Production Of Cyclic Lipopeptides Research Articles

Co-Culture of White Rot Fungi Pleurotus ostreatus P5 and Bacillus amyloliquefaciens B2: A Strategy to Enhance Lipopeptide Production and Suppress of Fusarium Wilt of Cucumber.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC), poses a serious threat to cucumber productivity. Compared to traditional chemical pesticides, biological control strategies have attracted more attention recently owing to their effectiveness against pathogens and their environmental safety. This study investigated the effect of white rot fungi Pleurotus ostreatus P5 on the production of cyclic lipopeptides (CLPs) of Bacillus amyloliquefaciens B2 and the potential co-culture filtrate of strains B2 and P5 to control cucumber Fusarium wilt. A PCR amplification of CLP genes revealed that B. amyloliquefaciens B2 had two antibiotic biosynthesis genes, namely, ituA and srf, which are involved in iturin A and surfactin synthesis. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) revealed that CLPs derived from strain B2 contained two families, iturin A (C14, C15) and surfactin (C12-C17). The co-culture exhibited an enhanced accumulation of iturin A and surfactin compared to the monoculture of strain B2. Furthermore, the gene expressions of ituA and srf were both significantly upregulated when co-cultured with the fungus compared to monocultures. In an in vitro experiment, the co-culture filtrate and monoculture filtrate of B. amyloliquefaciens B2 inhibited mycelial growth by 48.2% and 33.2%, respectively. In a greenhouse experiment, the co-culture filtrate was superior to the monoculture filtrate in controlling cucumber Fusarium wilt disease and in the promotion of plant growth. Co-culture filtrate treatment significantly enhanced the microbial metabolic activity and decreased the abundance of FOC in the rhizosphere soil. These results show that the co-culture of P. ostreatus P5 and B. amyloliquefaciens B2 has great potential in cucumber Fusarium wilt disease prevention by enhancing the production of bacterial CLPs.

Nematicidal lipopeptides from Bacillus paralicheniformis and Bacillus subtilis: A comparative study.

The aim of this work was to develop a comparative study between Bacillus paralicheniformis TB197 and B. subtilis ATCC 21332 strains in terms of growth, cyclic lipopeptide production, nematicidal activity, and active lipopeptide characteristics. Crude lipopeptide extracts (CLEs) from their fermentation broths were obtained, and their nematicidal activity (NA) was estimated as the mean lethal dose (LD50), employing Caenorhabditis elegans. Using a bioguided approach, CLE components were fractionated by semipreparative thin layer chromatography, and active lipopeptides were characterized by mass spectrometry. Both strains produced similar concentrations of CLEs (p ≥ 0.05) (0.99 ± 0.11 and 1.14 ± 0.15mg/mL by TB197 and ATCC 21332, respectively). The estimated LD50 values of CLEs from the TB197 and ATCC 21332 strains were 3.88 and 8.15mg/mL, respectively, showing that the NA of the TB197 strain CLE was 2.1-fold higher (p ≤ 0.05). Mass spectrometry revealed that strain TB197 synthesizes several families of lipopeptides, namely, fengycin A (C14-C17), fengycin B (C16-C17), surfactin (C15-C17), and lichenysin (C12, C13, C14, and C16), from which fengycins and lichenysins possess the highest NA (100 and 60% mortality in C. elegans larvae, respectively), while the ATCC 21332 strain produces mainly surfactin (C13-C17) (NA 63% mortality). The main differences found in this study were that the TB197 strain has a higher tolerance to inhibition by the product, and the lipopeptides they synthesize have a higher nematicidal activity due to the diversity of families compared to ATCC 21332. Likewise, it was shown that more polar lipopeptides (fengycins) are more effective at causing mortality in C. elegans larvae. KEY POINTS: • The nematicidal activity of lipopeptides from TB197 is higher than from ATCC 21332 • TB197 produces surfactin, lichenysin, and fengycin, while ATCC 21332 mainly produces surfactin • The most polar lipopeptides (fengycins) cause more mortality in C. elegans L2.

