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.     

New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction

Bacillus strains produce non-ribosomal lipopeptides that can be grouped into three families: surfactins or lichenysins, iturins and fengycins or plispastatins. These biosurfactants show a broad spectrum of biological activities. To detect strains able to produce these lipopeptides, a new polymerase chain reaction screening approach was developed using degenerated primers based on the intraoperon alignment of adenylation and thiolation nucleic acid domains of all enzymes implicated in the biosynthesis of each lipopeptide family. The comparative bioinformatics analyses of each operon led to the design of four primer pairs for the three families taking into account the differences between open reading frames of each synthetase gene. Tested on different Bacillus sp. strains, this technique was used successfully to detect not only the expected genes in the lipopeptide producing strains but also the presence of a plispastatin gene in Bacillus subtilis ATCC 21332 and a gene showing a high similarity with the polyketide synthase type I gene in the B. subtilis ATCC 6633 genome. It also led to the discovery of the presence of non-ribosomal peptide synthetase genes in Bacillus thuringiensis serovar berliner 1915 and in Bacillus cereus LMG 2098. In addition, this work highlighted the differences between the fengycin and plipastatin operon at DNA level.     

Novel whole-cell Reporter Assay for Stress-Based Classification of Antibacterial Compounds Produced by Locally Isolated Bacillus spp.

Reporter bacteria are beneficial for the rapid and sensitive screening of cultures producing peptide antibiotics, which can be an addition or alternative to the established antibiotics. This study was carried out to validate the usability of specific reporter strains for the target mediated identification of antibiotics produced by native Bacillus spp. isolated from different food sources. During preliminary classification, cell wall stress causing Bacillus isolates were screened by using reporter strain Bacillus subtilis BSF2470. The isolates which induced cell wall stress were further characterized for their specific mode of action by using other B. subtilis reporter strains (TMB 488, TMB 299 and TMB 279). The isolate B. licheniformis N12 was found to produce bacitracin confirmed by the response to reporter strain B. subtilis TMB 279 and by putative identification of bacitracin biosynthetic loci. The other isolate B. subtilis EC1 also induced B. subtilis TMB 279, but does not possess the bacitracin gene cluster indicating that it can be a novel, bacitracin like antibiotic. The different but related subsets of peptide antibiotics that bind the pyrophosphate moiety of the lipid carrier of cell wall biosynthesis can be identified using this whole cell based reporter strains.     

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.     

The plant-associated Bacillus amyloliquefaciens strains MEP2 18 and ARP2 3 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease

Aims: This work was conducted to identify the antifungal compounds produced by two previously isolated Bacillus sp. strains: ARP₂3 and MEP₂18. Both strains were subjected to further analysis to determine their taxonomic position and to identify the compounds responsible for their antifungal activity as well as to evaluate the efficiency of these strains to control sclerotinia stem rot in soybean. Methods and Results: The antifungal compounds were isolated by acid precipitation of cell‐free supernatants, purified by RP‐HPLC and then tested for antagonistic activity against Sclerotinia sclerotiorum. Mass spectra from RP‐HPLC eluted fractions showed the presence of surfactin C₁₅, fengycins A (C₁₆–C₁₇) and B (C₁₆) isoforms in supernatants from strain ARP₂3 cultures, whereas the major lipopeptide produced by strain MEP₂18 was iturin A C₁₅. Alterations in mycelial morphology and sclerotial germination were observed in the presence of lipopeptides‐containing supernatants from Bacillus strains cultures. Foliar application of Bacillus amyloliquefaciens strains on soybean plants prior to S. sclerotiorum infection resulted in significant protection against sclerotinia stem rot compared with noninoculated plants or plants inoculated with a nonlipopeptide‐producing B. subtilis strain. Conclusions: Both strains, renamed as B. amyloliquefaciens ARP₂3 and MEP₂18, were able to produce antifungal compounds belonging to the cyclic lipopeptide family. Our data suggest that the foliar application of lipopeptide‐producing B. amyloliquefaciens strains could be a promising strategy for the management of sclerotinia stem rot in soybean. Significance and Impact of the Study: Sclerotinia stem rot was ranked as one of the most severe soybean disease in Argentina and worldwide. The results of this study showed the potential of B. amyloliquefaciens strains ARP₂3 and MEP₂18 to control plant diseases caused by S. sclerotiorum.     

