and purified surfactin A from these strains against rice bakanae disease. Biologically active surfactin fractions were purified by HPLC, and surfactin A variants with chain lengths from C12 to C16 were confirmed by LCMS-ESI. In hemolytic assays, a positive correlation between surfactin A production and halo zone formation was observed. The purified surfactin A had strong antifungal activity against Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride and T. reesei. Maximum fungal growth suppression (84%) was recorded at 2000 ppm against F. moniliforme. Surfactin A retained antifungal activity at different pH levels (5-9) and temperatures (20, 50 and 121 °C). Hydroponic and pot experiments were conducted to determine the biocontrol activity of SPB strains and the purified surfactin A from these strains on Super Basmati rice. Surfactin production in the rice rhizosphere was detected by LCMS-ESI at early growth stages in hydroponics experiments inoculated with SPB strains. However, the maximum yield was observed with a consortium of SPB strains (T4) and purified surfactin A (T5) treatments in the pot experiment. The outcomes of the present study revealed that surfactin A significantly reduced rice bakanae disease by up to 80%. These findings suggest that purified surfactin A could be an effective biocontrol agent against bakanae disease in rice and should be incorporated into strategies for disease management., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

A new cyclic hexapeptide, cyclo-(Gly-Leu-Val-IIe-Ala-Phe), named bacicyclin (1), was isolated from a marine Bacillus sp. strain associated with Mytilus edulis. The sequences of the amino acid building blocks of the cyclic peptide and its structure were determined by 1D- and 2D-NMR techniques. Marfey's analysis showed that the amino acid building blocks had L-configuration in all cases except for alanine and phenylalanine, which had D-configuration. Bacicyclin (1) exhibited antibacterial activity against the clinically relevant strains Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentration values of 8 and 12 µM, respectively. These results demonstrate the potential of marine bacteria as a promising source for the discovery of new antibiotics., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cyclic lipopeptides (CLPs) are non-ribosomal biosurfactants produced by Bacillus species that exhibit outstanding interfacial activity. The synthesis of CLPs is under genetic and environmental influence, and representatives from different families are generally co-produced, generating isoforms that differ in chemical structure and biological activities. This study to evaluate the effect of low and high NaCl concentrations on the composition and surface activity of CLPs produced by Bacillus strains TIM27, TIM49, TIM68, and ICA13 towards microbial enhanced oil recovery (MEOR). The strains were evaluated in mineral medium containing NaCl 2.7, 66, or 100 g L -1 and growth, surface tension and emulsification activity were monitored. Based on the analysis of 16S rDNA, gyrB and rpoB sequences TIM27 and TIM49 were assigned to Bacillus subtilis, TIM68 to Bacillus vallismortis, and ICA13 to Bacillus amyloliquefaciens. All strains tolerated up to 100-g L -1 NaCl, but only TIM49 and TIM68 were able to reduce surface tension at this concentration. TIM49 also showed emulsification activity at concentrations up to 66-g L -1 NaCl. ESI-MS analysis showed that the strains produced a mixture of CLPs, which presented distinct CLP profiles at low and high NaCl concentrations. High NaCl concentration favored the synthesis of surfactins and/or fengycins that correlated with the surface activities of TIM49 and TIM68, whereas low concentration favored the synthesis of iturins. Taken together, these findings suggest that the determination of CLP signatures under the expected condition of oil reservoirs can be useful in the guidance for choosing well-suited strains to MEOR.

Characterized as one of the most potent biosurfactants, surfactin is a cyclic lipopeptide synthesized by several strains of Bacillus genus. The aim of this review was to present the physicochemical and structural properties of surfactin and to demonstrate advances and applications of this biosurfactant for health and environmental biotechnology. Further, this review also focused on toxicological effects of surfactin on in vivo and in in vitro systems. The hydrophobic nature of surfactin enables interaction with membrane-bound phospholipids and indicates the ability of the molecule to act as a new weapon with respect to therapeutic and environmental properties. Seeking to avoid environmental contamination produced by widespread use of synthetic surfactants, surfactin emerges as a biological control agent against pathogen species owing to its antibacterial and antiviral properties. In addition, the mosquitocidal activity of surfactin was suggested as new strategy to control disease vectors. The current findings warrant future research to assess the toxicity of surfactin to enable an optimizing anticancer therapy and to seek refined methodologies, including nanotechnology techniques, to allow for an improved delivery of the biogenic molecule on target cells.