PLANT GROWTH-PROMOTING TRAITS OF ANTARCTIC ENDOPHYTIC BACTERIA

Successful colonization of Antarctic lands by vascular plants Deschampsia antarctica and Colobanthus quitensis and their adaptation to stressful environments is associated not only with climate change but also with the functioning of microbial groups of phylo- and endosphere of these plants. The aim of our study was to screen plant growth-promoting traits in endophytic bacteria of antarctic vascular plants. Materials and methods. We have studied 8 bacterial cultures isolated from D. antarctica collected during the 25th Ukrainian Antarctic Expedition (January-April 2020) along the Western part of the Antarctic Peninsula. Overnight liquid cultures were obtained on Nutrient Broth medium (HiMedia, Ltd.) in a shaking incubator (26 ℃, 160 rpm). Bacterial isolates were grown on Ashby's combined-nitrogen-free medium with sucrose. Drop collapse assay for cyclic lipopeptide production (CLP), motility assay, exoprotease production and phosphate solubilizing ability were performed using generally accepted methods. Results. All studied isolates have shown plant growth-promoting traits. The most abundant were nitrogen-fixing activity and motility. Both these play important role in plant colonization and promoting the growth of plants in harsh environments. The evidences of CLP were shown by two strains only. There was no notice of phosphate solubilizing ability and exoprotease production. Conclusions. Endophytic bacteria of antarctic vascular plants could support the growth and nutrition needs of the plants.

Diversity of cyclic antimicrobial lipopeptides from Bacillus P34 revealed by functional annotation and comparative genome analysis

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.

Bioactive Secondary Metabolites from Bacillus subtilis: A Comprehensive Review

Bacillus subtilis is widely underappreciated for its inherent biosynthetic potential. This report comprehensively summarizes the known bioactive secondary metabolites from B. subtilis and highlights potential applications as plant pathogen control agents, drugs, and biosurfactants. B. subtilis is well known for the production of cyclic lipopeptides exhibiting strong surfactant and antimicrobial activities, such as surfactins, iturins, and fengycins. Several polyketide-derived macrolides as well as nonribosomal peptides, dihydroisocoumarins, and linear lipopeptides with antimicrobial properties have been reported, demonstrating the biosynthetic arsenal of this bacterium. Promising efforts toward the application of B. subtilis strains and their natural products in areas of agriculture and medicine are underway. However, industrial-scale availability of these compounds is currently limited by low fermentation yields and challenging accessibility via synthesis, necessitating the development of genetically engineered strains and optimized cultivation processes. We hope that this review will attract renewed interest in this often-overlooked bacterium and its impressive biosynthetic skill set.

Enhanced Tolerance of Chinese Cabbage Seedlings Mediated by Bacillus aryabhattai H26-2 and B. siamensis H30-3 against High Temperature Stress and Fungal Infections.

Two rhizobacteria Bacillus aryabhattai H26-2 and B. siamensis H30-3 were evaluated whether they are involved in stress tolerance against drought and high temperature as well as fungal infections in Chinese cabbage plants. Chinese cabbage seedlings cv. Ryeokgwang (spring cultivar) has shown better growth compared to cv. Buram-3-ho (autumn cultivar) under high temperature conditions in a greenhouse, whilst there was no difference in drought stress tolerance of the two cultivars. In vitro growth of B. aryabhattai H26-2 and B. siamensis H30-3 were differentially regulated under PEG 6000-induced drought stress at different growing temperatures (30, 40 and 50°C). Pretreatment with B. aryabhattai H26-2 and B. siamensis H30-3 enhanced the tolerance of Chinese cabbage seedlings to high temperature, but not to drought stress. It turns out that only B. siamensis H30-3 showed in vitro antifungal activities and in planta crop protection against two fungal pathogens Alternaria brassicicola and Colletotrichum higginsianum causing black spots and anthracnose on Chinese cabbage plants cv. Ryeokgwang, respectively. B. siamensis H30-3 brings several genes involved in production of cyclic lipopeptides in its genome and secreted hydrolytic enzymes like chitinase, protease and cellulase. B. siamensis H30-3 was found to produce siderophore, a high affinity iron-chelating compound. Expressions of BrChi1 and BrGST1 genes were up-regulated in Chinese cabbage leaves by B. siamensis H30-3. These findings suggest that integration of B. aryabhattai H26-2 and B. siamensis H30-3 in Chinese cabbage production system may increase productivity through improved plant growth under high temperature and crop protection against fungal pathogens.

