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.     

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.     

A left-handed alpha-helix containing both L- and D-amino acids: the solution structure of the antimicrobial lipodepsipeptide tolaasin

The 18-amino acid cytolytic lipodepsipeptide tolaasin, produced in culture by virulent strains of Pseudomonas tolaasii, is the causal agent of the brown blotch disease of the cultivated mushroom. Tolaasin has a sequence of D-amino acids in its N-terminal region, then alternates L- and D-amino acids, and bears a C-terminal lactone macrocycle composed of 5-residues. The solution structure of tolaasin in sodium dodecyl sulfate was studied by 2D-NMR spectroscopy and molecular dynamics simulated annealing calculations. Tolaasin forms an amphipathic left-handed alpha-helix in the regionDPro2-DalloThr14 comprising the sequence of seven D-amino acids and the adjacent L-D-L-D-D-region. To the best of our knowledge, this is the first recognized example of a left-handed alpha-helix including both D- and L-amino acids. The lactone macrocycle adopts a "boat-like" conformation and is shifted from the helical axis as to form a "golf-club" overall conformation. These structural features will be of importance in understanding, and preventing, tolaasin's role in the bacterial colonization of the host plant, and its toxic action on cells. Furthermore, the observed antimicrobial activity together with the potential resistance to enzymatic degradation and the increased antigenicity (both due to the presence of L- and D-amino acids) strongly suggests for tolaasin a potential role as a template model for the design of new therapeutic antibacterial molecules.     

Temperature‐dependent hemolytic activity of membrane pore‐forming peptide toxin,tolaasin

Tolaasin, a pore‐forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin‐induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 °C, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 °C and the temperature was decreased, complete hemolysis was observed even at 4 °C. These results indicate that tolaasin binding to cell membrane is temperature‐sensitive while tolaasin‐induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore‐forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose‐dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin‐induced hemolysis can be explained by a multi‐hit model. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Genetic characterization of Pseudomonas 'NZI7'--a novel pathogen that results in a brown blotch disease of Agaricus bisporus

AIMS: To characterize a novel pseudomonad isolate capable of causing brown blotch disease of Agaricus bisporus. METHODS AND RESULTS: Using the white-line-in-agar (WLA) assay, fluorescent pseudomonads isolated from a New Zealand mushroom farm were screened for the lipodepsipeptide tolaasin, a characteristic marker of Pseudomonas tolaasii. One isolate, NZI7, produced a positive WLA assay and caused brown lesions of A. bisporus comparable with those produced by Ps. tolaasii. However, genetic analysis suggested that Ps. tolaasii and NZI7 were genetically dissimilar, and that NZI7 is closely related to Pseudomonas syringae. Nucleotide sequence analyses of a gene involved in tolaasin production indicated that similar genes are present in both NZI7 and Ps. tolaasii. CONCLUSION: NZI7 represents a novel Pseudomonas species capable of causing brown blotch disease of A. bisporus. SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic identification of Ps. tolaasii based on A. bisporus browning and positive WLA may have limited specificity.     

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 microM). This effect was not detected for tolaasin I at the concentrations tested (< 28 microM). 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.     

Left handed alpha-helix formation by a bacterial peptide

The alpha-helix is a common element of secondary structure in proteins and peptides. In eukaryotic organisms, which exclusively incorporate L-amino acids into such molecules, stereochemical interactions make such alpha-helices, invariably right-handed. Pseudomonas tolaasii Paine is the causal organism of the economically significant brown blotch disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach. P. Tolaasii proceduces an extracellular lipodepsipeptide toxin, tolaasin, which causes the brown pitted lesions on the mushroom cap. Circular dichroism studies on tolaasin in a membrane-like environment indicate the presence of a left-handed alpha-helix, probably formed by a sequence of 7 D-amino acids in the peptide. P. tolaasii represents the first reported example of an organism which has evolved the ability to biosynthesize a left-handed alpha-helix.     

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.     

