Characterization of the surfactin synthetase isolated from the Bacillus subtilis strain producing iturin

Summary The lipopeptides, surfactin and iturin, are co-produced by B. subtilis. In this work, the three subunits of surfactin synthetase have been characterized by affinity chromatography on affigel columns where the ligand is one of the amino acid components of surfactin.     

Optimization of Sterilization of Salmonella enteritidis in Meat by Surfactin and Iturin Using a Response Surface Method

In this paper, the sterilization of surfactin and iturin to Salmonella enteritidis was observed, and the optimization of the inactivation of surfactin and iturin to S. enteritidis in meat by a response surface methodology was studied. Results showed that surfactin and iturin had high sterilization to S. enteritidis, whose minimal inhibitory concentration was 6.25 and 12.50 μM respectively. The optimization result indicated that S. enteritidis could be inactivated by five orders of magnitude when the temperature was 4.83°C, the action time was 17.20 h, and the concentration (surfactin/iturin molar ratio 1:2) was 0.69 MIC.     

Studies on lipopeptide biosynthesis by Bacillus subtilis: Isolation and characterization of iturin−, surfactin+ mutants

Abstract Two mutant strains, M35 and M89, were obtained by UV irradiation from a wild-type Bacillus subtilis producing iturin and surfactin. Sporulation and surfactin production were similar in both mutants and in the parent strain, while the iturin production of M35 was 300-fold less than that of the wild-type strain; M89 did not produce any iturin. The analysis of the incorporation of sodium [1-¹⁴C]acetate into cellular lipids and lipopeptides showed that M89 still synthesized β-amino fatty acids, the lipid moiety of iturin.     

Optimization of Antifungal Effect of Surfactin and Iturin to <Emphasis Type="Italic">Penicillium notatum</Emphasis> in Syrup of Peach by RSM

In the paper, the sensitivity of Penicillium notatum to surfactin and iturin was determined, and the optimization of the antifungal of surfactin and iturin to Penicillium notatum in syrup of peach by a response surface methodology (RSM) was researched. Results demonstrated that Penicillium notatum was sensitive to them, whose minimal inhibitory concentration (MIC) was 62.5 and 31.25 μg ml⁻¹ respectively. In the optimization experiment, when the temperature was 2.37°C, the action time was 25.21 h, and the concentration (surfactin/iturin weight ratio 1:1) was 40.26 μg ml⁻¹, Penicillium notatum could be sterilized by 5 orders of magnitude. All the results in the experiment indicated surfactin and iturin could kill remarkably Penicillium notatum in syrup of peach.     

Surfactin and iturin A effects on Bacillus subtilis surface hydrophobicity

The synthesis of extracellular molecules such as biosurfactants should have major consequences on bacterial adhesion. These molecules may be adsorbed on surfaces and modify their hydrophobicities. Certain strains of Bacillus subtilis synthesize the lipopeptides, which exhibit antibiotic and surface active properties. In this study the high-performance liquid chromatography (HPLC) analysis of the culture supernatants of the seven B. subtilis strains, showed that the lipopeptide profile varied greatly according to the strain. Among the three lipopeptide types, only iturin A was produced by all B. subtilis strains. Bacterial hydrophobicity, evaluated by the water contact angle measurements and the hydrophobic interaction chromatography, varied according to the strain. Two strains (ATCC 15476 and ATCC 15811) showing extreme behaviors in term of hydrophobicity were selected to study surfactin and iturin A effects on bacterial hydrophobicity. The two lipopeptides modified the B. subtilis surface hydrophobicity. Their effects varied according to the bacterial surface hydrophobic character, the lipopeptide type and the concentration. Lipopeptide adsorption increased the hydrophobicity of the hydrophilic strain but decreased that of the hydrophobic. Comparison of lipopeptide effects on B. subtilis surface hydrophobicity showed that surfactin was more effective than iturin A for the two strains tested.     

芽孢杆菌几种重要抗菌脂肽研究进展

多种芽孢杆菌为益生菌,能分泌多种天然抗菌活性物质,其中脂肽是重要的一类。目前已鉴定的脂肽约有90多种,多数为环脂肽。脂肽中表面活性素(surfactin)、伊枯草菌素(iturin)、芬原素(fengycin)、杆菌霉素(bacillomycin)、多粘菌素(polymyxins)等是研究最广泛的脂肽。其中surfactin、iturin、fengycin由于其具有表面活性剂特性及抗真菌、抗细菌、抗病毒、抗肿瘤、抗炎症等功能,应用潜力巨大。本文对surfactin、iturin及fengycin的结构、功能、合成调控及其分离纯化和生产等方面的研究进展进行了评述。合成生物学是提高脂肽产量的重要手段,未来脂肽可用于种植业、养殖业、食品、医药、石油工业和环保等领域,因此需要在新型脂肽的发现、高产活性脂肽的生产、脂肽低廉生产技术的研发及安全性的评估等方面加强研究。     

Coproduction of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis

Of the 13 strains of Bacillus subtilis tested for the coproduction of the lipopeptide surfactin and the antifungal lipopeptides of the iturin family, only 1 produced both lipopeptides with a high yield. The cultures were made in a synthetic medium. Several L-amino acids and various carbon sources were good substrates for the lipopeptide production. The maximum yield of surfactin was about 110 mg/liter and that of iturin A about 39 mg/liter/absorbance unit for the best strain, B. subtilis S 499.     

Characterization of the srfA locus of Bacillus subtilis: only the valine-activating domain of srfA is involved in the establishment of genetic competence

srfA is a locus required for the production of the lipopeptide antibiotic surfactin. This locus is also necessary for efficient sporulation and competence development. Mutations in the 5′ portion of the srfA operon affect all three of these processes, whereas mutations in the 3′ portion of srfA only affect sporulation and surfactin production. Analysis of the proteins encoded by the srfA locus revealed seven large domains which are likely to be responsible for the activation and binding of the seven amino acids of surfactin. Identification of the amino acid that is activated by the srfA domains was determined by amino acid-dependent pyrophosphate exchange reactions on partially purified cell extracts of strains carrying different srfA mutations. These results indicate colin-earity between the order of the domains in the srfA locus and the amino acid sequence of surfactin. The minimal genetic element of srfA required for the establishment of competence was shown to be the 5′ region of the second open reading of srfA, which encodes the valine activation domain. This portion of srfA, when cloned on a plasmid, complemented the competence deficiency of a srfA deletion mutant in trans.     

Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping

Analysis of the primary structure of peptide synthetases involved in the non-ribosomal synthesis of peptide antibiotics has revealed a highly conserved and ordered modular arrangement. A module contains at least two domains, involved in ATP-dependent substrate activation and thioester formation. The occurrence and arrangement of these functional building blocks is associated with the number and order of the amino acids incorporated in the peptide product. In this study, we present data on the targeted exchange of the leucine-activating module within the three-module surfactin synthetase 1 (SrfA-A) of Bacillus subtilis. This was achieved by engineering several hybrid srfA-A genes, which were introduced into the surfactin biosynthesis operon by in vivo recombination. We examined the hybrid genes for expression and investigated the enzymatic activities of the resulting recombinant peptide synthetases. For the first time, we demonstrate directly that an individual minimal module, of bacterial or fungal origin, confers its amino acid-specific activity on a multi-modular peptide synthetase. Furthermore, it is shown that directed incorporation of ornithine at the second position of the peptide chain induces a global alteration in the conformation of surfactin and may result in premature cyclization or a branched cyclic structure. Received: 10 July 1997 / Accepted: 11 September 1997     

Isolation of new variants of surfactin by a recombinant Bacillus subtilis

A recombinant Bacillus subtilis MI113(pC115), carrying a gene responsible for the production of surfactin and iturin A cloned from B. subtilis RB14C, produced new surfactin variants, in addition to the already reported surfactin, when MI113(pC115) was cultured in solid-state fermentation of soybean curd residue (okara) as a substrate. All variants isolated by HPLC were characterized. Received: 18 December 1996 / Received revision: 20 February 1997 / Accepted: 28 February 1997     

Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A.

Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A. This synergistic effect seems to be in relation with interactions between iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.     

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine

Non‐self‐recognition of microorganisms partly relies on the perception of microbe‐associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA‐regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long‐lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP‐non‐producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.     

Cell-free biosynthesis of surfactin, a cyclic lipopeptide produced by Bacillus subtilis

The lipopeptide antibiotic surfactin is a potent extracellular biosurfactant produced by various Bacillus subtilis strains. Biosynthesis of surfactin was studied in a cell-free system prepared from B. subtilis ATCC 21332 and OKB 105, which is a transformant producing surfactin in high yield [Nakano, M. M., Marahiel, M. A., & Zuber, P. (1988) J. Bacteriol. 170, 5662-5668]. Cell material was disintegrated by treatment with lysozyme and French press. A cell-free extract was prepared by ammonium sulfate fractionation, which catalyzed the formation of surfactin at the expense of ATP. Lipopeptide biosynthesis required the L-amino acid components of surfactin and D-3-hydroxytetradecanoyl-coenzyme A thioester. D-Leucine which is present in surfactin was not utilized but inhibited the biosynthetic process. The structure of surfactin, synthesized enzymatically in vitro, was confirmed by chromatographic comparison with the authentic compound and by amino acid analyses. An enzyme fraction was prepared by gel permeation chromatography which catalyzed ATP/pyrophosphate exchange reactions dependent on the component amino acids of surfactin. This enzyme fraction was capable of binding substrate amino acids covalently, probably via thioester linkages. The formation of these intermediates was inhibited by various thiol blocking reagents and phenylmethanesulfonyl fluoride. De novo synthesis of the lipopeptide was not observed with this partially purified enzyme fraction most likely due to the lack of an acyltransferase activity required for linking the beta-hydroxy fatty acid to the peptide moiety.     

Second stage production of iturin A by induced germination of Bacillus subtilis RB14

Bacillus subtilis RB14, a dual producer of lipopeptide antibiotics iturin A and surfactin undergoes sporulation in the submerged fermentation and the production of these secondary metabolites becomes halted. In this study, production of lipopeptide antibiotics was investigated by induced germination of the spores by heat-activation and nutrient supplementation. The induced spores became metabolically active vegetative state and produced lipopeptide antibiotic iturin A that added up the total production at the end of the fermentation. However, additional production of surfactin was not observed. This second time iturin A production by the germinated cells from the spores was defined as second stage production.     

微生物源脂肽的生物合成和分子调控

微生物源脂肽具有抑制真菌和细菌的生长、抗病毒和抗肿瘤等多种生物活性,在农业生物防治、临床医疗、环境治理等多种领域具有巨大的应用潜力。然而,低产量一直是影响其推广应用的瓶颈。深入了解脂肽合成的关键因素和调控策略对于提高其产量和纯度至关重要。本文概括了3大家族脂肽surfactin、fengycin和iturin的结构、功能及应用前景,介绍了NRPS和NRPS-PKS两种合成系统的结构域和功能,阐释了脂肽生物合成过程中侧链脂肪酸的合成、脂肪酸的活化及与氨基酸的连接、肽链的延伸和环化三个阶段的模块组装和酶催化活动,以及三大家族脂肽合成操纵子开放阅读框的组成;总结了导入或缺失关键基因、定点突变、模块替换、强启动子替换、修饰前体路径等多种遗传操作对脂肽产量的影响,以及群体感应肽信息素、sigma因子等全局调控因子对脂肽合成基因表达的调节。指出利用多组学联用深入探讨脂肽合成的全局分子调控机制和加强结构域蛋白互作和分子动力学研究是提高脂肽产量和纯度以及创造新脂肽的理论基础,提出了利用基因组装和编辑等合成生物学方法及代谢工程技术提高脂肽产量和挖掘新型脂肽靶向性的可能途径,为推进脂肽的生产和应用进程提供科学参考。     

Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8

Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides. Received: 14 July 1995 / Accepted: 22 December 1995     

Molecular and Biochemical Approaches for Characterization of Antifungal Trait of a Potent Biocontrol Agent Bacillus subtilis RP24