The LuxR Regulators PcoR and RfiA Co-regulate Antimicrobial Peptide and Alginate Production in Pseudomonas corrugata.

Cyclic lipopeptides (CLPs) are considered as some of the most important secondary metabolites in different plant-associated bacteria, thanks to their antimicrobial, cytotoxic, and surfactant properties. In this study, our aim was to investigate the role of the Quorum Sensing (QS) system, PcoI/PcoR, and the LuxR-type transcriptional regulator RfiA in CLP production in the phytopatogenic bacterium, Pseudomonas corrugata based on our previous work where we reported that the pcoR and rfiA mutants were devoid of the CLPs cormycin and corpeptin production. Due to the close genetic link between the QS system and the RfiA (rfiA is co-transcribed with pcoI), it was difficult to ascertain the specific regulatory role in the expression of target genes. A transcriptional approach was undertaken to identify the specific role of the PcoR and RfiA transcriptional regulators for the expression of genes involved in CLP production. The RNA-seq-based transcriptional analysis of the wild-type (WT) strain CFBP 5454 in comparison with GL2 (pcoR mutant) and GLRFIA (rfiA mutant) was performed in cultural conditions favoring CLP production. Differential gene expression revealed that 152 and 130 genes have significantly different levels of expression in the pcoR and rfiA mutants, respectively. Of these, the genes linked to the biosynthesis of CLPs and alginate were positively controlled by both PcoR and RfiA. Blast homology analysis showed that 19 genes in a large CLP biosynthetic cluster involved in the production of three antimicrobial peptides, which span approximately 3.5% of the genome, are strongly over-expressed in the WT strain. Thus, PcoR and RfiA function mainly as activators in the production of bioactive CLPs, in agreement with phenotype analysis of mutants. RNA-seq also revealed that almost all the genes in the structural/biosynthetic cluster of alginate exopolysaccharide (EPS) are under the control of the PcoR–RfiA regulon, as supported by the 10-fold reduction in total EPS yield isolated in both mutants in comparison to the parent strain. A total of 68 and 38 gene expressions was independently regulated by PcoR or RfiA proteins, respectively, but at low level. qPCR experiments suggest that growth medium and plant environment influence the expression of CLP and alginate genes.

Fungal Competitors Affect Production of Antimicrobial Lipopeptides in Bacillus subtilis Strain B9-5.

Bacillus subtilis has shown success in antagonizing plant pathogens where strains of the bacterium produce antimicrobial cyclic lipopeptides (CLPs) in response to microbial competitors in their ecological niche. To gain insight into the inhibitory role of these CLPs, B. subtilis strain B9-5 was co-cultured with three pathogenic fungi. Inhibition of mycelial growth and spore germination was assessed and CLPs produced by B. subtilis B9-5 were quantified over the entire period of microbial interaction. B. subtilis B9-5 significantly inhibited mycelial growth and spore germination of Fusarium sambucinum and Verticillium dahliae, but not Rhizopus stolonifer. LC-MS analysis revealed that B. subtilis differentially produced fengycin and surfactin homologs depending on the competitor. CLP quantification suggested that the presence of Verticillium dahliae, a fungus highly sensitive to the compounds, caused an increase followed by a decrease in CLP production by the bacterium. In co-cultures with Fusarium sambucinum, a moderately sensitive fungus, CLP production increased more gradually, possibly because of its slower rate of spore germination. With co-cultures of the tolerant fungus Rhizopus stolonifer, B. subtilis produced high amounts of CLPs (per bacterial cell) for the duration of the interaction. Variations in CLP production could be explained, in part, by the pathogens' overall sensitivities to the bacterial lipopeptides and/or the relative growth rates between the plant pathogen and B. subtilis. CLP production varied substantially temporally depending on the targeted fungus, which provides valuable insight concerning the effectiveness of B. subtilis B9-5 protecting its ecological niche against the ingress of these pathogens.

Biocontrol activity of surfactin A purified from Bacillus NH-100 and NH-217 against rice bakanae disease

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.

