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Skeletonema costatum is a diatom widely distributed in red tide microalgae blooms and as one of the main algae causing harmful algal blooms, because of their rapid reproduction and production of toxic and harmful substances, often play a negative role in aquatic ecosystems, and human health and wellbeing. Bacillomycin D is a nonribosomal cyclic antifungal lipopeptide in the iturins family. In this study, synthetic Bacillomycin D was tested for its ability to inhibit the growth of S. costatum. The EC50. 24h of Bacillomycin D on S. costatum was 24.70 μg/mL. The chlorophyll fluorescence parameters Fv/Fm, Fv/Fo, and yield of the diatoms decreased significantly with increasing concentrations of Bacillomycin D. Study of the mechanism showed that Bacillomycin D induced cell death by changing cell membrane permeability, promoting the release of cellular contents. In this study, transcriptomic analysis showed Bacillomycin D significantly inhibited the photosynthesis and metabolism of S. costatum. These findings investigated the inhibitory effect of Bacillomycin D on the growth of S. costatum and provided a theoretical foundation for the development of new environmentally friendly biological algicide.

In wild strains of Bacillus, a handful of extracellular natural products act as signals that can regulate multicellular behavior, but relatively little is known about molecular mechanisms’ detail. We proposed a previously unreported molecular mechanism for triggering multicellularity in B. velezensis Bs916 by an endogenous cyclic lipopeptide, bacillomycin L. The genome-wide effect on gene expression was caused by the disruption of bacillomycin L gene cluster, and 100 µg/mL bacillomycin L was revealed by quantitative transcriptomics. A total of 878 differentially expressed genes among Bs916, Δbl, and Δbl + 100BL were identified and grouped into 9 functional categories. The transcription levels of 40 candidate genes were further evaluated by RT-qPCR analysis. The expression of eight candidate genes regulated by bacillomycin L in a dose-dependent manner was revealed by LacZ fusion experiment. Although the addition of bacillomycin L could not completely restore the expression levels of the differentially regulated genes in △bl, our results strongly suggest that bacillomycin L acts as a tuning signal of swarming motility and complex biofilm formation by indirectly regulating the expression levels of some two-component systems (TCSs) connector genes, particularly including several Raps that potentially regulate the phosphorylation levels of three major regulators ComA, DegU, and Spo0A. Key points • Proposed model for bacillomycin L regulation in B. velezensis Bs916. • Bacillomycin L can act as an extracellular signal to regulate the phosphorylation levels of three major regulators, ComA, DegU, and Spo0A and control the multicellular processes of vegetative growth, competent, motility, matrix production, sporulation, and autolysis.

To evaluate the role of the biocontrol agent Bacillus subtilis CtpxS2-1 in inducing lupin systemic resistance against anthracnose caused by Colletotrichum acutatum by lipopeptide production. First, growth inhibition and thin layer chromatography-bioautography analysis confirmed that CtpxS2-1 cultures and their lipopeptide extracts, specifically fengycin, have strong antifungal activity against C. acutatum. Subsequent microscopic examination of these fungal inhibition zones showed mycelial pathogen deformations. PCR amplification of CtpxS2-1 confirmed the presence of genes encoding fengycins E and C, bacillomycin C, iturin A, and surfactins B and C. Based on this evidence, the effect of CtpxS2-1 and its lipopeptides on the induction of the lupin defence- and growth-related genes PR-1, PR-4, SOD-2, PIN-1 and PIN-3 was evaluated by RT-qPCR. In seedlings from roots treated with CtxpS2-1, a significant increase in the expression of these genes was induced. Efficacy assays showed that CtpxS2-1 treatment completely controlled anthracnose incidence (0.0%) compared with the untreated control. Furthermore, root and shoot growth in treated seedlings with CtpxS2-1 significantly increased due to disease control, as did the synthesis of the defence enzymes catalase, peroxidase and superoxide dismutase. B. subtilis CtpxS2-1 is a key factor enhancing Andean lupin health by producing lipopeptides that damage C. acutatum cellular structures and inhibit their growth, as well as by inducing the expression of defence-related genes of lupin plants involved in systemic acquired resistance (SAR) against anthracnose.

Bacillus amyloliquefaciens belongs to the genus Bacillus and family Baciliaceae. It is ubiquitously found in food, plants, animals, soil, and in different environments. In this review, the application of B. amyloliquefaciens in probiotic and prebiotic microbes in fermentation, synthesis, and hydrolysis of food compounds is discussed as well as further insights into its potential application and gaps. B. amyloliquefaciens is also a potential microbe in the synthesis of bioactive compounds including peptides and exopolysaccharides. In addition, it can synthesize antimicrobial compounds (e.g., Fengycin, and Bacillomycin Lb), which makes its novelty in the food sector greater. Moreover, it imparts and improves the functional, sensory, and shelf life of the end products. The hydrolysis of complex compounds including insoluble proteins, carbohydrates, fibers, hemicellulose, and lignans also shows that B. amyloliquefaciens is a multifunctional and potential microbe which can be applied in the food industry and in functional food processing.