Bacillus subtilis strain RP24, isolated from rhizoplane of field grown pigeon pea, exhibited in vitro antagonism against a wide range of phytopathogenic fungi. An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the strain RP24 and its negative mutant (NM) in potato dextrose medium. High performance liquid chromatography (HPLC) of partially purified extract of RP24 showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (5.230 min) from Sigma–Aldrich whereas the corresponding peak was absent in extract of NM. The structure was further confirmed by liquid chromatographic mass spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of surfactin and fengycin besides iturin A. Amplification of the lpa-14 (encodes the 4′-phosphopantetheinyl transferase required for the maturation of template enzyme of iturin A) and ituD (encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production) genes of iturin operon of strain RP24 was carried out and the sequences obtained were compared with the existing database of NCBI. The sequences of lpa-14 and ituD gene of RP24 showed 98% and 97% homology with lpa-14 and ituD genes of B. subtilis in the existing database. The results indicated that strain RP24 harbors iturin operon in its genome and a chemical mutation in this operon might have resulted in loss of antifungal activity in the negative mutant.     

Investigation of optimal conditions for foam separation of iturin,an antifungal peptide produced by Bacillus subtilis

An antifungal peptide, iturin, was produced by a newly-isolated Bacillus subtilis, which contains cyclic 7 d- and l-α amino acids and β-amino acids with aliphatic side chains. It consists of 6 derivatives with different length side chains. When the bacterium was cultivated in a jar fermentor, all the iturin produced was entrained in the foam. Thus, continuous separation and condensation of the product iturin is possible only by collecting the foam produced during fermentation. The controlling factors for foaming, namely the aeration rate, temperature and medium composition, especially the ratio of glucose to Polypepton, were investigated in relation to iturin production. The most effective conditions for the condensation of iturin were an aeration rate of 0.1 vvm, a temperature of 30°C, and a glucose to Polypepton ratio of 0.3–0.4. The total amount of iturin produced, and the ratio of peaks 3,4 and 5 in the iturin were maximized at 50 g/l of Polypepton by using a fermentor equipped with a basket-shaped agitation unit.     

Influence of the culture medium on the production of iturin A by Bacillus subtilis

The production of iturin A by Bacillus subtilis was studied with respect to the composition of the culture medium. Increasing phosphate concentrations did not modify the antibiotic yield. Fructose, sucrose and mannitol were better carbon sources than glucose for antibiotic production. The nature of the nitrogen source was an important factor in the production of antibiotic. Among the amino acids which are components of iturin A, L-asparagine was the best substrate for the biosynthesis of iturin A; L-glutamine and L-serine were rather poor substrates while L-proline and D-tyrosine gave no antibiotic. Ammonium salts permitted good synthesis of antibiotic but the addition of calcium ions to the culture medium inhibited the excretion of antibiotic from the cells.     

黑水虻肠道细菌抗菌筛选及其活性物质分子鉴定

【目的】从昆虫黑水虻分离的肠道细菌进行抗植物病原菌的拮抗菌筛选,对获得有拮抗活性的肠道细菌进行活性物质的分子鉴定。【方法】用稀释涂布法从水虻肠道中分离菌株,采用平板对峙法进行抗菌筛选,对有抗菌活性的菌株通过生理生化实验、16S rRNA鉴定和进化树分析确定其种属。参考已知脂肽合成关键基因设计引物,以拮抗菌总DNA为模板进行PCR扩增,对目的片段进行测序。【结果】通过抗菌筛选获得一株对水稻黄单胞菌以及小麦纹枯病病原菌等有很强抑制效果的水虻肠道细菌BSF-CL,经鉴定为枯草芽胞杆菌。脂肽合成关键基因PCR结果显示BSF-CL菌株具有脂肽Iturin和Surfactin合成的关键基因。推测BSF-CL很可能合成脂肽Iturin和Surfactin。【结论】从水虻肠道中分离出对水稻黄单胞菌有很强抑菌活性的菌株,分离菌被鉴定为一种枯草芽胞杆菌,通过活性物质的分子克隆鉴定初步推测其活性物质可能为脂肽Iturin和Surfactin。