PCR assays for specific and sensitive detection of Pseudomonas tolaasii,the cause of brown blotch disease of mushrooms

AIMS: The present study describes PCR assays to detect specifically Pseudomonas tolaasii from various samples. METHODS AND RESULTS: Two sets of PCR primers were developed to amplify genes required for tolaasin production. Only a PCR product of 449 bp or 249 bp was produced in PCR reactions with the Pt-1A/Pt-1D1 or Pt-PM/Pt-QM primer sets, respectively, and DNA and cells of Ps. tolaasii. Nested and immunocapture-nested PCR could detect to 3 cells of Ps. tolaasii and amplify the Ps. tolaasii-specific DNA from a sample containing 10 000 times more other bacterial cells than Ps. tolaasii, respectively. CONCLUSIONS: The PCR assays are simple, rapid and reliable methods for detection and identification of Ps. tolaasii. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocols can effectively distinguish Ps. tolaasii from other bacteria and detect Ps. tolaasii from various samples for studying ecology of the bacterium and preventing the use of contaminated water or spawn or medium in mushroom cultivation.     

Evidence for genotypic differences between the two siderovars of Pseudomonas tolaasii,cause of brown blotch disease of the cultivated mushroom Agaricus bisporus

Sixteen representative isolates of Pseudomonas tolaasii, the causal agent of brown blotch of the cultivated mushroom Agaricus bisporus, were previously assigned to two siderovars (sv1 and sv2) on the basis of pyoverdines synthesized. Each isolate was pathogenic and produced a typical white line precipitate when cultured adjacent to Pseudomonas "reactans" strain LMG 5329. These 16 isolates of P. tolaasii, representing sv1 and sv2, were further characterized using genotypic methods to examine the relationships between the isolates. Rep-PCR studies revealed two distinct patterns from these isolates, which were consistent with the siderovar grouping. Ribotyping differentiated P. tolaasii LMG 2342T (sv1) and PS 3a (sv2) into two distinct ribotypes. A pair of primers, targeted to a 2.1-kb fragment of tl1 (encoding a tolaasin peptide synthetase), yielded the same PCR product from P. tolaasii LMG 2342T (sv1) and PS 22.2 (sv1), but not from PS 3a (sv2). Southern blot analysis indicated that homologues of tl1 are present in PS 3a, but the pattern of hybridization differed from PS 22.2 and LMG 2342T. Sequence determination and analysis of the internally transcribed spacer region ITSI for P. tolaasii LMG 2342T, LMG 6641, and PS 3a strains further supported the presence of the two siderovars. It is concluded that considerable genotypic differences exist among Finnish isolates of P. tolaasii causing brown blotch disease on the cultivated mushroom, which is in agreement with the phenotypic diversity highlighted through previous siderotyping studies.     

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.     

Preliminary description of biocidal (syringomycin) activity in fluorescent plant pathogenic Pseudomonas species

Strains representing the fluorescent plant pathogenic Pseudomonas spp., Ps. agarici , Ps. asplenii , Ps. avellanae , Ps. beteli , Ps. caricapapayae , Ps. cichorii , Ps. corrugata , Ps. ficuserectae , Ps. flectens , Ps. fuscovaginae , Ps. marginalis , Ps. meliae , Ps. savastanoi , Ps. syringae , Ps. tolaasii and Ps. viridiflava were tested for biocidal activity using Aspergillus niger as assay organism. Inhibitory behaviour was found in strains of Ps. asplenii , Ps. blatchfordae , Ps. cichorii , Ps. corrugata , Ps. fuscovaginae , Ps. marginalis , Ps. marginalis pv. pastinacea , Ps. syringae pv. syringae , Ps. syringae pv. aptata , Ps. syringae pv. atrofaciens , Ps. syringae pv. lapsa , Ps. tolaasii , and strains of a Pseudomonas sp. pathogenic to Actinidia , in the Ps. savastanoi genomic sp. Antifungal activity could be identified with the production of members of the syringomycin family of toxins by strains in Ps. syringae , Ps. asplenii and Ps. fuscovaginae . These toxin reactions support suggestions made elsewhere of the synonymy of the latter two species. In a preliminary characterization using tests for stability to heat, protease, acid and alkaline treatments, unknown toxins consistent with syringomycin-like toxins the strains from Actinidia speciesColour RGB 0,0,128. The toxins from Ps. cichorii and from Ps. corrugata differed in their reactions from all other agents. Pseudomonas tolaasii produces the antifungal compound tolaasin. The white line reaction with ' Ps. reactans ', a test for tolaasin production by strains of Ps. tolaasii , was confirmed as specific for this compound. Some of these low molecular weight toxins may be produced by some of these plant pathogenic strains.     

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.     

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 .     