Bacillus methylotrophicus DCS1 strain was isolated from diesel contaminated soil and screened for its ability to produce biosurfactants; it was found effective for the production of surface active molecules. The structural characterization of the isolated lipopeptides was studied by a variety of analytical techniques. The organic extract of DCS1 lipopeptides was fractionated by silica gel column chromatography (60Mesh). Fractions containing lipopeptides were collected and identified by tandem mass spectrometry MALDI-TOF-MS and MALDI-TOF MS 2 . The crude biosurfactants contains a mixture of homologous lipopeptides with molecular weights between 1016 and 1556Da. Mass spectrometry analysis of partially purified lipopeptides revealed that it contains different isoforms belonging to three families: surfactin, iturin and fengycin. To identify lipopeptides isoforms, MALDI-TOF MS 2 was used and ions representing characteristic fragmentations were detected. The mass spectrometry characterization revealed the presence of four variants of surfactin lipopeptides, four variants of pumilacidin that differ according to the β-hydroxy fatty acid chain length as well as the type of amino acid at position 7, five variants of iturin A/mycosubtilin varying in the β-amino fatty acid chain length from C12 to C16, C16 iturin C1, five isoforms of bacillomycin D varying in the β-amino fatty acid chain length from C14 to C18, and six fengycin isoforms that differ according to the length of the β-hydroxy fatty acid side chain as well as the amino acid at position 6. The capacity of B. methylotrohicus DCS1 strain to produce many lipopeptides isoforms belonging to different families and having a structural diversity is a very interesting characteristic that allows them to be used in various fields of biotechnological applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Surfactin and iturin are antimicrobial lipopeptides produced from Bacillus spp. and have significant prospective applications in many fields. Therefore, accurate analysis of these lipopeptides in the fermented product of some Bacillus strains is important. A sensitive method for simultaneous quantitative determination of surfactin and iturin fermented by Bacillus natto NT-6 was developed and validated using liquid chromatography-tandem mass spectrometry. Crude extracts of antimicrobial lipopeptide samples were dissolved in a mixture of acetonitrile/water (7:3, v/v) in 0.1 % (v/v) formic acid and eluted with acetonitrile/water (7:3, v/v) containing 5 mmol L -1 ammonium acetate and 0.1 % (v/v) formic acid. The target compounds were detected by mass spectrometry (ESI+) using selective ion monitoring. A good linear regression in the range of 0.20-10.0 mg L -1 for both surfactin and iturin (R 2  ≥ 0.9995) was observed with spiked recoveries of 93.3-108.2 %, RSD values less than 15 %, precision 4.14-13.30 %, and a detection limit of 0.374 mg L -1 . This method has a simple preprocessing operation, good repeatability, and provides an accurate quantitative analysis of surfactin and iturin. Graphical Abstract Surfactin and iturin from Bacillus natto NT-6 extraction and detection procedure.

This work concerns the study of the enhancement of surfactin and fengycin production by B. mojavensis A21 and application of the produced product in diesel biodegradation. The influences of the culture medium and cells immobilization were studied. The highest lipopeptides production was achieved after 72 hours of incubation in a culture medium containing 30 g/L glucose as carbon source and a combination of yeast extract (1 g/L) and glutamic acid (5 g/L) as nitrogen sources with initial pH 7.0 at 30°C and 90% volumetric aeration. The study of primary metabolites production showed mainly the production of acetoin, with a maximum production after 24 h of strain growth. The use of immobilized cells seemed to be a promising method for improving lipopeptides productivity. In fact, the synthesis of both lipopeptides, mainly fengycin, was greatly enhanced by the immobilization of A21 cells. An increase of diesel degradation capacity of approximately 20, 27, and 40% in the presence of 0.5, 1, and 2 g/L of produced lipopeptides, respectively, was observed. Considering these properties, B. mojavensis A21 strain producing a lipopeptide mixture, containing both surfactin and fengycin, may be considered as a potential candidate for future use in bioremediation and crop protection.

Aim: To identify the different antifungal biomolecules produced by isolate Bacillus vallismortis R2 and analyse their effect on Alternaria alternata, a common pathogen causing black point disease of wheat., Methods and Results: The different antifungal molecules produced by isolate R2 were purified by column chromatography. The liquid chromatography-mass spectrometry of purified fractions confirmed the ability of R2 to produce biomolecules putatively similar to three different families of cyclic lipopeptides (CLPs) viz. surfactins, iturins and fengycins. The synergistic interaction among CLPs was evident from significant increase in the antifungal activity of mixture of purified fractions as compared to that of the individual fractions by agar well diffusion assay. The evaluation of antifungal activity of purified fractions by 96-well microtitre plate assay showed that the fengycin-like molecules supported significantly higher antifungal activity against A. alternata than iturin A and no antifungal activity was supported by surfactin., Conclusions: The isolate R2, producing different CLPs, can be used as an environmental friendly alternative to chemical fungicides. Among the three different CLPs viz. surfactin, iturin A and fengycin produced by R2, the fengycins were the most active lipopeptides., Significance and Impact of the Study: This is the first report on co-production of three different types of CLPs by the cells of B. vallismortis., (© 2016 The Society for Applied Microbiology.)