Improvement of biomass and cyclic lipopeptides production in Bacillus amyloliquefaciens MEP218 by modifying carbon and nitrogen sources and ratios of the culture media

Bacillus amyloliquefaciens MEP218 is a native isolate with a broad spectrum of antifungal activity against plant pathogenic fungi. The ability of strain MEP218 to antagonize phytopathogens is due to the production of cyclic lipopeptides (CLPs). In this work, different carbon (C) and nitrogen (N) sources and C to N ratios were evaluated in order to improve both, biomass and CLPs production by strain MEP218. Among the C and N sources and C to N ratios tested, glucose and NH4NO3 at the C to N ratio of 10:1 enhanced significantly biomass and CLPs yield. Moreover, CLPs produced in this medium exhibited higher antibacterial activity against Xanthomonas axonopodis pv. vesicatoria (Xav) than those obtained in the recommended medium for CLPs production. Interestingly, CLPs addition influenced the development of Xav biofilm on biotic and abiotic surfaces. A comparison of HPLC-chromatograms of CLPs obtained in the optimized medium versus the ones obtained in the recommended medium showed a notable increase of surfactin in the CLPs obtained in the optimized medium. Furthermore, two peaks with antibacterial activity against Xav, identified by mass spectrometry analysis as fengycins A and B, were detected in the CLPs from the strain MEP218, grown in the optimized culture medium. The results obtained in this work suggest that changes in C and N sources and C to N ratios affect the yield and type of CLPs produced by B. amyloliquefaciens MEP218. To the best of our knowledge, this is the first study to report the finding of fengycins with antibacterial activity. CLPs produced by the strain MEP218 are potential candidates for controlling bacterial spot disease in tomato and pepper.

Biosynthesis of the antimicrobial cyclic lipopeptides nunamycin and nunapeptin by Pseudomonas fluorescens strain In5 is regulated by the LuxR-type transcriptional regulator NunF.

Nunamycin and nunapeptin are two antimicrobial cyclic lipopeptides (CLPs) produced by Pseudomonas fluorescens In5 and synthesized by nonribosomal synthetases (NRPS) located on two gene clusters designated the nun–nup regulon. Organization of the regulon is similar to clusters found in other CLP‐producing pseudomonads except for the border regions where putative LuxR‐type regulators are located. This study focuses on understanding the regulatory role of the LuxR‐type‐encoding gene nunF in CLP production of P. fluorescens In5. Functional analysis of nunF coupled with liquid chromatography–high‐resolution mass spectrometry (LC‐HRMS) showed that CLP biosynthesis is regulated by nunF. Quantitative real‐time PCR analysis indicated that transcription of the NRPS genes catalyzing CLP production is strongly reduced when nunF is mutated indicating that nunF is part of the nun–nup regulon. Swarming and biofilm formation was reduced in a nunF knockout mutant suggesting that these CLPs may also play a role in these phenomena as observed in other pseudomonads. Fusion of the nunF promoter region to mCherry showed that nunF is strongly upregulated in response to carbon sources indicating the presence of a fungus suggesting that environmental elicitors may also influence nunF expression which upon activation regulates nunamycin and nunapeptin production required for the growth inhibition of phytopathogens.

Key Impact of an Uncommon Plasmid on Bacillus amyloliquefaciens subsp. plantarum S499 Developmental Traits and Lipopeptide Production.

The rhizobacterium Bacillus amyloliquefaciens subsp. plantarum S499 (S499) is particularly efficient in terms of the production of cyclic lipopeptides, which are responsible for the high level of plant disease protection provided by this strain. Sequencing of the S499 genome has highlighted genetic differences and similarities with the closely related rhizobacterium B. amyloliquefaciens subsp. plantarum FZB42 (FZB42). More specifically, a rare 8008 bp plasmid (pS499) harboring a rap-phr cassette constitutes a major distinctive element between S499 and FZB42. By curing this plasmid, we demonstrated that its presence is crucial for preserving the typical physiology of S499 cells. Indeed, the growth rate and extracellular proteolytic activity were significantly affected in the cured strain (S499 P−). Furthermore, pS499 made a significant contribution to the regulation of cyclic lipopeptide production. Surfactins and fengycins were produced in higher quantities by S499 P−, whereas lower amounts of iturins were detected. In line with the increase in surfactin release, bacterial motility improved after curing, whereas the ability to form biofilm was reduced in vitro. The antagonistic effect against phytopathogenic fungi was also limited for S499 P−, most probably due to the reduction of iturin production. With the exception of this last aspect, S499 P− behavior fell between that of S499 and FZB42, suggesting a role for the plasmid in shaping some of the phenotypic differences observed in the two strains.

