Surfactin inhibits the growth of Propionibacterium acnes by destroying the cell wall and membrane

Propionibacterium acnes plays a major role in acne vulgaris. In the pre-experiment, the growth of P. acnes was inhibited effectively using surfactin; however, the antibacterial mechanism has not been described. Therefore, the aim of this study was to evaluate antibacterial activity and analyse the mechanism of surfactin against P. acnes. Minimum inhibitory concentration, time-killing kinetics and scanning electron microscopy were used to evaluate the activity of surfactin against P. acnes, which showed that 128 μg ml⁻¹ effectively inhibited growth. Cell wall permeability was evaluated by detecting the extracellular alkaline phosphatase activity, which increased to 1·83- and 2·32-fold after incubating with 128 and 256 μg ml⁻¹ of surfactin for 10 h, respectively. Propidium iodide fluorescence, leakage of nucleic acid, protein, K⁺, and Ca²⁺, membrane potential and the leakage of calcein from small unilamellar vesicles all increased after incubation with surfactin, indicating that its strong biological activities act mainly by altering membrane integrity. In a mouse model of acne, surfactin significantly reduced P. acnes–induced epidermal swelling and erythema. These results indicate that surfactin effectively inhibited the growth of P. acnes by destroying the cell wall and membrane, and is a potential candidate for acne treatment.     

Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer

The antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer were investigated. The MIC (minimal inhibitory concentration) of fengycin without commercial surfactin added was 0.4 mg/ml while the MIC of fengycin with commercial surfactin added was 2.0 mg/ml. Fengycin acted on cell membrane and cellular organs and inhibited DNA synthesis. The antifungal effect of fengycin was reduced after commercial surfactin was added. All these results suggest that the fungal cell membrane may be the primary target of fengycin action and commercial surfactin may reduce the antifungal activity of fengycin.     

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.     

Rapid Detection and Characterization of Surfactin-Producing <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and Closely Related Species Based on PCR

The surfactin production genetic locus (sfp) is responsible for the ability of Bacillus subtilis to produce the lipopeptide biosurfactant, surfactin. This report demonstrates the utility of using PCR of the sfp gene as a means of identifying Bacillus species that produce surfactin. We carried out a hemolysis zone assay, quantitative HPLC and NMR in parallel to ensure that the PCR provided correct results. PCR analyses were performed for the sfp gene on 15 standard strains and 20 field-collected Bacillus spp. isolates native to Taiwan. Among the 15 standard strains, surfactin was produced by seven strains of B. subtilis and two closely related species, B. amyloliquefaciens B128 and B. circulans ATCC 4513. Of the 20 field-collected Bacillus spp. isolates; 16 strains yielded surfactin- positive results with PCR and HPLC. A good correlation was observed. Within the 16 field isolates, B. amyloliquefaciens S13 (452.5 mg/L) and B. subtilis S15 (125.6 mg/L) had high productivity of surfactin. The technique is valuable for finding out potential good yields of surfactin-producing strains. The PCR method we used could also be used to find different species or genera containing homologous genes. This is the first report of the detection of surfactin production by B. amyloliquefaciens and B. circulans based on PCR screening.     

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.     

Biosynthesis of iturin and surfactin byBacillus subtilis: Evidence for amino acid activating enzymes

Summary Enzymes catalyzing ATP-PPi exchange reactions mediated by specific amino acids were found inB. subtilis lysates partially purified by gel permeation. The nature of these amino acids varied according to the stage of cell growth: activation of surfactin amino acids was observed during surfactin synthesis; activation of iturin amino acids was not detected at the begining of surfactin synthesis but appeared during iturin synthesis.     

Effect of pps disruption and constitutive expression of srfA on surfactin productivity,spreading and antagonistic properties of Bacillus subtilis 168 derivatives

Aims: To analyse the effects of plipastatin operon disruption and constitutive expression of surfactin operon in Bacillus subtilis 168 on surfactin productivity, in vitro invasive growth and antagonism against fungi. Methods and Results: The srfA native promoter was replaced by the constitutive promoter P_repU in B. subtilis 168 after integration of a functional sfp gene. Moreover, the plipastatin synthesis was further disrupted in the B. subtilis 168 derivatives. In liquid media, an earlier and higher expression of P_repU, than that found with P_srfA, led to a specific surfactin production fivefold higher after 6 h of culture. On solid media, not only the invasive growth and the haemolytic activity but also the antifungal activity of the constitutive strains were improved when compared to the parental strain BBG111. As expected, the disruption of the plipastatin operon strongly reduced in vitro antifungal properties but, interestingly, enhanced specific surfactin production (1·47 g g^?1 of biomass), spreading behaviour and haemolytic activity of the strains. Conclusions: This work demonstrates for the first time the interdependency of surfactin and plipastatin regarding their biosynthesis as well as their influence on the biological activities of the producing strain. Significance and Impact of the Study: The constitutive overproduction of surfactin enhances the invasive growth and the in vitro antagonistic activity of the mutant strain. Both properties are known to play an important role in the biocontrol of plant diseases. Plipastatin operon disruption increases the surfactin productivity of mutant strains. These mutants are interesting for use in continuous bioprocesses for surfactin production or in bioremediation.     

Optimization of Inactivation of Endospores of <Emphasis Type="Italic">Bacillus cereus</Emphasis> in Milk by Surfactin and Fengycin Using a Response Surface Method

In this paper, the sterilization of surfactin and fengycin to Bacillus cereus was observed, and the optimization of the inactivation of surfactin and fengycin to spores of B. cereus by a response surface methodology was studied. Results showed that surfactin and fengycin had high sterilization to B. cereus, whose minimal inhibitory concentration was 31.25 μM and 62.5 μM respectively. The optimization result indicated that spores of B. cereus could be inactivated by two orders of magnitude when the temperature was 20.41°C, the action time was 21.13 h, and the concentration (surfactin/fengycin molar ratio 1:1) was 54.20 μM.     

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     

生物表面活性剂Surfactin生产菌株的定向改造策略

表面活性素(Surfactin)是芽胞杆菌属(Bacillussp.)代谢产生的脂肽类生物表面活性剂,是由非核糖体肽合成酶(NRPS)催化而得的一种次级代谢产物。由于surfactin具有稳定性好、可被降解、表面活性好等理化性质以及抑菌、抗肿瘤等生物活性,在医药、农业、食品、化妆品、石油开采等方面都具有很大的应用潜力。但是,天然菌株产率低、生产成本高等特点限制了surfactin的规模化应用。本文对surfactin的合成机理进行了简要阐述,并针对目前提升surfactin产量和改变结构组分的4种定向改造策略(启动子工程、强化外排分泌、改造NRPS结构域和脂肪酸链合成酶系)进行了综述,最后对surfactin的研究方向进行了展望。     

Chromosomal integration of sfp gene in Bacillus subtilis to enhance bioavailability of hydrophobic liquids

Bacillus subtilis C9 effectively degrades aliphatic hydrocarbons up to a chain length of C₁₉ and produces a lipopeptide-type biosurfactant, surfactin, yet it has no genetic competency. Therefore, to obtain a transformable surfactin producer, the sfp gene cloned from B. subtilis C9 was integrated into the chromosome of B. subtilis 168, a non-surfactin producer, by homologous recombination. The transformants reduced the surface tension of the culture broth from 70.0 mN/m to 28.0 mN/m, plus the surface-active compound produced by the transformants exhibited the same R_f value as that from B. subtilis C9 and authentic surfactin in a thin-layer chromatographic analysis. The integration of the sfp gene into the chromosome of B. subtilis 168 was confirmed by Southern hybridization. Like B. subtilis C9, the transformants readily degraded n-hexadecane, although the original strain did not. It was also statistically confirmed that the hydrocarbon degradation of the transformants was highly correlated to their surfactin production by the determination of the correlation coefficient (r²=0.997, P<0.01). Therefore, these results indicate that the surfactin produced from B. subtilis enhances the bioavailability of hydrophobic liquids.     