Algicidal bacteria have been turned out to be available for inhibiting Phaeocystis globosa which frequently caused harmful algal blooms and threatened to economic development and ecological balance. A marine bacterium Bacillus sp. Ts-12 exhibited significant algicidal activity against P. globosa by indirect attack. In present study, an algicidal compound was isolated by silica gel column, Sephadex G-15 column and HPLC, further identified as hexahydropyrrolo[1,2-a]pyrazine-1,4-dione, cyclo-(Pro-Gly), by GC-MS and (1)H-NMR. Cyclo-(Pro-Gly) significantly increased the level of reactive oxygen species (ROS) within P. globosa cells, further activating the enzymatic and non-enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and ascorbic acid (AsA). The increase in methane dicarboxylic aldehyde (MDA) content showed that the surplus ROS induced lipid peroxidation on membrane system. Transmission electron microscope (TEM) and flow cytometry (FCM) analysis revealed that cyclo-(Pro-Gly) caused reduction of Chl-a content, destruction of cell membrane integrity, chloroplasts and nuclear structure. Real-time PCR assay showed that the transcriptions of photosynthesis related genes (psbA, psbD, rbcL) were significantly inhibited. This study indicated that cyclo-(Pro-Gly) from marine Bacillus sp. Ts-12 exerted photosynthetic inhibition and oxidative stress to P. globosa and eventually led to the algal cells lysis. This algicidal compound might be potential bio-agent for controlling P. globosa red tide.

Salterns, one of the most extreme natural hypersaline environments, are a rich source of halophilic and halotolerant microorganisms, but they remain largely underexplored ecological niches in the discovery of bioactive secondary metabolites. In continued efforts to investigate the metabolic potential of microbial populations from chemically underexplored sites, three new lipopeptides named iturin F₁, iturin F₂ and iturin A₉ (1-3), along with iturin A₈ (4), were isolated from Bacillus sp. KCB14S006 derived from a saltern. The structures of the isolated compounds were established by 1D-, 2D-NMR and HR-ESIMS, and their absolute configurations were determined by applying advanced Marfey's method and CD spectroscopy. All isolates exhibited significant antifungal activities against various pathogenic fungi and moderate cytotoxic activities toward HeLa and src(ts)-NRK cell lines. Moreover, in an in vitro enzymatic assay, compound 4 showed a significant inhibitory activity against indoleamine 2,3-dioxygenase.

Bacterially-produced small molecules demonstrate a wide range of structural and functional diversity. A new diketopiperazine, cyclo(D-trans-Hyp-L-Leu) (1), and five other known diketopiperazines (2-6), were isolated and purified from the fermented broth of a Kenyan bacterium Bacillus licheniformis LB 8CT. The structure of 1 was elucidated by a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS, and the absolute configuration was determined by a combination of NOESY analysis and Marfey's method. The known compounds were identified as cyclo(D-cis-Hyp-L-Leu) (2), cyclo(D-cis-Hyp-L-Phe) (3), cyclo(D-Pro-L-Tyr) (4), cyclo-(D-Trp-L-Leu) (5), and cyclo(L-Tyr-Gly) (6) by comparison of their spectroscopic and physical data with reported values. Compounds 1-6 were tested for antifungal and antimicrobial properties.

Seven cyclic lipopeptide biosurfactants (1-7) were isolated for the first time from the fermentation broth of endophytic Bacillus clausii DTM1 and were identified as anteisoC13[Val7] surfactin-(L-Glu)-O-methyl-ester (1), anteisoC12[Val7] surfactin (2), anteisoC15[Val7] surfactin (3), isoC14[Leu7] surfactin (4), anteisoC12[Leu7] surfactin (5), nC13[Leu7] surfactin (6), and anteisoC14[Leu7] surfactin-(L-Glu)-O-methyl-ester (7); 1 has not been isolated before as a natural product from any source. Plate-based herbicide and insecticide bioassays showed that all compounds exhibited interesting insecticidal and herbicidal activities.

The motility of zoospores is critical in the disease cycles of the peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabolites regulating the motility of zoospores of Phytophthora capsici, we discovered two new inhibitors from the ethyl acetate extract of the fermentation broth of a marine-derived strain Bacillus sp. 109GGC020. The structures of these novel metabolites were elucidated as new cyclic lipopeptides and named gageopeptins A (1) and B (2) by spectroscopic analyses including high resolution MS and extensive 1D and 2D NMR. The stereoconfigurations of 1 and 2 were assigned based on the chemical derivatization studies and reviews of the literature data. Although compounds 1 and 2 impaired the motility of zoospores of P. capsici in dose- and time-dependent manners, compound 1 (IC50 = 1 μg/ml) was an approximately 400-fold stronger motility inhibitor than 2 (IC50 = 400 μg/ml). Interestingly, the zoospores halted by compound 1 were subsequently lysed at higher concentrations (IC50 = 50 μg/ml). Compounds 1 and 2 were also tested against some bacteria and fungi by broth dilution assay, and exhibited moderate antibacterial and good antifungal activities., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bacillus amyloliquefaciens L-H15 with broad spectrum antifungal activity was used as a biocontrol agent to suppress Fusarium oxysporum and other soil-borne fungal plant pathogens. Two antifungal fractions were isolated by bioactivity-guided reversed-phase high-performance liquid chromatography. The two compounds were identified by tandem Q-TOF mass spectroscopy as C15 Iturin A (1) and a novel cyclic peptide with a molecular weight of 852.4 Da (2). Both compounds showed good inhibitory activities against three plant fungal pathogens in cylinder-plate diffusion assay. To our best knowledge, this is the first report on a cyclic antifungal peptide with a molecular weight of 852.4 Da. The strong antifungal activity suggests that the B. amyloliquefaciens L-H15 and its bioactive components might provide an alternative resource for the biocontrol of plant diseases and sustainable agriculture.