A note on ginger blotch, a new bacterial disease of the cultivated mushroom, Agaricus bisporus

Ginger blotch, a new bacterial disease of the cultivated mushroom, Agaricus bisporus , is described from farms in the UK. The symptoms are distinct from the classical blotch disease caused by Pseudomonas tolaasii. The causative organism has been isolated and identified as a new member of the Pseudomonas fluorescens complex which can be distinguished from Pseudomonas tolaasii by several simple tests.     

Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops: numbers of the bacterium and symptom development on mushrooms grown in various environments after artificial inoculation

The recovery of Pseudomonas tolaasii applied to peat, limestone and mushroom caps, is very difficult, recovery rates being 0.2–16.0%. Without Agaricus bisporus mycelium, inoculated Ps.tolaasii disappears in the casing layer. As mushroom primordia grew in size on inoculated mushroom beds, the number of detectable cells of the pathogen increased. Symptoms of blotch disease became visible when 5.4 times 10⁶ cfu were detectable, when the mushroom primordia were 6 mm in diameter; 60% of mushrooms showed symptoms before they were 15 mm in diameter. Application of Ps.tolaasii cells as low as 20 cfu/cm² of bed gave epidemics of this severity. Neither size nor age of mushrooms affects their susceptibility. When Ps.tolaasii was placed directly onto caps, 6 times 10⁷ cfu were necessary to produce a blotch lesion (though only 3.5 times 10⁶ cfu could be recovered). Changes in r.h. and temperature did not affect the numbers of cells of Ps.tolaasii on inoculated caps; very frequent watering did so. Increased severity of the disease was seen only on over-watered mushrooms; this occurred by increase in the size of lesions seen at the primordium stage. The number of cells of Ps.tolaasii present on the early primordial stages of mushroom growth controls the extent of blotch disease seen at harvesting, whereas variations in r.h. or temperature during growing do not do so. An illustrated disease symptom measurement key (of general application for assessing severity of blotch disease) is included in the text.     

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.     

Pseudomonas tolaasii control by kasugamycin in cultivated mushrooms (Agaricus bisporus)

Pseudomonas tolaasii , causing brown blotch disease on the edible mushroom Agaricus bisporus , was effectively controlled by kasugamycin. An artificial infection was first established in the first flush, by inoculating the button-sized mushrooms of the first flush with a suspension of Ps. tolaasii. A 1% aqueous solution of kasugamycin supplied on the button-sized mushrooms of the second flush drastically reduced bacterial blotch symptoms on these mushrooms at picking stage. Disease incidence in the second flush in the control treatment (inoculated with Ps. tolaasii ) was composed of 18% lightly, 29% moderately and 10% heavily affected mushrooms, which totalled up to 57% affected. The 1% kasugamycin treatment significantly reduced total disease incidence to only 9% (lightly) affected. Single sodium hypochlorite treatments showed no result.     

Pseudomonas tolaasii in cultivated mushroom (Agaricus bisporus) crops: effects of sodium hypochlorite on the bacterium and on blotch disease severity

Sodium hypochlorite killed Pseudomonas tolaasii in water in 30 s at pH 6.0 when 5 mg/1 free available chlorine (FAC) was used. On glass beads 62.5 mg/1 FAC was necessary to kill the pathogen in 30 s. Peat and limestone mixture ('casing') prevented some cells of the pathogen being killed by chlorine. Casing treated with 50 and 100 mg/1 FAC still contained some Ps. tolaasii cells which were later able to multiply. Although some viable cells of the pathogen survived the use of 150 mg/1 FAC these were apparently unable to multiply. Mushroom tissue is more 'disinfectant-wasting' than casing, the pathogen on it surviving 250 mg/1 FAC for 10 min. In controlled environmental experiments, use of 150 mg/1 FAC at mushroom 'pinning' (2.5 mm diameter primordia) gave as much control of blotch disease as was obtainable if chlorination began after casing. Delay in starting chlorination until the mushrooms were 10 to 15 mm in diameter resulted in blotch disease incidence and severity as severe as in unchlorinated controls. Disease incidence was not reduced when 50, 100 and 150 mg/1 FAC was used, but disease severity was significantly reduced when 150 mg/1 was used. Adjusting the pH of the water did not affect these results. On commercial farms, routine watering with 150 mg/1 FAC starting at pinning, checked frequently by the sodium arsenite titrimetric method, for 3 years, reduced the percentage of mushrooms discarded because of very severe Ps. tolaasii blotch from 5.2% to 0.6% on one farm and from 7.4% to 0.5% on another, but did not eliminate the disease completely.