Characteristics and antimicrobial activity of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains isolated from soil

Antagonistic Bacillus strains were isolated from soil and analyzed for the purpose of determining whether they could be used as natural biological agents. Primary in vitro screening for antagonism of the isolates was performed against five phytopathogenic mould fungi. Strains TS 01 and ZR 02 exhibited the most pronounced inhibitory effects. They were identified as Bacillus subtilis on the basis of their morphological, cultural and physiology-biochemical properties as well as their hierarchical cluster analysis conducted by means of computer program SPSS. The antimicrobial activity of the strains from cultural medium and sterile filtrate were determined in vitro against a great number of predominantly phytopathogenic fungi and bacteria. TS 01 and ZR 02 strains exhibited very broad and at the same time degree varying antibiotic spectra of activities against both Gram-positive and Gram-negative microorganisms. Many of them were tested against sensitivity to the antimicrobial action of B. subtilis for the very first time. B. subtilis TS 01 and ZR 02 showed highest antifungal activity (sterile zone in diameter over 37 mm) against Alternaria solani, Botrytis cinerea, Monilia linhartiana 869, Phytophthora cryptogea 759/1 and Rhizoctonia sp. The most sensitive bacterial species were found to be Pseudomonas syringae pv. tomato Ro and Xanthomonas campestris with sterile zones 48.0 and 50.0 mm in diameter, respectively. The latter draws a conclusion that the isolated and identified Bacillus subtilis strains are promising natural biocontrol agents and should be further studied and tested for control of numerous plant diseases.     

Characterization of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> inhibitory lipopeptide from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> IB

Bacillus subtilis strain IB exhibiting inhibitory activity against the Fusarium head blight disease fungus Fusarium graminearum was isolated and identified. The major inhibitory compound was purified from the culture broth through anion exchange, hydrophobic interaction, and reverse phase high-performance liquid chromatography (RP-HPLC) steps. It was a 1,463-Da lipopeptide and had an amino acid composition consisting of Ala, Glx, Ile, Orn, Pro, Thr, and Tyr at a molar ratio of 1:3:1:1:1:1:2. Electrospray ionization mass spectrometry/mass spectrometry (ESI MS/MS) analyses of the natural and the ring-opened peptides showed the antagonist was fengycin, a kind of macrolactone molecule with antifungal activity produced by several Bacillus strains. Fluorescence microscopic analysis indicated this peptide permeabilized and disrupted F. graminearum hyphae.     

Analysis of the genome of an alkaliphilic Bacillus strain from an industrial point of view

Bacillus species and other microbes with pH optima for growth higher than pH 9 are defined as alkaliphiles. A large number of alkaliphilic Bacillus strains producing useful enzymes, have been isolated from various environments. Some of these enzymes, such as proteases and cellulases from alkaliphilic Bacillus strains, have been commercialized and have brought great advantages to industry and domestic life. To support further development of the enzyme industry, we initiated analysis of the genome of Bacillus halodurans C-125, which is 4.25 Mb in size, and constructed a physical and genetic map for comparison with the Bacillus subtilis chromosome. Systematic sequencing of the whole genome of Bacillus halodurans C-125 has been automated since the beginning of May 1998, and sequencing of 98% of the whole genome has been done so far. Through genome analysis, it became apparent that the genome organization of alkaliphilic Bacillus halodurans C-125 is totally different from that of B. subtilis orthologues. Received: July 11, 1999 / Accepted: December 27, 1999     

Wet and dry density of Bacillus anthracis and other Bacillus species

Aims: To determine the wet and dry density of spores of Bacillus anthracis and compare these values with the densities of other Bacillus species grown and sporulated under similar conditions. Methods and Results: We prepared and studied spores from several Bacillus species, including four virulent and three attenuated strains of B. anthracis, two Bacillus species commonly used to simulate B. anthracis (Bacillus atrophaeus and Bacillus subtilis) and four close neighbours (Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis and Bacillus stearothermophilus), using identical media, protocols and instruments. We determined the wet densities of all spores by measuring their buoyant density in gradients of Percoll and their dry density in gradients of two organic solvents, one of high and the other of low chemical density. The wet density of different strains of B. anthracis fell into two different groups. One group comprised strains of B. anthracis producing spores with densities between 1·162 and 1·165 g ml^?1 and the other group included strains whose spores showed higher density values between 1·174 and 1·186 g ml^?1. Both Bacillus atrophaeus and B. subtilis were denser than all the B. anthracis spores studied. Interestingly and in spite of the significant differences in wet density, the dry densities of all spore species and strains were similar. In addition, we correlated the spore density with spore volume derived from measurements made by electron microscopy analysis. There was a strong correlation (R² = 0·95) between density and volume for the spores of all strains and species studied. Conclusions: The data presented here indicate that the two commonly used simulants of B. anthracis, B. atrophaeus and B. subtilis were considerably denser and smaller than all B. anthracis spores studied and hence, these simulants could behave aerodynamically different than B. anthracis. Bacillus thuringiensis had spore density and volume within the range observed for the various strains of B. anthracis. The clear correlation between wet density and volume of the B. anthracis spores suggest that mass differences among spore strains may be because of different amounts of water contained within wet dormant spores. Significance and Impact of the Study: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefense against B. anthracis. The similarities and difference in density and volume that we found should assist in the selection of simulants that better resemble properties of B. anthracis and, thus more accurately represent the performance of countermeasures against this threat agent where spore density, size, volume, mass or related properties are relevant.     