We previously reported that Bacillus amyloliquefaciens biocontrol strain SD-32 produces powerful antifungal lipopeptides, C17 bacillomycin D homologues. In the course of the investigation we found that the antifungal activity of the culture supernatant of this bacterium was not ascribed exclusively to bacillomycin D. We attempted to identify metabolites other than bacillomycin D to gain insight into the mechanism for the biocontrol by this bacterium. After purifying the fractions of the culture supernatant exhibiting synergistic activity with bacillomycin D, we isolated two new cyclic lipodepsipeptides, anteiso-C13 and iso-C13 [Ile(7)]surfactins, together with three known [Ile(7)]surfactins. Interestingly, [Ile(7)]surfactins showed synergistic activities with bacillomycin D to gray mold disease on cucumber leaves but not to Botrytis cinerea itself in vitro, suggesting that the synergistic effects might be on infection processes of the fungus. Actually, we observed that they did not show synergistic actions on conidial germination or mycelial growth of B. cinerea on the leaves.

Lichenysin produced by 53 different Bacillus licheniformis strains has been structurally examined with a qualitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using quadrupole-time-of-flight mass spectrometry. The same lichenysin isoforms are produced from all strains, indicating that the growth conditions have a stronger influence on the lipopeptide production than the genotype. A rapid method for the quantification of lichenysin from bacterial cell cultures with LC-MS/MS after a simple methanol extraction has been refined. For the first time commercially available lichenysin has been used as calibrant, making quantification more accurate. The trueness for C15-lichenysin has been improved to 94% using matrix-matched calibration with lichenysin compared with 30% using solvent calibration with surfactin. The quantitative method was fully validated based on Commission Decision 2002/657/EC. The LOD of the method was below 1 µg g(-1) and the repeatability ranged from 10% to 16%.

While attempting to improve production of fluoro-iturin A in Bacillus sp. CS93 new mono- and di-fluorinated fengycins were detected in culture supernatants by (19)F NMR and tandem mass spectrometry, after incubation of the bacterium with 3-fluoro-L-tyrosine. The fluorinated amino acid was presumably incorporated in place of one or both of the tyrosyl residues in fengycin. Investigations to generate additional new fluorinated derivatives were undertaken using commercially available fluorinated phenylalanines and 2-fluoro- and 2,3-difluoro-tyrosine that were synthesised by Negishi cross-coupling of iodoalanine and fluorinated bromo-phenols. The anti-fungal activity of the fluorinated lipopeptides was assayed against Trichophyton rubrum and found to be similar to that of the non-fluorinated metabolites.

Since Streptococcus mutans is the principal etiologic agent causing dental caries, by encompassing an array of unique virulence traits, emerging treatment strategies that specifically target the virulence of this pathogen may be promising as alternative approaches compared to conventional antibiotic therapy. In this perspective, we investigated chloroform extract of cell-free culture supernatant from mangrove rhizosphere bacterium Bacillus amyloliquefaciens (MMS-50) in terms of anticariogenic properties of S. mutans, without suppressing its viability. Crude chloroform extract of MMS-50 was subjected to column and high performance liquid chromatographic techniques to obtain the active fraction (AF), and MMS-50 AF was used for all further assays. GC-MS and FT-IR were carried out to identify the major components present in MMS-50 AF. Comparative gene expression analysis of some biofilm-forming and virulence genes (vicR, comDE, gtfC, and gbpB) was done by real-time PCR. Cyclo(L-leucyl-L-prolyl) was found to be the chief compound in MMS-50 AF responsible for bioactivity. The minimum and maximum inhibitory concentrations of MMS-50 AF against S. mutans were found to be 100 and 250 μg/mL, respectively. Anti-virulence assays performed using below-sub-MIC levels of MMS-50 AF (30 μg/mL) resulted in significant reduction in adherence (68%), acid production, acid tolerance, glucan synthesis (32%), biofilm formation (53.5%) and cell surface hydrophobicity, all devoid of affecting its viability. The micrographs of CLSM and SEM further confirmed the antibiofilm and anti-virulence efficacies of MMS-50 AF. Expression data showed significant reduction in expression of all studied virulence genes. Thus, the current study unveils the anticariogenic potential of cyclo(L-leucyl-L-prolyl) from B. amyloliquefaciens, as well as its suitability as a novel and alternative anticariogenic agent against dental caries., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The broad-spectrum fungal antagonist, Bacillus amyloliquefaciens 6B (BA6B), isolated from the Jakhao coast of Kutch, India, was investigated for its antifungal metabolites using mass spectrometry. The cyclic lipopeptides harvested from the cell-free fermentation broth of BA6B by acid precipitation and subsequently dissolved in methanol were subjected to liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS/MS) for their identification and sequence determination. The 26 types of surfactin variants were identified from the methanolic extract by LC-ESI-MS/MS analysis. Among 26 surfactin species, several new cyclic as well as acyclic surfactin variants based on the variation in the β-hydroxy fatty acid (β-OH FA) chain length and/or in amino acid positions 4, 5, 6, and 7 were identified. The mass spectrometric analysis of crude extract also enabled the identification of 11 unique molecular mass ions with minimum two or maximum four types of isobaric peptide variants., (© 2013 International Union of Biochemistry and Molecular Biology, Inc.)

