WLIP and tolaasin I, lipodepsipeptides from Pseudomonas reactans and Pseudomonas tolaasii, permeabilise model membranes

The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 μM). This effect was not detected for tolaasin I at the concentrations tested (< 28 μM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.     

In silico study of the ion channel formed by tolaasin I produced by Pseudomonas tolaasii

A toxin produced by Pseudomonas tolaasii, tolaasin, causes brown blotch disease in mushrooms. Tolaasin forms pores on the cellular membrane and destroys cell structure. Inhibiting the ability of tolaasin to form ion channels may be an effective method to protect against attack by tolaasin. However, it is first necessary to elucidate the three-dimensional structure of the ion channels formed by tolaasin. In this study, the structure of the tolaasin ion channel was determined in silico based on data obtained from nuclear magnetic resonance experiments.     

Purification of a pore-forming peptide toxin, tolaasin, produced by Pseudomonas tolaasii 6264

Tolaasin, a pore-forming peptide toxin, is produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms. P. tolaasii 6264 was isolated from the oyster mushroom damaged by the disease in Korean. In order to isolate tolaasin molecules, the supernatant of bacterial culture was harvested at the stationary phase of growth. Tolaasin was prepared by ammonium sulfate precipitation and three steps of chromatograpies, including a gel permeation and two ion exchange chromatographies. Specific hemolytic activity of tolaasin was increased from 1.7 to 162.0 HU mg(-1) protein, a 98-fold increase, and the purification yield was 16.3%. Tolaasin preparation obtained at each purification step was analyzed by HPLC and SDS-PAGE. Two major peptides were detected from all chromatographic preparations. Their molecular masses were analyzed by MALDI-TOF mass spectrometry and they were identified as tolaasin I and tolaasin II. These results demonstrate that the method used in this study is simple, time-saving, and successful for the preparation of tolaasin.     

Biological characterization of white line-inducing principle (WLIP) produced by Pseudomonas reactans NCPPB1311

The biological activities of the lipodepsipeptides (LDP) white line-inducing principle (WLIP), produced by Pseudomonas reactans NCPPB1311, and tolaasin I, produced by R tolaasii NCPPB2192, were compared. Antimicrobial assays showed that both LDP inhibited the growth of fungi-including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.--chromista, and gram-positive bacteria. Assays of the two LDP on blocks of Agaricus bisporus showed their capacity to alter the mushrooms' pseudo-tissues though WLIP was less active than that of tolaasin I. Contrary to previous studies, tolaasin I was found to inhibit the growth of gram-negative bacteria belonging to the genera Escherichia, Erwinia, Agrobacterium, Pseudomonas, and Xanthomonas. The only gram-negative bacterium affected by WLIP was Erwinia carotovora subsp. carotovora. Both WLIP and tolaasin I caused red blood cell lysis through a colloid-osmotic shock mediated by transmembrane pores; however, the haemolytic activity of WLIP was greater than that of tolaasin I. Transmembrane pores, at a concentration corresponding to 1.5 x C50, showed a radius between 1.5 and 1.7 +/- 0.1 nm for WLIP and 2.1 +/- 0.1 nm for tolaasin I. The antifungal activity of WLIP together with the finding that avirulent morphological variants of P. reactans lack WLIP production suggests that WLIP may play an important role in the interaction of the producing bacterium P. reactans and cultivated mushrooms.     

Fuscopeptins, antimicrobial lipodepsipeptides from Pseudomonas fuscovaginae, are channel forming peptides active on biological and model membranes.

FP-A and FP-B are LDPs produced by the plant pathogen Pseudomonas fuscovaginae. As expected from their primary structure, they shared a similar mechanism of action with the better characterized SPs, synthesized by strains of Pseudomonas syringae pv. syringae. Indeed, they displayed hemolytic activity on human erythrocytes and were able to induce calcein release from LUVs: the effect was dependent on the concentration of the FPs and the lipid composition of the liposome and, in particular, it increased with the SM content of the membrane. The permeabilizing activity was further investigated on PLMs. FPs were able to open pores on pure POPC membranes. Pore opening was strongly voltage dependent: by switching the potential from negative to positive values, an increase in the absolute amplitude of transmembrane current was induced with simultaneous closure of pores. In 0.1 M KCl both FPs' pores had a conductance of 4 and 9 pS at - 140 mV and + 140 mV, respectively. Studies on ion selectivity indicated that FPs formed cation-selective channels.     