花生侵脉新赤壳菌果腐病生防芽孢杆菌的分离鉴定及防病效果

【目的】为了分离鉴定对花生侵脉新赤壳菌果腐病病原菌Neocosmospora vasinfecta具有抑制作用的根际芽孢杆菌。【方法】利用平板稀释法从花生的根际土壤分离芽孢杆菌,再采用平板对峙法筛选出对N.vasinfecta具有抑制作用的根际芽孢杆菌,通过形态观察、生理生化特性和分子生物学相结合的多相分类方法对生防根际芽孢杆菌进行分类鉴定,检测脂肽类抗生素合成基因类型,并进行花生侵脉新赤壳菌果腐病的田间防治试验。【结果】从花生根际土壤中分离到28株芽孢杆菌,其中对花生果腐病病原菌具有明显抑制作用有8株。多相分类法结果显示2株为枯草芽孢杆菌(Bacillus subtilis),6株为解淀粉芽孢杆菌(B.amyloliquefaciens)。脂肽类抗生素合成基因检测显示,8株生防芽孢杆菌含有至少1种脂肽类抗生素,其中所有生防菌均含有丰原素B合成基因,推测这些芽孢杆菌对N.vasinfecta的抑制机制可能与脂肽类抗生素的合成相关。田间防病实验结果显示,B.amyloliquefaciens GF-3和GF-22制备的生物有机肥均能有效降低NPRP的发病指数,其防治效率分别为32.35%和79.41%,增产率分别为19.12%和25.85%。【结论】分离鉴定了2株对花生侵脉新赤壳菌果腐病具有明显防治效果的根际芽孢杆菌,这不仅为花生侵脉新赤壳菌果腐病的生防制剂研制提供了菌株,还为研究防治机理奠定了基础。     

Cyclic lipopeptide profile of three <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strains; antagonists of <Emphasis Type="Italic">Fusarium</Emphasis> head blight

The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.     

Development of selective media for Bacillus sphaericus and Bacillus thuringiensis var. israelensis

Summary The resistance of 8 strains of Bacillus sphaericus and of 2 strains of Bacillus thuringiensis var. israelensis (B.t.i.) to various antibiotics and antibiotic combinations were tested. All B. sphaericus strains were resistant to streptomycin, lincomycin and bacitracin, and six strains were resistant to combinations of these antibiotics. This antibiotic resistance could be utilised to establish selective media to identify and follow the fate of B. sphaericus and of B.t.i. in the field.     

Enhancement of alkaline protease production in Bacillus circulans using plasmid transformation

Plasmid transformation is an efficient and crucial biotechnological tool that enables the enhancement of many important microbial characters that would be beneficial in a lot of industrial, agricultural and environmental applications. In the present study, five Bacillus species (B. subtilis, B. cereus, B. alvei, B. circulans and B. pumilus) were investigated. They were isolated from agricultural soils of different local arid environments of the Kingdom of Saudi Arabia, identified and characterized for their plasmid content. The main objective of the present study was to enhance the production of alkaline protease in Bacillus circulans (the recipient strain) through plasmid transformation from B. subtilis (the donor strain). All the tested Bacillus strains successfully produced unique multiple (3, 4 and 5) spontaneous antibiotic resistant mutants against chloramphenicol, neomycin, rifampicin, streptomycin, kanamycin and tetracycline and all of which were mutated to Rif_r strains. B. pumilus showed the highest resistance against five of the six tested antibiotics while both of B. alvei and B. circulans showed the lowest resistance to only three of the tested antibiotics. Results revealed that B. subtilis was the best among the tested species concerning the production of alkaline protease (90.2 U/ml) while B. pumilus was the lowest in activity (40.3 U/ml). Screening of plasmid content revealed the presence of one or two mega indigenous plasmids in all the tested species. The four transformant strains BC ₁ , BC ₂ , BC ₃ and BC ₄ resulting from plasmid transformation exhibited significant increases in the activity of alkaline protease and recorded 2.31- to 3-fold increases compared to the parent B. circulans cells and 2.11- to 2.75-fold increases compared to the donor cells of B. subtilis. They also acquired antibiotic resistance to tetracycline and chloramphenicol that was completely absent in the parent cells of B. circulans. Results revealed that plasmid transformation among the tested Bacillus spp. is a powerful technique that can be efficiently exploited to enhance alkaline protease production in the transformed Bacillus spp. compared to their wild strains and we recommend using the improved transformant strains for commercial and industrial purposes.     