Fusarium wilt of watermelon caused by Fusarium oxysporum f. sp. niveum (FON) is a destructive soil-borne disease. The objective of this study was to screen antagonistic bacteria with potential applications for controlling this disease. Strain IBFCBF-4 with obvious antifungal activity against FON and seven other phytopathogens was successfully isolated from a rhizosphere soil. Based on the morphological, physiological, biochemical characteristics and 16S rDNA sequence analysis, IBFCBF-4 was identified as Bacillus subtilis. Under greenhouse condition, IBFCBF-4 could significantly reduce the incidence of Fusarium wilt of watermelon by 51.1% compared to the control. The studies on the antagonistic mechanism showed that IBFCBF-4 could cause abnormal swelling and increased branching to the hyphae of FON. Meanwhile, the volatile and non-volatile compounds produced by IBFCBF-4 were able to suppress the mycelial growth of FON. Furthermore, PCR analysis confirmed the presence of ituA, ituB, ituC, ituD, bamD, bmyB, fenB, srfAB, surf, bacA, bacD, bacAB, tasA, mrsA and mrsM genes involved in the biosynthesis of iturin A, bacillomycin, fengycin, surfactin, bacilysin, tasA and mersacidin, respectively. Taken together, B. subtilis IBFCBF-4 can be used as a potential biocontrol agent against Fusarium wilt of watermelon, but further work is required to improve its efficacy.

Objective: To investigate the inhibitory effect on Burkholderia pseudomallei (B. pseudomallei) strain HNBP001 of a bacillomycin D-like cyclic lipopeptide compound named bacillomycin DC isolated from Bacillus amyloliquefaciens HAB-2. Methods: The antibacterial effect of bacillomycin DC on B. pseudomallei was determined using the disk diffusion method. The minimum inhibitory concentrations were evaluated by microdilution assay. In addition, transmission electron microscopy was performed and quantitative real-time polymerase chain reaction assay was carried out to determine the expression of MexB, OprD2, and qnrS genes. Results: Bacillomycin DC produced an inhibition zone against B. pseudomallei with minimum inhibitory concentration values of 12.5 μg/mL 24 h after treatment and 50 μg/mL at 48 and 72 h. Transmission electron microscopy showed that bacillomycin DC resulted in roughening cell surface and cell membrane damage. Quantitative real-time polymerase chain reaction analysis showed low expression of MexB, OprD2 and qnrS genes. Conclusions: Bacillomycin DC inhibits the growth of B. pseudomallei and can be a new candidate for antimicrobial agents of B. pseudomallei.

Biological control is a method of applied ecology that uses one organism, or its product, to inhibit another pathogenic organism. This method in plant protection should be more environmentally friendly than commonly used pesticides. The rhizobacterium Bacillus velezensis FZB42, which can suppress the growth of other microorganisms, can be used as a biological control agent. Xanthomonas campestris pv. campestris is a phytopathogen that causes significant losses in agricultural production. This diploma thesis proved an antagonistic effect of the bacterial strain B. velezensis FZB42 on the phytopathogenic bacteria Xanthomonas campestris pv. campestris SU in mixed culture. The study confirmed that the antibiotic effect of B. velezensis strain FZB42 against X. campestris pv. campestris SU in mixed culture depends on the inoculation ratios of both strains. The interaction of the two bacterial strains was characterized by metabolomics analysis, it was confirmed that B. velezensis FZB42 produces four dominant secondary metabolites: lipopeptides surfactin, fengycin, and bacillomycin and siderophore bacillibactin, which show antifungal and antibiotic activity. The inhibitory activity of B. velezensis strain FZB42 was further monitored by SEM analysis, which showed damage to X. campesris pv. campestris SU...

Lipopeptides (such as iturin, fengycin, and surfactin) from Bacillus possess antibacterial, antifungal, and antiviral activities and have important application in agriculture and pharmaceuticals. Although unremitting efforts have been devoted to improve lipopeptide production by designing gene regulatory circuits or optimizing fermentation process, little attention has been paid to utilizing multi-omics for systematically mining core genes and proteins during the bacterial growth cycle. Here, lipopeptide bacillomycin Lb from new Bacillus amyloliquefaciens X030 was isolated and first found to have anticancer activity in various cancer cells (such as SMMC-7721 and MDA-MB-231). A comprehensive genomic and growth proteomic analysis of X030 revealed bacillomycin Lb biosynthetic gene cluster, key enzymes and potential regulatory proteins (PerR, PhoP, CcpA, and CsfB), and novel links between primary metabolism and bacillomycin Lb production in X030. The antitumor activity of the fermentation supernatant supplemented with amino acids (such as glutamic acid) and sucrose was significantly increased, verifying the role of key metabolic switches in the metabolic regulatory network. Quantitative real-time PCR analysis confirmed that 7 differential expressed genes exhibited a positive correlation between changes at transcriptional and translational levels. The study not only will stimulate the deeper and wider antitumor study of lipopeptides but also provide a comprehensive database, which promotes an in-depth analysis of pathways and networks for complex events in lipopeptide biosynthesis and regulation and gives great help in improving the yield of bacillomycin Lb (media optimization, genetic modification, or pathway engineering).

The inhibitory effect of Bacillomycin D, a cyclic lipopeptide, on Rhizopus stolonifer colonization of cherry tomato was studied, and its possible mechanism of action was explored. Bacillomycin D showed a direct inhibitory effect on R. stolonifer spore germination and mycelial growth in vitro. It conferred both a direct inhibitory effect on R. stolonifer growth in cherry tomato in vivo and induced host resistance in cherry tomato. Moreover, Bacillomycin D treatment significantly increased the activities of plant defense-related enzymes, including chitinase (CHI), β-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and peroxidase (POD). Real-time PCR (RT-PCR) showed that defense-related genes involved in the salicylic acid defense signaling pathway and genes encoding pathogenesis-related proteins were up-regulated in Bacillomycin D treatment. Furthermore, Bacillomycin D-C16 resulted in direct inhibition and a remarkable induced resistance to R. stolonifer which was higher than as induced by Bacillomycin D-C14. Together, the data indicated that Bacillomycin D can control the growth of R. stolonifer through both the direct inhibition of the fungus and the activation of defense-related genes and enzymes in cherry tomato.