Identification of non-pseudomonad bacteria from fruit bodies of wild agaricales fungi that detoxify tolaasin produced by Pseudomonas tolaasii

Bacterial isolates from wild Agaricales fungi detoxified tolaasin, the inducer of brown blotch disease of cultivated mushrooms produced by Pseudomonas tolaasii. Mycetocola tolaasinivorans and Mycetocola lacteus were associated with fruit bodies of wild Pleurotus ostreatus and wild Lepista nuda, respectively. Tolaasin-detoxifying bacteria belonging to other genera were found in various wild mushrooms. An Acinetobacter sp. was isolated from fruit bodies of Tricholoma matsutake, Bacillus pumilus was isolated from Coprinus disseminatus, and Sphingobacterium multivorum was isolated from Clitocybe clavipes. A Pedobacter sp., which seemed not be identifiable as any known bacterial species, was isolated from a Clitocybe sp. Tolaasin-detoxifying bacteria identified thus far were attached to the surface of mycelia rather than residing within the fungal cells. M. tolaasinivorans, M. lacteus, B. pumilus, the Pedobacter sp., and S. multivorum efficiently detoxified tolaasin and strongly suppressed brown blotch development in cultivated P. ostreatus and Agaricus bisporus in vitro, but the Acinetobacter sp. did so less efficiently. These bacteria may be useful for the elucidation of mechanisms involved in tolaasin-detoxification, and may become biological control agents of mushroom disease.     

The white-line-in-agar test is not specific for the two cultivated mushroom associated pseudomonads, Pseudomonas tolaasii and Pseudomonas "reactans"

A sharply defined white line in vitro forms between the pathogenic form of Pseudomonas tolaasii and another Pseudomonas bacterium, referred to as "reactans". This interaction has been considered as highly specific. However, results presented in this study rise doubt about the strict specificity of this interaction, as some other pseudomonads, associated with the cultivated mushroom Agaricus bisporus, also yielded a white line precipitate when they were streaked towards Pseudomonas tolaasii LMG 2342T. Moreover, some Finnish isolates inducing brown blotch symptoms on mushrooms like P. tolaasii(T), produced a typical white precipitate when streaked towards P. "reactans" LMG5329, even though phenotypical and genotypical features exclude these isolates from the species P. tolaasii. We propose that the white-line-in-agar (WLA) test should no longer be considered as an unequivocal diagnostic trait of P. tolaasii.     

Pseudomonas tolaasii and tolaasin: comparison of symptom induction on a wide range of Agaricus bisporus strains

Abstract A wide range of Agaricus bisporus , including commercial, wild and hybrid strains, were tested for resistance to brown blotch disease caused by Pseudomonas tolaasii . Effects of toxin and living bacteria were compared. Wild and hybrid A. bisporus ranged in the same order from very poorly to highly susceptible whatever the inoculum type used, tolaasin or bacteria. Symptom aspects induced by both inocula were visually identical, but some differences occurred in response intensity. The data suggest that toxin is probably not the only factor involved in symptom development.     

Identification of compounds exhibiting inhibitory activity toward the Pseudomonas tolaasii toxin tolaasin I using in silico docking calculations,NMR binding assays,and in vitro hemolytic activity assays

Using in silico docking calculations, NMR analysis of target–ligand binding, and hemolytic activity assays, we searched a 30,000-compound library for an effective inhibitor of tolaasin I, a Pseudomonas tolaasii toxin that causes virulent infection in mushrooms. Of more than 30,000 compounds screened in silico, two compounds were selected. One of these compounds, sorbitololeic acid, bound to tolaasin I and inhibited its hemolytic activity in vitro. Therefore, sorbitololeic acid can be a potential inhibitor of tolaasin I.     