Translocation of the thioesterase domain for the redesign of plipastatin synthetase.

Non-ribosomal peptide synthetases (NRPSs) are large enzymatic complexes that catalyse the synthesis of biologically active peptides in microorganisms. Genetic engineering has recently been applied to reprogram NRPSs to produce lipopeptides with a new sequence. The carboxyl-terminal thioesterase (TE) domains from NRPSs catalyse cleavage products by hydrolysis or complex macrocyclization. In this study, we modified plipastatin synthetase by moving the intrinsic TE region to the end of the internal thiolation (T) domains, thus generating Bacillus subtilis strains that could produce new truncated cyclic or linear peptides of the predicted sequence, which further provided an important insight into the regioselectivity of plipastatin TE. The TE was capable of recognizing and catalysing the lactone formation between L-Try3 with the last few residues L-Pro7 and L-Gln8 at the C-terminus. Additionally, the unmatched linkers connecting the TE region and T domain resulted in nonproduction strains, suggesting that the native T–TE linker is necessary and sufficient for the TE domain to release the products from the hybrid enzymes. This is the first report to demonstrate truncated cyclic lipopeptides production and module skipping by simply moving the TE domain forward in an NRPS system.

Role of phenazines and cyclic lipopeptides produced by pseudomonas sp. CMR12a in induced systemic resistance on rice and bean.

Pseudomonas sp. CMR12a produces two different classes of cyclic lipopeptides (CLPs) (orfamides and sessilins), which all play a role in direct antagonism against soilborne pathogens. Here we show that Pseudomonas sp. CMR12a is also able to induce systemic resistance to Magnaporthe oryzae on rice and to the web blight pathogen Rhizoctonia solani AG2-2 on bean. Plant assays with biosynthesis mutants of Pseudomonas sp. CMR12a impaired in the production of phenazines and/or CLPs and purified metabolites revealed that distinct bacterial determinants are responsible for inducing systemic resistance in these two pathosystems. In rice, mutants impaired in phenazine production completely lost their ability to induce systemic resistance, while a soil drench with pure phenazine-1-carboxamide (PCN) at a concentration of 0.1 or 1 μM was active in inducing resistance against M. oryzae. In bean, mutants that only produced phenazines, sessilins or orfamides were still able to induce systemic resistance against Rhizoctonia web blight, but a balanced production of these metabolites was needed. This study not only shows that Pseudomonas sp. CMR12a can protect rice to blast disease and bean to web blight disease, but also displays that the determinants involved in induced systemic resistance are plant, pathogen and concentration dependent.

식물 성장 촉진에 사용에 있어 Bacillus mojavensis KJS-3의 특징

Scientists have recently shown an interest in the characteristics of Bacillus mojavensis strains because of their increasing use in plants as a defense against diseases and mycotoxins. We have shown here that B. mojavensis KJS-3 possesses the typical characteristics of B. mojavensis strains including a strong resistance to high temperatures (≤50°C), tolerance to high salt concentrations (7% NaCl), ethanol tolerance (40% ethanol), and pH range for growth (pH 5-9). B. mojavensis KJS-3 has been used for the production of cyclic lipopeptides including important antifungal substances such as surfactin, iturin, and fengycin. Polymerase chain reaction analysis in this study showed that B. mojavensis KJS-3 can be used for the production of fengycin and the findings of LC-MS/MS analyses suggest that B. mojavensis KJS-3 can be used to produce iturin and surfactin. Antifungal activity analys is confirmed that B. mojavensis KJS-3 has antifungal effects on Botrytis cinerea, Rhizoctonia solani AG-4, Sclerotinia sclerotiorum, and Colletotricum goeosporioides. A microscopy assessment of the roots of wild ginseng plants planted together with B. mojavensis KJS-3 revealed that the roots contained B. mojavensis KJS-3, confirming the bacteria to be a plant growth promoting endophyte (PGPE) which acts against plant diseases and mycotoxins. Our findings lead us to conclude that B. mojavensis KJS-3 can be produced at an industrial level as a microbial pesticide or microbial fertilizer.

Biological control of wheat root diseases by the CLP-producing strain Pseudomonas fluorescens HC1-07.