Whole-genome sequencing of bacteria Bacillus velezensis BIM B-439D has revealed that its genome is represented by a single circular chromosome 3 978 954 bp in size and contains 3969 predicted genes. Chromosome has 10 conserved loci that determine the production of antimicrobial compounds of varying chemical nature, namely lipopeptides (surfactin, bacillomycin, fengycin), polyketides (bacillaene, difficidin/oxydifficidin, macrolactin), bacillibactin siderophore, dipeptide bacilysin, a protein/polyketide of uncertain composition, and amylocyclicin bacteriocin. Mutants with impaired surfactin synthesis have been obtained by direct mutagenesis; they were characterized by an increased production of bacillomycin and fengycin. The obtained mutants demonstrated high antimicrobial activity towards a number of bacterial and fungal pathogens (Penicillium expansum, Alternaria tenuis, Botrytis cinerea,Bipolaris sorokiniana), but they inhibited the growth of the Fusarium genus fungi to a lesser degree.

Bacillus strains are well studied for antagonistic effect against fungal pathogens, but the selection of potential antifungal strains is laborious and time-consuming process. Newly developed genetic markers and LC-MS based detection was undertaken simultaneously to detect eight antimicrobial compounds viz., surfactin, bacillomycin, iturin, fengycin/plipastatin, bacilysin, bacillaene, bacillibactin and plantazolicin in forty-seven Bacillus strains. Out of these strains, 19 were positive for the presence of marker genes encoding antimicrobial compounds. Bacillus strains FZB42, EZ1509, EZ1507, VM10, GBAC46, VM49 and LLCG43 possessed genes for maximum number of antimicrobial compounds. LC-MS analysis of antimicrobial compounds showed corresponding results except OKB105 and 168. Contrary to marker-based detection of genes, LC-MS analysis revealed that OKB105 can produce surfactin but unable to synthesize fengycin, while 168 was deficit in both compounds. To assay antifungal potential, 19 Bacillus strains and their methanolic extracts were tested in vitro to inhibit mycelial growth of S. sclerotiorum. Results revealed that EZ1509, VM10, GBAC46, VM49 and FZB42 showed highest inhibitory activity. A bioassay on detached rapeseed leaves demonstrated that strains VM10, EZ1509, FZB42 and GBAC46 were excellent in reducing lesion diameter, while, OKB105 and 168 were completely ineffective to control S. sclerotiorum. Interestingly, antifungal activity of Bacillus strains was positively co-related to the number of antimicrobial genes, indicating their role in antifungal activity of Bacillus strains. Our findings suggest that combining genetic markers and LC-MS analysis can rapidly screen Potential Bacillus strains with antifungal attributes and this screening method can serve as foundation for the development of new biopesticides.

Blossom blight caused by Botrytis cinerea is one among the most devastating diseases that cause complete post-harvest loss in flower crops. The present study focuses on the development of effective bioformulation towards suppression of blossom blight and plant growth promotion in rose. Bacillus amyloliquefaciens (VB2) and Bacillus subtilis (AP) effectively inhibited mycelial growth of B. cinerea in vitro. Genome screening of VB2 and AP revealed the presence of antimicrobial peptide genes including, ituD, ipa14, bacA, bacD, srfA, sfP, spaC, spaS responsible for the biosynthesis of antibiotics such as iturin, bacilysin, bacillomycin, surfactin and subtilin. Further, the presence of volatile antifungal compounds in the bacterial secretome was identified through gas chromatography–mass spectrometry (GC/MS) analysis. Upon treatment, AP accelerated the metabolite profile of the plants and a rise in peak area of antifungal compounds such as, pentadecanoic acid, n-hexadecanoic acid, octadecanoic acid (stearic acid) and tetradecanoic acid was observed. In vitro, VB2 produced maximum indole acetic acid (9.17 µg/ml) and gibberellic acid (8.20 µg/ml) in nutrient broth. Under field conditions, foliar spray of VB2 at 0.5% (5 ml/l), four times at weekly interval suppressed blossom blight incidence (64% reduction over control) and also promoted yield. Future research towards development of an effective bioformulation with extended shelf life will aid in the management of various fungal, bacterial and viral diseases in different crop plants.

Bacillomycin D can inhibit the growth of Aspergillus ochraceus in food samples. In addition, it can induce apoptosis in and inhibit the proliferation of cancer cells, although the details of this mechanism are unknown. In this study, we separated bacillomycin D-C14, D-C15, D-C16 monomers from the Bacillus subtilis strain fmbJ. The bacillomycin D monomers containing longer fatty acid chains better induced apoptosis in Bgc-823, Sgc-7901, and Hgc-27 gastric cancer cells. The Bgc-823 cell line was the most sensitive. Acridine orange-ethidium bromide staining indicated that bacillomycin D-C16-induced Bgc-823 cell death by triggering apoptosis, characterized by membrane blebbing, cellular shrinkage, and DNA fragmentation. Flow cytometric analysis showed a bacillomycin D-C16 dose-dependent trigger of Bgc-823 apoptosis. Bacillomycin D-C16-induced the mitochondrial pathway, as indicated by a reduced Bcl-2/Bax expression ratio, enhanced cytochrome C release, and higher levels of cleaved caspase-3. Furthermore, bacillomycin D-C16 effectively repressed phosphorylation of the serine-threonine protein kinase Akt at Ser-473 and increased the levels of the FoxO3a protein. The combination of the PI3K/Akt-inhibitor BEZ235 with bacillomycin D-C16 enhanced the apoptosis of Bgc-823 cells. Together, these findings indicated that bacillomycin D-C16 induces apoptosis through the PI3K/Akt and FoxO3a signaling pathways.