Application of siderotyping for characterization of Pseudomonas tolaasii and "Pseudomonas reactans" isolates associated with brown blotch disease of cultivated mushrooms

Pyoverdine isoelectric focusing analysis and pyoverdine-mediated iron uptake were used as siderotyping methods to analyze a collection of 57 northern and central European isolates of P. tolaasii and "P. reactans." The bacteria, isolated from cultivated Agaricus bisporus or Pleurotus ostreatus mushroom sporophores presenting brown blotch disease symptoms, were identified according to the white line test (W. C. Wong and T. F. Preece, J. Appl. Bacteriol. 47:401-407, 1979) and their pathogenicity towards A. bisporus and were grouped into siderovars according to the type of pyoverdine they produced. Seventeen P. tolaasii isolates were recognized, which divided into two siderovars, with the first one containing reference strains and isolates of various geographical origins while the second one contained Finnish isolates exclusively. The 40 "P. reactans" isolates divided into eight siderovars. Pyoverdine isoelectric focusing profiles and cross-uptake studies demonstrated an identity for some "P. reactans" isolates, with reference strains belonging to the P. fluorescens biovars II, III, or V. Thus, the easy and rapid methods of siderotyping proved to be reliable by supporting and strengthening previous taxonomical data. Moreover, two potentially novel pyoverdines characterizing one P. tolaasii siderovar and one "P. reactans" siderovar were found.     

Two types of ion channel formation of tolaasin,a Pseudomonas peptide toxin

Tolaasin is a peptide toxin produced by Pseudomonas tolaasii and causes brown blotch disease of the cultivated mushrooms. Two types of ion channels were identified by the incorporation of tolaasin into lipid bilayer. The slope conductance of type 1 channel measured in the buffer containing 100 mM KCl was 150 pS with a linear current vs. voltage relationship. The type 2 tolaasin channel had two subconductance states of 300 and 500 pS. Both channels were inhibited by Zn(2+). Ion channel formations of tolaasin were concentration-dependent and single channel currents were successfully obtained at 0.6 unit tolaasin, 15.9 nM. The type 1 channel was obtained more frequently than the type 2 channel and the ratio of their appearance was approximately 4:1, respectively.     

Pseudomonas tolaasii: extra-genomic factor mediates toxin production and efficiency

Pseudomonas tolaasii is responsible for brown blotch symptoms on Agaricus bisporus. Investigations among 15 P. tolaasii wild-type strains revealed the existence of an extra-genomic factor. Pseudomonas tolaasii CNBP2152D, a spontaneous derivative of the wild-type strain CNBP2152 which has lost the factor, exhibited a significant decline in pathogenicity. Comparison of blotch symptoms induced by toxins extracted from CNBP2152 and CNBP2152D showed that the extra-genomic factor mediates toxin production and efficiency.     

Interaction between gfp-tagged Pseudomonas tolaasii P12 and Pleurotus eryngii

Pseudomonas sp., (formerly reported as strain P12) which produces brown blotch disease symptoms on Pleurotus eryngii, has been identified as P. tolaasii based on its biochemical, physiological properties and 16S rDNA sequence analysis. This pathogen is able to infect basidiocarps when surface-inoculated on mushroom casing soil. However, infected basidiocarps develop the brown blotch disease symptoms when the pathogen concentration in the fruiting body tissues is higher than 10(4) cfu/g d.w. Using gfp-tagged cells and confocal laser scanning microscopy, it was possible to show that the pathogen has the ability to tightly attach to the hyphae of Pleurotus eryngii.     

Pseudomonas lipodepsipeptides and fungal cell wall-degrading enzymes act synergistically in biological control

Pseudomonas syringae pv. syringae strain B359 secreted two main lipodepsipeptides (LDPs), syringomycin E (SRE) and syringopeptin 25A (SP25A), together with at least four types of cell wall-degrading enzymes (CWDEs). In antifungal bioassays, the purified toxins SRE and SP25A interacted synergistically with chitinolytic and glucanolytic enzymes purified from the same bacterial strain or from the biocontrol fungus Trichoderma atroviride strain P1. The synergism between LDPs and CWDEs occurred against all seven different fungal species tested and P. syringae itself, with a level dependent on the enzyme used to permeabilize the microbial cell wall. The antifungal activity of SP25A was much more increased by the CWDE action than was that of the smaller SRE, suggesting a stronger antifungal role for SP25A. In vivo biocontrol assays were performed by using P. syringae alone or in combination with T. atroviride, including a Trichoderma endochitinase knock-out mutant in place of the wild type and a chitinase-specific enzyme inhibitor. These experiments clearly indicate that the synergistic interaction LDPs-CWDEs is involved in the antagonistic mechanism of P. syringae, and they support the concept that a more effective disease control is given by the combined action of the two agents.     