Recent trends in the biochemistry of surfactin

The name surfactin refers to a bacterial cyclic lipopeptide, primarily renowned for its exceptional surfactant power since it lowers the surface tension of water from 72 mN m⁻¹ to 27 mN m⁻¹ at a concentration as low as 20 μM. Although surfactin was discovered about 30 years ago, there has been a revival of interest in this compound over the past decade, triggered by an increasing demand for effective biosurfactants for difficult contemporary ecological problems. This simple molecule also looks very promising as an antitumoral, antiviral and anti-Mycoplasma agent. Structural characteristics show the presence of a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a β-hydroxy fatty acid with 13–15 C atoms. In solution, the molecule exhibits a characteristic “horse saddle” conformation that accounts for its large spectrum of biological activity, making it very attractive for both industrial applications and academic studies. Surfactin biosynthesis is catalysed non-ribosomally by the action of a large multienzyme complex consisting of four modular building blocks, called the surfactin synthetase. The biosynthetic activity involves the multicarrier thiotemplate mechanism and the enzyme is organized in structural domains that place it in the family of peptide synthetases, a class of enzymes involved in peptidic secondary-metabolite synthesis. The srfA operon, the sfp gene encoding a 4′-phosphopantetheinyltransferase and the comA regulatory gene work together for surfactin biosynthesis, while the gene encoding the acyltransferase remains to be isolated. Concerning surfactin production, there is no indication whether the genetic regulation, involving a quorum-sensing mechanism, overrides other regulation factors promoted by the fermentation conditions. Knowledge of the modular arrangement of the peptide synthetases is of the utmost relevance to combinatorial biosynthetic approaches and has been successfully used at the gene level to modify the surfactin template. Biosynthetic and genetic rationales have been described for building variants. A fine study of the structure/function relationships associated with the three-dimensional structure has led to the recognition of the specific residues required for activity. These studies will assist researchers in the selection of molecules with improved and/or refined properties useful in oil and biomedical industries. Received: 9 October 1998 / Received revision: 29 January 1999 / Accepted: 31 January 1999     

Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis

The chromosomal region of Bacillus subtilis comprising the entire srfA operon, sfp and about four kilo-bases in between have been completely sequenced and functionally characterized. The srfA gene codes for three large subunits of surfactin synthetase, 402, 401 and 144 kDa, respectively, arranged in a series of seven amino acid activating domains which, as shown in the accompanying communication, recognize and bind the seven amino acids of the surfactin peptide. The srfA amino acid activating domains share homologies with similar domains of other peptide synthetases; in particular, regions can be identified which are more homologous in domains activating the same amino acid. A fourth gene in srfA encodes a polypeptide homologous to grsT. Four genes are positioned between srfA and sfp, the disruption of which does not affect surfactin biosynthesis.     

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.     

Identification and characterization of a mosquito pupicidal metabolite of a Bacillus subtilis subsp. subtilis strain

The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC₅₀ dose of 1 μg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13–C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.     

Tuning micelles of a bioactive heptapeptide biosurfactant via extrinsically induced conformational transition of surfactin assembly