Pseudomonas fluorescens HC1-07, previously isolated from the phyllosphere of wheat grown in Hebei province, China, suppresses the soilborne disease of wheat take-all, caused by Gaeumannomyces graminis var. tritici. We report here that strain HC1-07 also suppresses Rhizoctonia root rot of wheat caused by Rhizoctonia solani AG-8. Strain HC1-07 produced a cyclic lipopeptide (CLP) with a molecular weight of 1,126.42 based on analysis by electrospray ionization mass spectrometry. Extracted CLP inhibited the growth of G. graminis var. tritici and R. solani in vitro. To determine the role of this CLP in biological control, plasposon mutagenesis was used to generate two nonproducing mutants, HC1-07viscB and HC1-07prtR2. Analysis of regions flanking plasposon insertions in HC1-07prtR2 and HC1-07viscB revealed that the inactivated genes were similar to prtR and viscB, respectively, of the well-described biocontrol strain P. fluorescens SBW25 that produces the CLP viscosin. Both genes in HC1-07 were required for the production of the viscosin-like CLP. The two mutants were less inhibitory to G. graminis var. tritici and R. solani in vitro and reduced in ability to suppress take-all. HC1-07viscB but not HC-07prtR2 was reduced in ability to suppress Rhizoctonia root rot. In addition to CLP production, prtR also played a role in protease production.

Synergistic Membrane Permeation by Antifungal Lipopeptides and Detergents

The fungicidal activity of Bacillus subtilis QST713, based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin (SFs), and Iturin (Its) families, has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. There has been evidence that the membrane perturbation by these these and other antimicrobial peptides follow a detergent-like mechanism. We have used the lifetime-based membrane leakage assay (Soft Matter, 2009, 5, 2849-51) to study the concerted action of the lipopeptides and detergents to induce leakage of liposomes made from POPC or yeast lipid extract. The results provide further insight into similarities and differences between detergent and peptide effects on membranes.

Biological Control of Rhizoctonia Root Rot on Bean by Phenazine- and Cyclic Lipopeptide-Producing Pseudomonas CMR12a

Pseudomonas CMR12a was previously selected as an efficient biocontrol strain producing phenazines and cyclic lipopeptides (CLPs). In this study, biocontrol capacity of Pseudomonas CMR12a against Rhizoctonia root rot of bean and the involvement of phenazines and CLPs in this ability were tested. Two different anastomosis groups (AGs) of Rhizoctonia solani, the intermediately aggressive AG 2-2 and the highly aggressive AG 4 HGI, were included in growth-chamber experiments with bean plants. The wild-type strain CMR12a dramatically reduced disease severity caused by both R. solani AGs. A CLP-deficient and a phenazine-deficient mutant of CMR12a still protected bean plants, albeit to a lesser extent compared with the wild type. Two mutants deficient in both phenazine and CLP production completely lost their biocontrol activity. Disease-suppressive capacity of CMR12a decreased after washing bacteria before application to soil and thereby removing metabolites produced during growth on plate. In addition, microscopic observations revealed pronounced branching of hyphal tips of both R. solani AGs in the presence of CMR12a. More branched and denser mycelium was also observed for the phenazine-deficient mutant; however, neither the CLP-deficient mutant nor the mutants deficient in both CLPs and phenazines influenced hyphal growth. Together, results demonstrate the involvement of phenazines and CLPs during Pseudomonas CMR12a-mediated biocontrol of Rhizoctonia root rot of bean.

All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713

The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only ≈ 1 μM free FE and in 15% of the vesicles at 10 μM. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance.

Showing the Potential of the Lifetime-Based Dye Leakage Assay: All-Or-None Membrane Permeabilization by Fungicidal Lipopeptides from Bacillus Subtilis QST 713

The fungicidal activity of Bacillus subtilis QST713, based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families, has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own but promote the membrane permeabilizing activity of the mixture. Fluorescence lifetime-based calcein (Softmatter, 2009, 5, 2849-51) efflux measurements, isothermal titration calorimetry and electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid induces stable, long-lived pores in 10% of the vesicles at only ≈ 1uM free FE and in 15% of the vesicles at 10 uM. We explain why this all-or-none leakage accounts for the killing of virtually all fungi whereas the same extent of graded leakage would likely be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10 mol-% fluid lipid resembles the behavior of detergent resistance.