In continuation of our search for new bioactive secondary metabolites from Bacillus cereus associated with entomopathogenic nematode (EPN), three cyclic dipeptides (CDPs), cyclo(L-Leu-D-Arg) (1), cyclo(2-hydroxy-Pro-L-Leu) (2), and cyclo(L-Val-L-Pro) (3) were purified from the ethyl acetate extract of B. cereus. The chemical structure of the compounds was identified by 1D, 2D NMR and HR-ESI-MS. Cyclo(L-Leu-D-Arg) recorded best antifungal activity and the highest activity was recorded against Cryptococcus neoformans (1 μg/mL), which is better than the standard antifungal agent amphotericin B. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used for finding cell proliferation inhibition and cyclo(L-Leu-D-Arg) recorded significant activity against breast cancer cell line (MDAM-B231) (IC50 value: 25 μM) and the three cyclic dipeptides recorded no toxicity against normal human cell (fore skin (FS) normal fibroblast) up to 50 μM except cyclo(L-Val-L-Pro). Cyclo(L-Leu-D-Arg) induced significant morphological changes and DNA fragmentation associated with apoptosis in MDAM-B231 cells by acridine orange/ethidium bromide staining and flow cytometry analysis. Out of three cyclic dipeptides tested only cyclo(2-hydroxy-Pro-L-Leu) recorded significant antioxidant activity. The hydroxyl radical scavenging activity of cyclo(2-hydroxy-Pro-L-Leu) is greater than BHA, the standard antioxidant agent. Cyclo(L-Leu-D-Arg) was isolated for the first time from a natural source with a d-arginine residue. To the best of our knowledge, this is the first time that the bioactivity of the isolated cyclic dipeptides is reported against medically important fungi and cancer cells. This study is a significant contribution to the knowledge of cyclo(L-Leu-D-Arg) from B. cereus as potential sources of new drugs in the pharmacological industry, especially as potent antifungal and anticancer agent., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A bioassay-guided fractionation of Bacillus amyloliquefaciens strain BO5A afforded the isolation of two new cyclic lipopeptides (1 and 2) as the major lipid constituents (>60%) of the CHCl3-MeOH (2:1) extract. The chemical structures of the isolated metabolites were elucidated by spectroscopic methods, including 1D and 2D NMR spectroscopy, mass spectrometry (MS), secondary ion mass spectrometry (MS1, MS2), and chemical degradation. The compounds are members of the surfactins family and are based on a heptapeptide chain composed by Glu-Val-Leu-Val-Asp-Leu-Leu. Its N-terminal end is N-acylated by an (R)-3-hydroxy fatty acid with linear alkyl chains of 16:0 and 15:0 (1 and 2, respectively). The 3-hydroxyl group closes a 25-membered lactone ring with the carboxylic group of the C-terminal amino acid. The isolated compounds were tested for their inhibitory activity against the four pathogenic fungi Fusarium oxysporum, Aspergillus niger, Botrytis cinerea, and Penicillium italicum and the biocontrol fungus Trichoderma harzianum. Compound 2 displayed activity against all tested pathogens.

The TrigoCor strain of Bacillus amyloliquefaciens provides consistent control against Fusarium head blight of wheat in controlled settings but there is a lack of disease and deoxynivalenol suppression in field settings. Since production of antifungal compounds is thought to be the main mode of action of TrigoCor control, we quantified levels of a key family of antifungal metabolites, iturins, as well as monitored Bacillus populations on wheat spikes over 14 days post-application in both the greenhouse and the field. We found that initial iturin levels on spikes in the greenhouse were three times greater than on spikes in the field, but that by 3 days post-application, iturin levels were equivalent and very low in both settings. We also determined that iturins declined rapidly over a 3-day post-application period on wheat spikes in both environments, despite the presence of significant Bacillus populations. Greenhouse trials and antibiosis tests indicated that the lower iturin levels on wheat spikes in the field could be a major factor limiting disease control in field settings. Future efforts to improve Bacillus disease control on wheat spikes and in the phyllosphere of various plants should focus on maintaining higher levels of iturins over critical infection periods.

Bacillus sp. associated with an entomopathogenic nematode is shown to produce diketopiperazine (DKP) that showed stronger antifungal activity against Colletotrichum gloeosporioides [minimum inhibitory concentration (MIC): 8 μg mL(- 1)] than commercial fungicide oligochitosan (MIC: 125 μg mL(- 1)). DKP identified as cyclo(D-Tyr-L-Leu) was isolated for the first time from a natural source with a d-tyrosine residue. This report also demonstrates for the first time an antifungal property exploration of cyclo(Tyr-Leu) class of dipeptides. The structural elucidation was carried out using 1D, 2D NMR methods and HPLC.

Three lipopeptides were isolated by bioactivity-guided fractionation from the fermentation broth of Bacillus mojavensis B0621A. A new iturinic lipopeptide, named mojavensin A, was tentatively characterized by 1D, 2D NMR and MS spectroscopy, Marfey's method containing a novel peptide backbone of L-Asn₁, D-Tyr₂, D-Asn₃, L-Gln₄, L-Pro₅, D-Asn₆, L-Asn₇ and an anteiso-type of the saturated β-fatty acid side chain. Compound 2 and 3 were tentatively identified as iso-C16 fengycin B and anteiso-C17 fengycin B, respectively. These lipopeptides displayed dose-dependent antifungal activity against a broad spectra of phytopathogens and were weakly antagonistic to Staphylococcus aureus. Moreover, they all revealed cytotoxic activities against the human leukemia (HL-60) cell line. Mojavensin A, iso-C16 fengycin B, and anteiso-C17 fengycin B inhibited the growth of HL-60 with IC₅₀ of 100, 100 and 1.6 μM, respectively.

Bacillus amyloliquefaciens LBM 5006 produces an antimicrobial factor active against Paenibacillus larvae, a major honeybee pathogen. The antagonistic effect and the mode of action of the antimicrobial factor were investigated. The antibacterial activity was produced starting at mid-logarithmic growth phase, reaching its maximum during the stationary phase. Exposure of cell suspensions of P. larvae to this antimicrobial resulted in loss of cell viability and reduction in optical density associated with cell lysis. Scanning electron microscopy showed damaged cell envelope and loss of protoplasmic material. The antimicrobial factor was stable for up to 80°C, but it was sensitive to proteinase K and trypsin. Mass spectrometry analysis indicates that the antimicrobial activity is associated with iturin-like peptides. The antimicrobial factor from B. amyloliquefaciens LBM 5006 showed a bactericidal effect against P. larvae cells and spores. This is the first report on iturin activity against P. larvae. This antimicrobial presents potential for use in the control of American foulbrood disease.

The lipopeptide surfactin secreted by plant-beneficial bacilli has crucial biological functions among which the ability to stimulate immune-related responses in host tissues. This phenomenon is important for biological control of plant diseases but its molecular basis is still poorly understood. In this work, we used various approaches to study the mechanism governing the perception of this biosurfactant at the plant cell surface. Combining data on oxidative burst induction in tobacco cells, structure/activity relationship, competitive inhibition, insertion kinetics within plant membranes and thermodynamic determination of binding parameters on model membranes globally indicates that surfactin perception relies on a lipid-driven process at the plasma membrane level. Such a sensor role of the lipid bilayer is quite uncommon considering that plant basal immunity is usually triggered upon recognition of microbial molecular patterns by high-affinity proteic receptors., (© 2011 Blackwell Publishing Ltd.)

Iturin A, a powerful antifungal surfactant, is a kind of bacterial lipopeptide produced by Bacillus strains. This study addresses the use of an aqueous two-phase system (ATPS) using ethanol/ammonium sulfate to extract iturin A from Bacillus amyloliquefaciens NJN-6 fermentation broth and the quantification of iturin A by HPLC. Baseline separation of iturin A homologues was performed using an RP-C(18) column with a mixture of water and acetonitrile. The results showed that the correlation coefficient between integral area and concentration was 0.9961 within the range of 20-140 mg/l. The RSD of the retention time and the peak area were 1.29% and 1.45%, respectively. The effects of some operating parameters in ATPS, e.g., pH, temperature and centrifugation time, were also studied. This method can be successfully used for the rapid quantification of iturin A., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Some of the most effective antibiotics (e.g. Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of MS-based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage MS, and show its advantages over single-stage MS. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three new lipopeptides (1-3) were isolated from the organic extract of Bacillus amyloliquefaciens strain (BO7). These compounds represented the major constituents (>60%) of the total cell lipids extractable with CHCl(3)/MeOH (2:1). Elucidation of their chemical structure was carried out by spectroscopic analyses, including 1D and 2D NMR spectroscopy, mass spectrometry (MS), and secondary ion mass spectrometry (MS/MS), along with chemical degradation. The compounds are members of the surfactins family and are based on the heptapeptide Glu-Leu-Leu-Ala-Asp-Leu-Leu, N-acylated to the N-terminal by an (R)-3-hydroxy fatty acid with linear alkyl chains from 16:0 to 18:0 (1-3, respectively). An ester bond between the 3-hydroxyl group of the fatty acid and the carboxylic group of the C-terminal amino acid closes a 13-membered lactone ring. The bacterial lipopeptides, particularly compound 3, displayed strong and dose-dependent antifungal activity against the plant pathogenic fungus Fusarium oxysporum.

Nosemosis, a disease caused by a microsporidian infection, is one of the most frequently observed parasitic pathologies affecting adult honeybees. Presently, Nosema ceranae seems to be the main microsporidian infection in Apis mellifera. The antibiotic fumagillin is the only compound available to treat Nosema diseases; however, it is no longer licensed in most EU member states; therefore, the need to identify new molecules/substances prevails. The intent of this paper is to test bacterial metabolites by Bacillus and Enterococcus strains, isolated from bee midgut and honey. The toxicity on bees and the antiparasitic activity on N. ceranae were assessed under laboratory conditions. Results did not yield toxicity for the administered surfactin or bacteriocin concentrations. Spores exposed to direct contact with a particular surfactin revealed a significant infectivity reduction when inoculated on bees. This surfactin, administered ad libitum from the individuals' emergence, led to a significant reduction in parasitosis development when bees were infected with untreated spores 7 days postemergence. Based on the results obtained, one of the surfactins is herein postulated as a molecule capable of reducing N. ceranae development, acting either by direct exposure to purified spores or incorporated into the digestive tract of the bee.

Aims: A Bacillus amyloliquefaciens strain, surviving epiphytically on the surface of fruit, was isolated while searching for naturally occurring biological control agents. This bacterial strain was characterized for its antifungal activity against seven selected fungal postharvest pathogens of citrus., Methods and Results: To understand the antifungal activity, seven postharvest fungal pathogens were screened for growth inhibition by B. amyloliquefaciens strain. Assays using B. amyloliquefaciens lipopeptide extracts showed a strong inhibitive activity. The inhibitory effect was observed in abnormal conidial germination and germ tube development when conidia were treated with different lipopeptide extract concentrations. Further analysis using PCR and chromatography confirmed the presence of fengycin, iturin and surfactine, of which iturin A showed the strongest and most common inhibitory effect. The results are supported by site-directed mutagenesis analysis, targeted to suppress the biosynthesis of iturin A production. Fruit trials confirmed disease development inhibition when the antagonist was applied 1 day prior to or 1 day after fungal application., Conclusions: We conclude that the iturin family of lipopeptides are vital in the antagonism of B. amyloliquefaciens against the seven citrus postharvest pathogenic fungi tested., Significance and Impact of the Study: We elucidated the principal mechanism used by B. amyloliquefaciens PPCB004 to suppress postharvest disease development on stored fruits.

To establish planar biomimetic membranes across large scale partition aperture arrays, we created a disposable single-use horizontal chamber design that supports combined optical-electrical measurements. Functional lipid bilayers could easily and efficiently be established across CO(2) laser micro-structured 8 x 8 aperture partition arrays with average aperture diameters of 301 +/- 5 microm. We addressed the electro-physical properties of the lipid bilayers established across the micro-structured scaffold arrays by controllable reconstitution of biotechnological and physiological relevant membrane peptides and proteins. Next, we tested the scalability of the biomimetic membrane design by establishing lipid bilayers in rectangular 24 x 24 and hexagonal 24 x 27 aperture arrays, respectively. The results presented show that the design is suitable for further developments of sensitive biosensor assays, and furthermore demonstrate that the design can conveniently be scaled up to support planar lipid bilayers in large square-centimeter partition arrays.

Bacillus mojavensis is an endophytic bacterium patented for control of fungal diseases in maize and other plants. Culture extracts and filtrates from this bacterium were antagonistic to the pathogenic and mycotoxic fungus Fusarium verticillioides. However, the identity of the inhibitory substance from extracts of this bacterium has not been determined. An HPLC-MS analysis of the culture filtrate showed a major ion peak that was identified as a cyclic lipopeptide. Furthermore, collisional ion dissociation (CID) analysis indicated that this lipopeptide was surfactin, a cyclic heptapeptide linked to a β-hydroxy fatty acid. A CID analysis of the peptide moiety was established by deduction and indicated that the peptide sequence consisted of two acidic amino acids and five hydrophobic amino acids with a sequence of Leu-Leu-Asp-Val-Leu-Leu-Glu. These spectra indicated that this bacterium produced Leu(7)-surfactin, which was toxic to F. verticillioides. Production of this cyclic lipopeptide is a characteristic of several species of Bacillus, but this is the first report of this very powerful biosurfactant from this endophytic species.

Cyclic lipopeptide, also named as acylpeptide, which was characteristic with novel structures, was paid more attention in the recent years. Cyclic lipopeptide showed various bioactivities including antibacterial, anti-tumor, anti-inflammatory, etc. Cyclic lipopeptide originated mainly from the second metabolites of microorganism, such as Cyanobacterium, Bacterium, Actinomyces, etc. The bacteria included the genus of Bacillus and Pseudomonas. In this account, the review has been made on the development of cyclic lipopeptide.