The study aimed to identify novel antagonistic microorganisms suitable for biological control of diseases in vegetables grown under tropical conditions. Forty seven Bacillus strains were isolated from rhizosphere soils of Trinidad and they were identified as B. amyloliquefaciens, B. subtilis, B. pumilus, B. cereus, B. megaterium, B. mycoides, B. aryabhattai based on 16S rRNA gene analysis. All Bacillus strains were screened for antibiotic lipopeptide genes viz., Iturin A synthetase C (ituC), Iturin A synthetase D (ituD), Bacillomycin D synthetase (BmyB), Bacilysin synthetase A (bacA), Fengycin synthetase (fenD), Surfactin (SrfC) and Zwittermycin A (ZmA). The study revealed the presence of iturin, bacillomycin, bacilysin, fengycin, surfactin and zwittermycin synthesizing genes in B. amyloliquefaciens, B. cereus, B. megaterium and B. pumilus strains. Testing of different Bacillus species against vegetable pathogens revealed greater antagonistic activity of lipopeptide producing B. amyloliquefaciens strains as compared with non-producers. Phylogenetic analysis of Bacillus species based on 16S rRNA gene clearly distinguished the lipopeptide producers from non-lipopeptide producers. The lipopeptide activity and biocontrol potential of B. amyloliquefaciens strains were further demonstrated in lettuce plants against Cercospora leaf spot under a protected cultivation system as a model. The study provided evidence for the biocontrol potential of lipopeptide producing B. amyloliqufaciens strains in the control of disease causing agents in vegetables. These results are promising for the development of bioagents suitable for disease management in tropical conditions.

Bacillus amyloliquefaciens strain 83 is a bacterium isolated from mango phyllosphere effective to control anthracnose in various tropical crops. The antifungal activity of bacillomycin D homologues, produced by this strain, against Colletotrichum gloeosporioides was characterized in this study. Minimum inhibitory concentration (MIC100) of bacillomycin D homologues revealed that slight changes in the fatty acid chain length (hydrophobicity) of bacillomycin D cause significant differences of its antifungal activity against spores but no significant differences were found against mycelium. These lipopeptides inhibit both spore germination and mycelial growth. Mycelium was more susceptible to bacillomycin D homologues than spores. Propidium iodide staining of spores and mycelium showed that bacillomycin D causes damage to the cell membranes which directly correlates with its viability. The present work explores the relationship between the chemical structure and biological activity of bacillomycin homologues. As synergism between lipopeptides produced by B. amyloliquefaciens was observed, the biosynthesis of diverse bacillomycin D homologues should play a major role in the biological control of fungal pathogens by this strain. These findings will contribute to the design of better antimicrobial lipopeptides with high selectivity and potency.

Aim In the study, we investigated the regulatory effects of these genes (codY, comA, degU and spo0A) on the biosynthesis of three lipopeptides (bacillomycin D, fengycin and surfactin) in Bacillus amyloliquefaciens. Methods and results The codY, comA, degU and spo0A genes in B. amyloliquefaciens fmbJ were knocked out. The results showed that the productions of bacillomycin D were significantly reduced compared with that of fmbJ. Their deletion induced great changes in the levels of transcripts specifying metabolic pathways, quorum sensing system and substance transport system in fmbJ. Moreover, overexpression of these genes improved the productions of bacillomycin D. In particular, the overexpression of spo0A enhanced bacillomycin D yield up to 648·9 ± 60·9 mg l-1 from 277·3 ± 30·5 mg l-1 . In addition, the yields of surfactin in fmbJΔcodY and fmbJΔdegU were significantly improved, and the regulatory factor CodY had no significant effect on the synthesis of fengycin. Conclusions These genes positively regulated the expression of bacillomycin D and fengycin synthase genes in strain fmbJ. However, codY and degU negatively regulated surfactin biosynthesis. Moreover, it was found that CodY had a concentration dependence on bacillomycin D synthesis. Spo0A might play a direct regulatory role in the synthesis and secretion of bacillomycin D. Significance and impact of the study This study indicated that genetic engineering of regulatory genes was an effective strategy to improve the yields of antimicrobial lipopeptides and provided promising strains for industrial production of lipopeptides.

Cyclic lipopeptides (CLPs) are a class of natural agricultural antibiotics produced by Bacillus, it plays an important role in the biological control using of Bacillus. In this study, 20 strains of Bacillus, including Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus siamese, and Bacillus cereus were determined the antagonistic ability against to pathogenic fungi such as Foc 4. PCR amplification was performed on 20 strains of Bacillus and 7 soil samples, by using the designed 14 pairs of primers for CLPs-related synthetase genes, including Bacillomycin, Fengycins, Iturins, Surfactin etc, screen CLPs primers that can be used for Bacillus and soil monitoring. The results showed that: a total of 16 Bacillus strains have the antagonistic activity against pathogens, and microscopic observation of the crude lipopeptide extract of fermentation broth of these Bacillus strains can destroy the biofilm system and cell wall of Foc4, causing the mycelium and meristems to expand and deform; 12 pairs primers of this study (BamD-F/R, BamA-F/R, FenA-F/R, FenB-F/R, 61FenA-F/R, 61FenB-F/R, ItuA-F/R, ItuB-F/R, ItuC-F/R, ItuD-F/R, SrfAA-F/R, SrfAB-F/R) could effective amplify CLPs gene, and find that all of test Bacillus strains has the ItuC gene, there is a positive relationship between the antagonistic ability of Bacillus and the diversity and richness of CLPs genes, through PCR amplification of total soil DNA, it was found that the designed CLPs primers were feasible to monitor Bacillus in soil environment. It would be used as an important supplement to conventional plate counting and high-throughput sequencing methods, monitor and guide the application of Bacillus control soil-borne diseases of plants to provide theoretical support for further field.

Verticillium dahliae is one of the most important soil pathogens, causing verticillium wilt. It is well known that the use of chemical products against this pathogen is not without side effects on the environment. In this regard, the present study was aimed to search for antagonistic rhizobacteria as an alternative of biological control against this causal agent. A total of 162 isolates were screened for their antagonistic activity according to the “double layer” and the “well diffusion” methods. Three of them (RS11, SF82 and ZO4), were subsequently selected as biological control agent (BCAs) according to their efficiency and were identified by 16S rRNA sequencing and Biolog microplate GEN III as Bacillus spp. Using 10 different lipopeptide gene primers, PCR reactions only revealed the involvement of genes responsible for iturins (ituA, ituD, ituC), bacillomycin (bmyA) and Bacilysin (bacA / B-F, bacA / B-R) biosynthesis. The Plant Growth Promoting Rhizobacteria traits [enzymatic activities, phytohormones production] of the three BCAs were also studied in vitro then on pepper plant (Capsicum annuum L.), indicating that Bacillus subtilis ZO4 was the most effective, enhancing leaf, stem and root growth comparing to the control.

Bacteria from the Bacillus group are microorganisms that inhabit a large number of different habitats. They are well known as producers of a wide array of antagonistic compounds of different structures, having between 5 to 8% of the total genome devoted to biosynthesis of secondary metabolites. Most important bioactive molecules from the genus Bacillus are non-ribosomally synthesized peptides and lipopeptides, polyketide compounds, bacteriocins and siderophores. Lipopeptides from Bacillus have very complex mechanisms of biosynthesis catalyzed by non-ribosomal peptide synthetases (NRPSs), large enzyme complexes with modular structure, with each module being in charge for the incorporation of a particular amino acid. In general, they have a broad spectrum of antagonistic activity against plant pathogenic bacteria, fungi and viruses. Most important molecules from this group, circular lipopeptides from surfactin, iturin and fengycin families affect the target cells on the membrane level. Bacillus strains exhibit their biocontrol capacity predominantly through inhibitory activity on the growth of plant pathogens, as well as inducing systemic resistance in plants and competing for ecological niches with plant pathogens. Our previous studies showed the presence of multiple biosynthetic operons for synthesis of non-ribosomal lipopeptides in the collection of natural isolates of Bacillus, with many strains having more than one of them. Several strains of Bacillus sp. that we have recently characterized showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed the presence of biosynthetic operons for iturin, bacillomycin, fengycin and surfactin in tested strains. Measurement of the kinetics of production of antimicrobial substances showed that, in most cases, synthesis started at the beginning of exponential phase of growth, reaching the maximum of antimicrobial activity at the beginning of the stationary growth phase and stayed at this level for the whole duration of observed period. Preparations of cell-free supernatants of tested strains were active against many fungal and bacterial pathogens, in vitro and in vivo. Mass spectrometry and HPTLC bioautography analysis of purified compounds confirmed the presence of lipopeptides of mentioned families, hence confirming the biocontrol capacity of Bacillus isolates.

The present study describes the identification and polyphasic characterization of salt tolerant bacilli displaying plant growth promoting and broad spectrum antifungal activities. A total of 110 strains were isolated, out of which nine were selected on the basis of halotolerance, in vitro plant growth promoting attributes and antagonism against multiple phytopathogens. Preliminary identification of strains was done on the basis of phylogenetic analysis and comparison of 16S RNA sequences with type strains. The test strains were identified as Bacillus pumilus, B. subtilis, B. licheniformis, B. safensis and B. cereus. These strains possess ability to tolerate high salt (8–10% NaCl), form endospore, showed broad spectrum antifungal activity and therefore might cope with adverse environments. Scanning electron microscopy of promising test strains (CG18 and CM25) and pathogen interaction indicated destruction of fungal mycelia by halotolerant antagonists due to cytoplasmic extrusion. Moreover, PCR amplification of bacillomycin (bmyB), chitinase (ChiA), and β-glucanase genes and production of hydrolytic enzymes suggested that test strains could have a role to manage plant pathogens under saline conditions. Finally, the selected strains exhibited some plant growth promotion traits. Overall, this study demonstrated that the evaluated strains could be useful in developing microbial products to enhance tolerance to biotic and abiotic stresses and boost plant growth.

Our study endeavors to evaluate the efficacy of endophytic Bacillus against Fusarium wilt disease and understand the resistance mechanism in tomato plants. A total of 21 endophytic bacterial strains were characterized at both biochemical and molecular levels and were screened against F. oxysporum f. sp. lycopersici by dual culture. Strain FZB24 recorded the maximum percent inhibition (48.3%) of F. oxysporum f. sp. lycopersici under in vitro conditions. Bacterial strains were evaluated for growth promoting characters and the presence of antibiotic genes through PCR analysis. FZB24 and TEB3 strains increased plant growth in the tomato plants in vitro. Strains FZB24, TEB3, TEB5, TEB6 and TEB9 had all the evaluated antibiotic biosynthetic genes viz., Iturin A, Iturin C, Surfactin, Bacillomycin A and Bacillomycin D. Corn starch-based formulation prepared with B. amyloliquefaciens (FZB24) was evaluated both under glass house and field conditions for Fusarium wilt disease management. B. amyloliquefaciens (FZB24) treatment induced defense enzymatic activities such as PO, PPO, PAL, CAT and SOD in the treated tomato plants. FZB 24 was found to be effective when applied as a combination of seed treatment @ 4 g/kg + seedling dip @ 4 g/l + soil application @ 500 g/ha + foliar spray @ 500 g/ha in reducing disease incidence. This treatment also recorded higher defense enzyme activities, maximum plant growth promotion and higher yield.

In the pursuit of new antifungal compounds, five coproduced lipopeptide variants (AF1 to AF5) from wild-type Bacillus subtilis RLID 12.1 were identified in our previous study. Out of five, AF4 was identified as a novel lead molecule belonging to the bacillomycin family showing less cytotoxicity at its respective minimum inhibitory concentrations (MIC) evaluated against 81 strains of Candida and Cryptococcus species (including clinical isolates); besides this, AF4 purified in the present study exhibited encouraging MIC values against 10 clinical mycelial fungi. Aiming for a selective production augmentation of AF4 lipopeptide variant, a new fermentation media comprising malt extract (1.01%), dextrose (0.55%), peptone (1.79%), MnSO4 (2 mM), and NaCl (0.5%) was formulated. Maximum production of 954.8 ± 10.8 mg/L was achieved with 44% selectivity at 30 °C compared to unoptimized conditions (186.4 ± 6.1 mg/L). Use of calcium alginate beads in the formulated media during the onset of lipopeptide production resulted in an augmentation in the selectivity of the most efficacious AF4 variant to about 72% presumably due to attenuation of other coproduced lipopeptide variants AF1 and AF2. Difference in yield of lipopeptides varied with bead size, bead preparation ratios, and sodium alginate concentrations. Use of Ca-alginate beads in the upstream production process of the lead AF4 variant may be considered as a novel strategy to address the potential challenge that may arise during the scale-up and downstream processing steps. Another significant finding derived from the study is that the proportion of bacillomycin variants of B. subtilis RLID 12.1 could be controlled by temperature and metal ions under static and shaking conditions.

Bacillus is the most widely studied biocontrol agent and has been extensively used in the development of biopesticides and fungicides. In this study, a new cyclic lipopeptide was isolated from Bacillus amyloliquefaciens HAB-2 by column chromatography on silica gel and Sephadex LH-20, and its structures was elucidated on the basis of spectroscopic analysis. This compound is a bacillomycin d-like compound, named as bacillomycin DC. The activity of bacillomycin DC was evaluated against C. gloeosporioides Penz. The median inhibitory concentration of bacillomycin DC was 1.21μg/mL. In addition, bacillomycin DC may have low toxicity to aquatic species as indicated by its 96h half maximum lethal concentration of 22.20μg/mL to zebrafish (Danio rerio). Our current study further provides evidence that bacillomycin DC is a potent fungicide against C. gloeosporioides Penz.

Background Lipopeptide synthetases play an important role in the production of lipopeptides. Lipopeptides are molecules made up of peptides and fatty acid moieties and have shown to have a broad range of antimicrobial activity. As infectious diseases have caused severe health problems mainly resulting from the development of antibiotic resistant strains of disease causing microorganisms there is a need of alternatives to antibiotics. The lipopeptide synthetase of the corresponding lipopeptides can be used as template to design these as drugs using computational techniques. Objective The objective of this study was homology modeling and molecular docking of two lipopeptide synthetases, bacillomycin D synthetase and iturin A synthetase, with their ligands as a means of drug design. Method Schrodinger software was used for homology modeling and molecular docking. Results After the identification of ligands, molecular docking of these ligands with the lipopeptide (bacillomycin and iturin) synthetases was performed. The docking was tested on the parameters of docking score and glide energy. 5 out of 21 ligands were found to dock with bacillomycin D synthetase whereas 8 out of 20 ligands docked with the iturin A synthetase. Conclusion The knowledge of the docking sites and docking characteristics of the lipopeptide synthetases mentioned in the paper with the ligands can provide advantages of high speed and reliability, reduced costs on chemicals and experiments and the ethical issues concerned with the use of animal models for screening of drug toxicity.

Take-all is one of the most destructive root diseases of wheat, however, effective biological control agents (BCAs) for controlling the causal organism, Gaeumannomyces graminis var. tritici (Ggt), are currently unavailable. In this study, 155 putative Bacillus strains isolated from soil were tested as potential BCAs against Ggt. The most effective was strain YB-57, which was identified as B. subtilis using 16S rDNA, morphology and physiology. Strain YB-57 showed as high as 60.9% growth inhibition of Ggt and also exhibited significantly inhibitory activity against seven other fungal phytopathogens, indicating its broad-spectrum antagonism to fungi. Greenhouse studies showed that YB-57 lowered the incidence of the take-all disease by 56.5%, which was similar to that of the fungicide, silthiofam. The antimicrobial activity of crude extracts from strain YB-57 fermentation liquor was relatively stable under a range of temperature, pH and UV light exposures and was also resistant to protease K digestion. The extract was purified, and five types of lipopeptides, iturin isomers, surfactin isomers, plipastatin isomers, bacillomycin isomers and difficidin, were identified by HPLC/ESI-MS/MS. These results suggested that B. subtilis strain YB-57 could be a useful BCA for take-all management.

Discovering new drugs and improving action mechanisms is a promising strategy to overcome chemotherapy ineffectiveness caused by cancer cell apoptosis resistance. Natural products (like cyclic lipopeptides, CLPs) are potential sources of nonapoptotic cell death inducers and can form diverse supramolecular structures, closely relating to their bioactivities. Herein, it is found for the first time that fatty chain is the key to maintain self-assembled form and antitumor activity of microbial-derived amphiphilic CLP bacillomycin Lb (B-Lb). Compared with B-Lb analogues assemblies without antitumor activity, B-Lb supramolecular self-assemblies (including nanomicelles, nanofibers, giant micrometer rods) can be generated in a multilevel and cross-scale manner and served as a methuosis-like cell death inducer triggered by cytoplasmic vacuolation through macropinocytosis in MDA-MB-231-Luc and MCF-7 cells and in vivo tumor-bearing mice. This study will promote constructing of customized CLP micro-/nanostructures with multipurposes and functions, and boost designing of new antitumor drugs as nonapoptotic cell death modulators based on structure-activity relationship.

The microbial ecology within the rhizosphere of Tylenchulus semipenetrans-infected Citrus L. trees was examined. Sixty bacterial strains were obtained from the roots of infected trees and from the eggs of T. semipenetrans. Among these strains some were obtained from the agar plates of two nematophagous fungi, Dactylellina gephyropaga (Drechsler) Ying Yang & Xing Z. Liu and Arthrobotrys conoides Drechsler. Bacterial strains were identified using 16S rDNA, gyrA and rpoB genes sequence analysis. Bacterial strains identified as Bacillus spp. were examined for their ability to synthesize surfactin, iturin, fengycin, kurstakin and bacillomycin using PCR amplification and sequencing of the encoding genes. Additionally, Bacillus strains were screened for their antifungal activity against F. solani, D. gephyropaga and A. conoides using the dual culture technique. Lipopeptide from whole cells and from supernatants of Bacillus spp. were screened using MALDI-TOF-MS analysis. The majority of the identified bacterial strains belong to the genus Bacillus with the predominance of B. cereus, B. thuringiensis, B. pumilus and B. subtilis. A total of fifteen Bacillus strains demonstrated an antifungal activity against F. solani, D. gephyropaga and A. conoides with the strongest effect found in B. amyloliquefaciens. The analysis of lipopeptides showed a high diversity of molecules, including majorly iturin C, bacillomycin D, fengycin A/B and Kurstakin found especially in B. subtilis strains. Moreover, MALDI-TOF-MS analysis showed that the responsible antibiotics for the antifungal Bacillus strains were associated with the presence of Surfactins/Pumilacidin and Fengycin A/B. Our results demonstrated the wide diversity of lipopeptides among Bacillus strains associated with citrus rhizosphere and demonstrated their antifungal ability. Our results extend the importance of Bacillus strains as potential candidates for antimicrobial activities due to their ability to synthesize and secrete cyclic lipopeptides.

The potential use of Bacillus velezensis FZB42 for biological control of various phytopathogens has been documented over the past few years, but its antagonistic interactions with xanthomonads has not been studied in detail. Novel aspects in this study consist of close observation of the death of Xanthomonas campestris pv. campestris cells in a co-culture with B. velezensis FZB42, and quantification of lipopeptides and a siderophore, bacillibactin, involved in the killing process. A new robust Xcc-SU isolate tolerating high concentrations of ferric ions was used. In a co-culture with the antagonist, the population of Xcc-SU was entirely destroyed within 24–48 h, depending on the number of antagonist cells used for inoculation. No inhibitory effect of Xcc-SU on B. velezensis was observed. Bacillibactin and lipopeptides (surfactin, fengycin, and bacillomycin) were present in the co-culture and the monoculture of B. velezensis. Except for bacillibactin, the maximum contents of lipopeptides were higher in the antagonist monoculture compared with the co-culture. Scanning electron microscopy showed that the death of Xcc-SU bacteria in co-culture was caused by cell lysis, leading to an enhanced occurrence of distorted cells and cell ghosts. Analysis by mass spectrometry showed four significant compounds, bacillibactin, surfactin, fengycin, and bacillomycin D amongst a total of 24 different forms detected in the co-culture supernatant: Different forms of surfactin and fengycin with variations in their side-chain length were also detected. These results demonstrate the ability of B. velezensis FZB42 to act as a potent antagonistic strain against Xcc.

The present study investigated the stimulation effect of L-glutamine (L-gln) on bacillomycin D biosynthesis of Bacillus subtilis fmbJ and the transcription of the synthetase genes and the signal proteins that related to the biosynthesis of bacillomycin D. It was found that L-gln significantly increased the production of bacillomycin D, in comparison with other amino acids. L-gln significantly enhanced the expression level of bmyA (BYA), bmyB (BYB), bmyC (BYC), the thioesterase gene (TE) and up-regulated DegU, σH and Spo0A, but down-regulated AbrB, therefore the yield of bacillomycin D significantly increased. Furthermore, when conjointly feeding 5 g/L of L-gln and 30 g/L of inulin, the production of bacillomycin D was up to 1.93 g/L.

Lipopeptides constitute a structurally diverse group of metabolites produced by various bacterial and fungal genera. In the past decades, research on lipopeptides has been fueled by their surfactant activities. However, natural functions of lipopeptides compounds have received considerably less attention. The aim of this study was to isolate and identify the lipopeptides from Bacillus amyloliquefaciens An6, and further evaluate their biological activities. An6 lipopeptides were detected by PCR using degenerated primers and MALDI-TOF-MS. An6 strain was found to produce surfactin, fengycin, and bacillomycin. Following their purification, the in vitro antioxidant activity of An6 lipopeptides was studied through different assays. The scavenging effect on 1,1-diphenyl-2-picrylhydrazyl radicals at a dosage of 0.75 mg/mL was 81%. Its reducing power was concentration-dependant and reached a maximum of 1.07 at 2.5 mg/mL. Moreover, they showed a strong inhibition of β-carotene bleaching. An6 lipopeptides mixture was also found to display significant antimicrobial activity against several Gram-positive, Gram-negative bacteria, and fungal strains. An6 lipopeptides were insensitive to proteolytic enzymes, stable between pH 4.0 and 12.0, and resistant to high temperature. Our results provided enough evidence proving that An6 lipopeptides could be used as functional-food components.

Cyclic lipopeptides (CLPs) from Bacillus strains have demonstrated a wide range of bioactivities making them interesting candidates for different applications in the pharmaceutical, food and biotechnological industries. Genome sequencing, together with phylogenetic analysis of the Bacillus sp. P34, isolated from a freshwater fish gut, showed that the bacterial strain belongs to the Bacillus velezensis group. In silico investigation of metabolic gene clusters of nonribosomal peptide synthetases (NRPS) revealed the genetic elements associated with the synthesis of surfactin, fengycin and iturin family component bacillomycin. Further, an assay was conducted to investigate the production of CLPs in the presence of heat inactivated bacterial cultures or fungal spores. Maximum fengycin concentration was observed at 24 h (2300-2700 mg/mL), while maximum iturin amounts were detected at 48 h (250 mg/mL) in the presence of heat-inactivated spores of Aspergillus niger. Heat-inactivated cells of Listeria monocytogenes caused a reduction of both fengycin and iturin amounts. The production of fengycins A and B and the iturin family component bacillomycin L was confirmed by mass spectrometry analyses. This study reinforces the potential of B. velezensis P34 as a valuable strain for biotechnological production of CLPs recognized as important antimicrobial substances.

International audience; An endophytic Bacillus amyloliquefaciens strain called C5, able to produce biosurfactant lipopeptides with a broad antibacterial activity spectrum, has been isolated from the roots of olive tree. Optimization of antibacterial activity was undertaken using grape seed flour (GSF) substrate at 0.02, 0.2, and 2% (w/v) in M9 medium. Strain C5 exhibited optimal growth and antimicrobial activity (MIC value of 60 μg/ml) when incubated in the presence of 0.2% GSF while lipopeptide production culminated at 2% GSF. Thin layer chromatography analysis of lipopeptide extract revealed the presence of at least three active spots at Rf 0.35, 0.59, and 0.72 at 0.2% GSF. Data were similar to those obtained in LB-rich medium. MALDI-TOF/MS analysis of lipopeptide extract obtained from 0.2% GSF substrate revealed the presence of surfactin and bacillomycin D. These results show that GSF could be used as a low-cost culture medium supplement for optimizing the production of biosurfactants by strain C5.

Highlights • Seven different lipopeptides (Tsushimycin, Daptomycin, Surfactin, Bacillomycin, Iturin, srfTE, LPD-12) are docked individually against spike (S)-glycoprotein of SARS-CoV-2. • Based on the maximum docked score and minimum atomic contact energy, LPD-12 is the best one for binding with the S glycoprotein which significantly interrupt its affinity of binding with angiotensin-converting enzyme-2 (ACE2), the only receptor molecule facilitates in disease development. • Results confirm a strong binding affinity of LPD-12 with ACE2 with a binding free energy of -1621.62 kcal which can also prevent the binding of S-protein, reciprocally. • Lipopeptides could serve as potential antiviral agents against SARS-CoV-2 by interacting or competing with viral fusion proteins. Lipid membrane alternation efficiency of small antimicrobial lipopeptides makes them able to block the viral membrane fusion to the host cell., Introduction : The recent pandemic outbreak of SARS-CoV-2 has been associated with a lethal atypical pneumonia, making COVID-19 an urgent public health issue with an increasing rate of mortality and morbidity. There are currently no vaccines or therapeutics available for COVID-19, which is causing an urgent search for a new drug to combat the COVID-19 pandemic. The lipid membrane alternation efficiency of small antimicrobial lipopeptides enables them to block viral membrane fusion to the host cell. Lipopeptides could serve as potential antiviral agents, by interacting or competing with viral fusion proteins. Methods : This study screened seven different lipopeptides (tsushimycin, daptomycin, surfactin, bacillomycin, iturin, srfTE, and LPD-12) and docked them individually against the spike (S)-glycoprotein of SARS-CoV-2. Results : Based on the maximum docked score and minimum atomic contact energy, LPD-12 (–1137.38 kcal) was the appropriate molecule for proper binding with the S-glycoprotein of SARS-CoV-2 and thus significantly interrupted its affinity of binding with angiotensin-converting enzyme-2 (ACE2), which is the only receptor molecule found to be facilitating disease development. The results confirmed a strong binding affinity of LPD-12 with ACE2, with a binding free energy of –1621.62 kcal, which could also reciprocally prevent the binding of S-protein. Conclustion : It can be concluded that LPD-12 may act as a potential therapeutic drug, by reducing the entry of SARS-CoV-2 to the human cells via the ACE2 receptor and related infections.