Comparing Methods for Identifying Bacillus Strains Capable of Producing the Antifungal Lipopeptide Iturin A

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

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     

Impact of Bacillus subtilis JA, a biocontrol strain of fungal plant pathogens, on arbuscular mycorrhiza formation in Zea mays

Summary  In the present study, the influence of Bacillus subtilis JA on arbuscular mycorrhizal fungi (AMF) was evaluated by either pot culture or in vitro conditions, respectively. Under the pot culture conditions, the inoculation of B. subtilis JA decreased the frequency (% F) of the root colonization by indigenous arbuscular mycorrhizal fungi and the shoot dry weight of maize (Zea mays L.), but had no apparent effect on the intensity (% I) of AM fungal root colonization. The unknown volatile emitted from the B. subtilis JA in vitro significantly inhibited spore germination and the hyphal growth in the dual-compartment experiments. Moreover, the data from the direct interaction between B. subtilis JA and Glomus etunicatum showed that soluble antifungal lipopeptides influenced the development of AMF. Therefore, the application of antifungal Bacillus strains should take the compatibility with the indigenous beneficial fungi into consideration.     

Restriction and modification in Bacillus species: genetic transformation of bacteria with DNA from different species, part I.

Summary Host specific restriction was detected in 13 Bacillus strains, when 63 strains of Bacillus subtilis and 15 other Bacillus strains were tested with phage 105C. These 13 strains were classified into 8 groups (M,H,C,N,E,F,G,P) by the type of restriction. M-type strains (B. subtilis Marburg 168, its derivatives, and two other strains) showed relatively weak restriction, restricting 105C from other groups of Bacillus by ratios of 10^-1 to 10^-3. Strains of groups H,C,N,E,F,G, and P restricted 105C from other groups by ratios of 10^-2 to 10^-8. It was confirmed with some of the strains that type-specific modification was endowed only by the last host. Furthermore, we isolated one restriction deficient mutant of B. subtilis marburg 168-YS11, which had also lost its modification phenotype.     

Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes

Fengycin is a biologically active lipopeptide produced by several Bacillus subtilis strains. The lipopeptide is known to develop antifungal activity against filamentous fungi and to have hemolytic activity 40-fold lower than that of surfactin, another lipopeptide produced by B. subtilis. The aim of this work is to use complementary biophysical techniques to reveal the mechanism of membrane perturbation by fengycin. These include: 1), the Langmuir trough technique in combination with Brewster angle microscopy to study the lipopeptide penetration into monolayers; 2), ellipsometry to investigate the adsorption of fengycin onto supported lipid bilayers; 3), differential scanning calorimetry to determine the thermotropic properties of lipid bilayers in the presence of fengycin; and 4), cryogenic transmission electron microscopy, which provides information on the structural organization of the lipid/lipopeptide system. From these experiments, the mechanism of fengycin action appears to be based on a two-state transition controlled by the lipopeptide concentration. One state is the monomeric, not deeply anchored and nonperturbing lipopeptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity. The mechanism, thus, appears to be driven mainly by the physicochemical properties of the lipopeptide, i.e., its amphiphilic character and affinity for lipid bilayers.     

脂肽类抗生素及其作用机制

脂肽类抗生素是由微生物代谢产生的一类具有很强表面活性的生物活性物质,含有由亲水性氨基酸和疏水性脂肪酸链组成的特殊分子结构,表现出亲水、亲脂两亲性的特性,具有抗菌、抗肿瘤和抗病毒等多种生物学活性,作为抗菌药物和肿瘤抑制剂在医药和临床应用领域具有良好的应用潜力和前景。本文对脂肽类抗生素的结构、生物学功能和作用机制等最新研究进展进行综述。