Aims: To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product-derived Bacillus amyloliquefaciens., Methods and Results: An unknown bacterial species cultured from the Yogu Farm probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell-free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp., Conclusion: The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis., Significance and Impact of the Study: This is the first report of intra-species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin's activity against bacterial vaginosis-associated pathogens.

Lipopeptides represent a unique class of bioactive microbial secondary metabolites, and iturin A shows attractive antibiotic properties among them. This study compares three methods, such as yeast/fungal growth inhibition assay, quantitative high-performance liquid chromatography (HPLC) and polymerase chain reaction (PCR) for identifying a number of Bacillus species that produce iturin A. We examined the feasibility of screening iturin A-producing Bacillus strains by PCR using specific primers for ituD and lpa-14 amplification. Twenty standard strains and 120 field-collected Bacillus spp. isolates were tested in this study. Four B. subtilis and one B. circulan strains from ATCC, and B. amyloliquefaciens B128, a known iturin A producer, exhibited positive results. Of the 120 field-collected isolates, 42 strains were positive. The potential of producing iturin A by these PCR-positive strains were then confirmed by conventional methods such as fungal growth inhibition assay and HPLC analysis. The consistency between results of PCR, HPLC, and fungal growth inhibition assay suggests that the PCR method could be used as an alternative tool for fast screening of iturin A-producing Bacillus strains from the environment. This is the first report of detecting iturin A production from B. circulans.

A new surfactin isomer (1) was isolated from a mangrove bacteria strain 'Bacillus sp'. Its structure was identified, and full assignments of (1)H and (13)C NMR spectral data were achieved for the first time by a combination of mass spectrometry and 1D and 2D NMR experiments including DEPT, (1)H-(1)H COSY, HSQC, HMBC, TOCSY, ROESY, and HSQC-TOCSY. The NMR spectral data of eight known analogs (2-9) are also reported.

Compounds that inactivate lipopolysaccharide (LPS) activity have the potential of being new anti-inflammatory agents. Therefore, we searched among microbial secondary metabolites for compounds that inhibited LPS-stimulated adhesion between human umbilical vein endothelial cells (HUVEC) and HL-60 cells. By this screening, we found a cyclic lipopeptide surfactin from the culture broth of Bacillus sp. BML752-121F2 to be inhibitory. The addition of the surfactin prior to the LPS stimulation decreased HL-60 cell-HUVEC adhesion without showing any cytotoxicity. We confirmed that surfactin inhibited LPS-induced expression of ICAM-1 and VCAM-1 in HUVEC. It also inhibited the cellular adhesion induced by lipid A, the active component of LPS; but it did not inhibit TNF-alpha or IL-1 beta-induced cell adhesion. Then, surfactin was shown to suppress the interaction of lipid A with LPS-binding protein (LBP) that mediates the transport of LPS to its receptors. Finally, surface plasmon resonance (SPR) analysis revealed the surfactin to interact reversibly with lipid A. Thus, this Bacillus surfactin was shown to be an inhibitor of LPS-induced signal transduction, directly interacting with LPS.

Three new cytotoxic cyclopeptides belonging to iturin class have been isolated from marine bacterium Bacillus sp. These three compounds have the cytotoxic activities. Their structures were determined based on chemical analysis and all kinds of spectroscopic techniques.

Fungicidic Bacillus amyloliquefaciens strains isolated from the indoor environment of moisture-damaged buildings contained heat-stable, methanol-soluble substances that inhibited motility of boar spermatozoa within 15 min of exposure and killed feline lung cells in high dilution in 1 day. Boar sperm cells lost motility, cellular ATP, and NADH upon contact to the bacterial extract (0.2 microg dry wt/ml). Two bioactive substances were purified from biomass of the fungicidal isolates. One partially characterized substance, 1,197 Da, was moderately hydrophobic and contained leucine, proline, serine, aspartic acid, glutamic acid and tyrosine, in addition to chromophore(s) absorbing at 365 nm. In boar sperm and human neural cells (Paju), the compound depolarized the transmembrane potentials of mitochondria (Delta Psi(m)) and the plasma membrane (Delta Psi(p)) after a 20-min exposure and formed cation-selective channels in lipid membranes, with a selectivity K(+):Na(+):Ca(2+) of 26:15:3.5. The other substance was identified as a plasma-membrane-damaging lipopeptide surfactin. Plate-grown biomass of indoor Bacillus amyloliquefaciens contained ca. 7% of dry weight of the two substances, 1,197 Da and surfactin, in a ratio of 1:6 (w:w). The in vitro observed simultaneous collapse of both cytosolic and mitochondrial ATP in the affected mammalian cell, induced by the 1,197-Da cation channel, suggests potential health risks for occupants of buildings contaminated with such toxins.