We have studied the effects of extrinsic environmental conditions on the conformation of surfactin, a heptapeptide biosurfactant from Bacillus subtilis, in aqueous solutions. It has been made clear that temperature, pH, Ca²⁺ ions and the synthetic nonionic surfactant hepta-ethylene glycol (C₁₂E₇) affect the conformation of surfactin in aqueous solutions. The β-sheet formation reached a maximum at 40°C both in presence and absence of (C₁₂E₇) and the nonionic surfactant enhances the β-sheet formation even at 25°C. Ca²⁺ induced the formation of a-helices and caused this transition at 0.3 mm with surfactin monomers or at 0.5 mm with surfactin micelles, but above these transition concentrations of Ca²⁺ β-sheets were observed. In micellar solution the β-sheet structure was stabilized at pH values below 7 or upon addition of Ca²⁺ in concentrations above 0.5 mm . Our results indicated that the bioactive conformation of surfactin is most likely the β-sheets when the molecules are assembled in micelles. The β-sheet structure in micelles could be retained by tuning the micelles. Surfactin micelles could be tuned in the bioactive conformation by manipulating pH, temperature, Ca²⁺ or (C₁₂E₇) concentrations in surfactin solutions. Our results strongly indicated that Ca²⁺ and other molecules (such as C₁₂E₇) may function as directing templates in the assembly and conformation of surfactin in micelles. Thus, we suggest environmental manipulation and template-aided micellation (TAM) as a new approach for preparing predesigned micelles, microemulsions or micro-spheres for specific application purposes. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Lipopeptides with Improved Properties: Structure by NMR,Purification by HPLC and Structure–Activity Relationships of New Isoleucyl-rich Surfactins

The biosynthesis of bacterial isoleucyl-rich surfactins was controlled by supplementation of L -isoleucine to the culture medium. Two new variants, the [Ile4,7]- and [Ile2,4,7]surfactins, were thus produced by Bacillus subtilis and their separation was achieved by reverse-phase HPLC. Amino acids of the heptapeptide moiety were analysed by chemical methods, and the lipid moiety was identified to β-hydroxy anteiso pentadecanoic acid by combined GC/MS. Sequences were established on the basis of two-dimensional NMR data. Because conformational parameters issuing from NMR spectra suggested that the cyclic backbone fold was globally conserved in the new variants, structure–activity relationships were discussed in details on the basis of the three-dimensional model of surfactin in solution. Indeed, both variants have increased surface properties compared with that of surfactin, and this improvement is assigned to an increase of the hydrophobicity of the apolar domain favouring micellization. Furthermore, the additional Leu-to-Ile substitution at position 2 in the [Ile2,4,7]surfactin leads to a substantial increase of its affinity for calcium, when compared with that of [Ile4,7]surfactin or surfactin. This effect is assigned, from the model, to an increase in the accessibility of the acidic side chains constituting the calcium binding site. Thus, the propensities of such active lipopeptides for both hydrophobic and electrostatic interactions were improved, further substantiating that they can be rationally designed. © 1997 European Peptide Society and John Wiley & Sons, Ltd     

Expression of sfp gene and hydrocarbon degradation by Bacillus subtilis

Bacillus subtilis C9 produces a lipopeptide-type biosurfactant, surfactin, and rapidly degrades alkanes up to a chain length of C₁₉. The nucleotide sequence of the sfp gene cloned from B. subtilis C9 was determined and its deduced amino acid sequence showed 100% homology with the sfp gene reported before [Nakano et al. (1992) Mol. Gen. Genet. 232: 313–321]. To transform a non-surfactin producer, B. subtilis 168, to a surfactin producer, the sfp gene cloned from B. subtilis C9 was expressed in B. subtilis 168. The transformed B. subtilis SB103 derivative of the strain 168 was shown to produce surfactin measured by its decrease in surface tension, emulsification activity, and TLC analysis of the surface active compound isolated from the culture broth. Like B. subtilis C9, B. subtilis SB103 containing sfp gene readily degraded aliphatic hydrocarbons (C_10–19), though its original strain did not. The addition of surfactin (0.5%, w/v) to the culture of B. subtilis 168 significantly stimulated the biodegradation of hydrocarbons of the chain lengths of 10–19; over 98% of the hydrocarbons tested were degraded within 24 h of incubation. These results indicate that the lipopeptide-type biosurfactant, surfactin produced from B. subtilis enhances the bioavailability of hydrophobic hydrocarbons.     

Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.