Structure,chemistry, and biological activity of pseudophomins A and B,new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens

Pseudophomins A and B are cyclic lipodepsipeptides isolated from Pseudomonas fluorescens strain BRG100, a bacterium with potential application for biocontrol of plant pathogens and weeds. Their chemical structures were established by a combination of spectroscopic data, X-ray crystallography, and selective chemical degradation. This unique chemical degradation allowed the unambiguous determination of the absolute configuration of the amino acid residue Leu-1, due to gamma-lactam formation followed by selective cleavage of the adjacent N(8)-C(7) bond. To the best of our knowledge this is the first application of gamma-lactam formation to the determination of absolute configuration of an adjacent amino acid. Pseudophomin B showed higher antifungal activity against the phytopathogens Phoma lingam/Leptosphaeria maculans and Sclerotinia sclerotiorum than pseudophomin A, and is likely to be the main component responsible for the antifungal activity of EtOAc extracts of strain BRG100. By contrast, pseudophomin A showed stronger inhibition of green foxtail (Setaria viridis) root germination than pseudophomin B.     

耐冷亚硝酸盐型反硝化菌Pseudomonas tolaasii Y-11的鉴定及其脱氮特性

摘要:【目的】反硝化细菌在生物脱氮中具有重要作用,而耐冷亚硝酸盐型反硝化细菌研究较少,本文从长期淹水的冬水田泥土分离获得一株耐冷高效去除亚硝酸盐氮和总氮的好氧反硝化细菌Y-11,明确其分类地位以及除氮特性,以期为后续利用该菌在初冬到春末处理亚硝酸盐水体污染奠定基础。【方法】通过形态学特征、特异性磷脂脂肪酸以及16S rRNA基因测序分析对该菌株进行鉴定;在好氧条件下以亚硝酸钠为唯一氮源,分别研究不同初始温度、转速、pH、碳源、接种量以及亚硝酸盐氮浓度对该菌去除亚硝酸盐氮和总氮的影响,确定最适降解条件。【结果】分离得到的菌株Y-11,经鉴定归于托拉斯假单胞菌(Pseudomonas tolaasii);在国内外尚无该种菌具有反硝化作用的报道,是对亚硝酸盐型反硝化细菌的进一步补充。Y-11菌株的最适脱氮条件为15 ℃,200 r/min,pH7.0,100 mL反硝化培养基中最适接种量为1.5×108 CFU,最佳碳源为乙酸 钠,亚硝酸盐氮为10 mg/L;以乙酸钠为电子供体,15 ℃、初始pH为7.2、150 r/min 振荡培养,48 h对亚硝酸盐氮和总氮的去除率分别为100%和61.28%。【结论】Y-11是一株具有较高反硝化能力的托拉斯假单胞菌,能高效地去除亚硝酸盐氮和总氮,其最适温度是15 ℃左右,是一株耐冷反硝化细菌。     

Structural determination of the O-specific chain of the lipopolysaccharide from the mushrooms pathogenic bacterium Pseudomonas tolaasii

The complete structure of the O-specific polysaccharide of the lipopolysaccharide isolated from the cultivated mushrooms pathogen Pseudomonas tolaasii is described. The structural determination, achieved by chemical and spectroscopical analyses, indicates a novel tetrasaccharide repeating unit built up of two units of 2-acetamido-2,6-di-deoxy-glucopyranose (Quinovosamine, QuipNAc) and two units of 2-acetamido-2-deoxy-gulopyranuronamide (GulpNAcAN), one of which is acetylated at C-3 position:     

O-Specific chain structure from the lipopolysaccharide fraction of Pseudomonas reactans: a pathogen of the cultivated mushrooms

An O-specific polysaccharide containing 2-acetamidino-2-deoxy-beta-D-glucopyranose (Glcp2Am), 2,4-diacetamido-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAc4NAc, bacillosamine) and 2,4-di-(N-acetyl-L-alanylamino)-2,4,6-trideoxy-beta-D-glucopyranose (QuipNAlaAc4NAlaAc) was isolated from the phenol-soluble lipopolysaccharide fraction of the mushroom-associated bacterium Pseudomonas reactans. The structure, determined by means of chemical analysis and 1D and 2D NMR spectroscopy, showed a linear trisaccharide-repeating unit, as shown below:-->3)-beta-D-QuipNAlaAc4NAlaAc-(1-->3)-alpha-D-Glcp2Am-(1-->3)-alpha-D-QuipNAc4NAc(1-->To our knowledge, this is the first complete O-chain structure reported for the lipopolysaccharide of a mushroom-associated bacterium.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .