White Line Inducing Principle Research Articles

WLIP, WLIPβ, and WLIPγ Produced from Pseudomonas canadensis Q3-1 via Precursor-Directed Biosynthesis and Their Roles on Biocontrol of Phytophthora Blight in Peppers.

White line-inducing principle (WLIP, 1), together with two new cyclic lipopeptides (CLPs) WLIPβ (2) and WLIPγ (3), were characterized from the supernatant of Pseudomonas canadensis Q3-1 via precursor-directed biosynthesis (PDB) in the current study. They were purified from the supernatant of P. canadensis Q3-1 by solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC), and their structures were mainly determined via bioinformatic analyses, spectrometric and spectroscopic techniques, as well as single crystal X-ray diffraction (XRD). These WLIPs share (R)-3-hydroxydecanoic acid (HDA), but they differ from each other in the composition of peptidic sequences. In addition, these CLPs showed biocontrol activities against Phytophthora blight (caused by Phytophthora capsici) in peppers. Collectively, this study has shown that PDB could be used for generating new CLPs in Pseudomonas spp. Moreover, we have confirmed that WLIP, WLIPβ, and WLIPγ could be used as lead agrochemicals to control Phytophthora blight in peppers.

Characterization of the biocontrol activity of three bacterial isolates against the phytopathogen Erwinia amylovora.

Antibiotics are sprayed on apple and pear orchards to control, among other pathogens, the bacterium Erwinia amylovora, the causative agent of fire blight. As with many other pathogens, we observe the emergence of antibiotic‐resistant strains of E. amylovora. Consequently, growers are looking for alternative solutions to combat fire blight. To find alternatives to antibiotics against this pathogen, we have previously isolated three bacterial strains with antagonistic and extracellular activity against E. amylovora, both in vitro and in planta, corresponding to three different bacterial genera: Here, we identified the inhibitory mode of action of each of the three isolates against E. amylovora. Isolate Bacillus amyloliquefaciens subsp. plantarum (now B. velezensis) FL50S produces several secondary metabolites including surfactins, iturins, and fengycins. Specifically, we identified oxydifficidin as the most active against E. amylovora S435. Pseudomonas poae FL10F produces an active extracellular compound against E. amylovora S435 that can be attributed to white‐line‐inducing principle (WLIP), a cyclic lipopeptide belonging to the viscosin subfamily (massetolide E, F, L, or viscosin). Pantoea agglomerans NY60 has a direct cell‐to‐cell antagonistic effect against E. amylovora S435. By screening mutants of this strain generated by random transposon insertion with decreased antagonist activity against strain S435, we identified several defective transposants. Of particular interest was a mutant in a gene coding for a Major Facilitator Superfamily (MFS) transporter corresponding to a transmembrane protein predicted to be involved in the extracytoplasmic localization of griseoluteic acid, an intermediate in the biosynthesis of the broad‐spectrum phenazine antibiotic d‐alanylgriseoluteic acid.

Biosynthesis and Antimicrobial Activity of Pseudodesmin and Viscosinamide Cyclic Lipopeptides Produced by Pseudomonads Associated with the Cocoyam Rhizosphere.

Pseudomonas cyclic lipopeptides (CLPs) are encoded non-ribosomally by biosynthetic gene clusters (BGCs) and possess diverse biological activities. In this study, we conducted chemical structure and BGC analyses with antimicrobial activity assays for two CLPs produced by Pseudomonas strains isolated from the cocoyam rhizosphere in Cameroon and Nigeria. LC-MS and NMR analyses showed that the Pseudomonas sp. COR52 and A2W4.9 produce pseudodesmin and viscosinamide, respectively. These CLPs belong to the Viscosin group characterized by a nonapeptidic moiety with a 7-membered macrocycle. Similar to other Viscosin-group CLPs, the initiatory non-ribosomal peptide synthetase (NRPS) gene of the viscosinamide BGC is situated remotely from the other two NRPS genes. In contrast, the pseudodesmin genes are all clustered in a single genomic locus. Nano- to micromolar levels of pseudodesmin and viscosinamide led to the hyphal distortion and/or disintegration of Rhizoctonia solani AG2-2 and Pythium myriotylum CMR1, whereas similar levels of White Line-Inducing Principle (WLIP), another member of the Viscosin group, resulted in complete lysis of both soil-borne phytopathogens. In addition to the identification of the biosynthetic genes of these two CLPs and the demonstration of their interaction with soil-borne pathogens, this study provides further insights regarding evolutionary divergence within the Viscosin group.

Pseudomonas Cyclic Lipopeptides Suppress the Rice Blast Fungus Magnaporthe oryzae by Induced Resistance and Direct Antagonism.

Beneficial Pseudomonas spp. produce an array of antimicrobial secondary metabolites such as cyclic lipopeptides (CLPs). We investigated the capacity of CLP-producing Pseudomonas strains and their crude CLP extracts to control rice blast caused by Magnaporthe oryzae, both in a direct manner and via induced systemic resistance (ISR). In planta biocontrol assays showed that lokisin-, white line inducing principle (WLIP)-, entolysin- and N3-producing strains successfully induced resistance to M. oryzae VT5M1. Furthermore, crude extracts of lokisin, WLIP and entolysin gave similar ISR results when tested in planta. In contrast, a xantholysin-producing strain and crude extracts of N3, xantholysin and orfamide did not induce resistance against the rice blast disease. The role of WLIP in triggering ISR was further confirmed by using WLIP-deficient mutants. The severity of rice blast disease was significantly reduced when M. oryzae spores were pre-treated with crude extracts of N3, lokisin, WLIP, entolysin or orfamide prior to inoculation. In vitro microscopic assays further revealed the capacity of crude N3, lokisin, WLIP, entolysin, xantholysin and orfamide to significantly inhibit appressoria formation by M. oryzae. In addition, the lokisin and WLIP biosynthetic gene clusters in the producing strains are described. In short, our study demonstrates the biological activity of structurally diverse CLPs in the control of the rice blast disease caused by M. oryzae. Furthermore, we provide insight into the non-ribosomal peptide synthetase genes encoding the WLIP and lokisin biosynthetic machineries.

Secondary Metabolites Production and Plant Growth Promotion by Pseudomonas chlororaphis and P. aurantiaca Strains Isolated from Cactus, Cotton, and Para Grass.

Fluorescent pseudomonads have been isolated from halophytes, mesophytes, and xerophytes of Pakistan. Among these, eight isolates, GS-1, GS-3, GS-4, GS-6, GS-7, FS-2 (cactus), ARS-38 (cotton), and RP-4 (para grass), showed antifungal activity and were selected for detailed study. Based on biochemical tests and 16S rRNA gene sequences, these were identified as strains of P. chlororaphis subsp. chlororaphis and aurantiaca. Secondary metabolites of these strains were analyzed by LC-MS. Phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine, Cyclic Lipopeptide (white line-inducing principle (WLIP)), and lahorenoic acid A were detected in variable amounts in these strains. P. aurantiaca PB-St2 was used as a reference as it is known for the production of these compounds. The phzO and PCA genes were amplified to assure that production of these compounds is not an artifact. Indole acetic acid production was confirmed and quantified by HPLC. HCN and siderophore production by all strains was observed by plate assays. These strains did not solubilize phosphate, but five strains were positive for zinc solubilization. Wheat seedlings were inoculated with these strains to observe their effect on plant growth. P. aurantiaca strains PB-St2 and GS-6 and P. chlororaphis RP-4 significantly increased both root and shoot dry weights, as compared with uninoculated plants. However, P. aurantiaca strains FS-2 and ARS-38 significantly increased root and shoot dry weights, respectively. All strains except PB-St2 and ARS-38 significantly increased the root length. This is the first report of the isolation of P. aurantiaca from cotton and cactus, P. chlororaphis from para grass, WLIP and lahorenoic acid A production by P. chlororaphis, and zinc solubilization by P. chlororaphis and P. aurantiaca.

First total synthesis of WLIP: on the importance of correct protecting group choice.

Cyclic lipodepsipeptides (CLPs) are a group of metabolites produced by Pseudomonas bacteria, involved in various biological functions and displaying a wide range of properties, including antibacterial and antifungal activities. The white line-inducing principle (WLIP) is a member of the viscosin group featuring a Glu2 amino acid. Recently, a total synthesis of pseudodesmin A - the Gln2 counterpart of WLIP - was described, and we here expand this route to Glu2 containing CLPs. We report the first total synthesis of WLIP and at the same time establish that the Gln2 to Glu2 substitution has an adverse impact on the crude purity and overall yield. A comparative study of different CLP analogues reveals the importance of the nature of the Glx2 protecting group in determining these outcomes. Replacement of the conventional tBu protecting group by the larger benzyl group for the Glu residue in our synthesis strategy indeed resulted in an improved conversion. Next to achieving the first WLIP total synthesis, we thus show the importance of a careful choice of protecting groups for the success of this type of solid-phase synthesis approaches towards CLPs.

WLIP derived from Lasiosphaera fenzlii Reich exhibits anti-tumor activity and induces cell cycle arrest through PPAR-γ associated pathways

White-line-inducing principle (WLIP), a lipodepsipeptide isolated from Lasiosphaera fenzlii Reich., which is one of the puffballs, for the first time, has been reported to exhibit anti-microbial activity. However, there are no reports regarding the anti-tumor effects of WLIP. In this study, we reported the anti-cancer effects of WLIP on K562 cells, and its potential effect on the PPAR-γ activation pathway. The obtained results showed that WLIP exerted strong anti-proliferative effect on K562 cells. Moreover, WLIP was found to increase apoptosis and induce G0/Gl arrest. Modeling results from the Surflex-Dock program suggested that PPAR-γ might be the potential anti-tumor target of WLIP, which was confirmed by the experiments that WLIP increased the activity in luciferase reporter assay and the expression of PPAR-γ in Western blot. Besides, WLIP was able to down-regulate the expression of Bcl-xL, Cyclin-D1 in K562 cells. In summary, our novel observations suggested that WLIP might have a potential implication in cancer prevention and treatment, and also showed for the first time that the anti-tumor effect of WLIP might be mediated through modulation of the PPAR-γ activation pathway.

Draft Genome Sequence of Pseudomonas fluorescens LMG 5329, a White Line-Inducing Principle-Producing Bioindicator for the Mushroom Pathogen Pseudomonas tolaasii

Pseudomonas tolaasii, the causative agent of Agaricus bisporus brown blotch disease, can be identified by the white line reaction, occurring upon confrontation of the tolaasin-producing mushroom pathogen with “Pseudomonas reactans,” producing the lipopeptide white line-inducing principle (WLIP). The draft genome sequence of the WLIP-producing indicator Pseudomonas fluorescens strain LMG 5329 is reported here.

Distinct lipopeptide production systems for WLIP (white line‐inducing principle) in Pseudomonas fluorescens and Pseudomonas putida

The interaction of WLIP (white line-inducing principle), a member of the viscosin group of Pseudomonas lipopeptides, with tolaasin, a lipopeptide mycotoxin secreted by Pseudomonas tolaasii, enables identification of the mushroom pathogen relying on formation of a lipopeptide coprecipitate between confronted colonies of an indicator strain (designated Pseudomonas 'reactans') and P. tolaasii. The WLIP non-ribosomal lipopeptide synthesis system of the mushroom isolate P. 'reactans' LMG 5329 (Wip) was identified and shown to be most similar to the Pseudomonas fluorescens SBW25 viscosin system (Visc), but remarkably different from the WLIP-generating Wlp system previously identified in the rice rhizosphere isolate Pseudomonas putida RW10S2. The Wlp machinery is composed of modules most similar to those recruited for biosynthesis of the non-viscosin-type lipopeptides putisolvin and entolysin by strains from the P. putida clade. In line with the pronounced synteny between the wip and visc flanking regions, strain LMG 5329 was identified as an authentic P. fluorescens closely related to strain SBW25. In both P. putida and P. fluorescens, WLIP production confers similar phenotypes of microbial antagonism and surface colonization. Genotypes other than wlp or wip were not identified among WLIP producers isolated from mushroom, maize rhizosphere or water.

Genetic and Functional Characterization of Cyclic Lipopeptide White-Line-Inducing Principle (WLIP) Production by Rice Rhizosphere Isolate Pseudomonas putida RW10S2

The secondary metabolite mediating the GacS-dependent growth-inhibitory effect exerted by the rice rhizosphere isolate Pseudomonas putida RW10S2 on phytopathogenic Xanthomonas species was identified as white-line-inducing principle (WLIP), a member of the viscosin group of cyclic lipononadepsipeptides. WLIP producers are commonly referred to by the taxonomically invalid name "Pseudomonas reactans," based on their capacity to reveal the presence of a nearby colony of Pseudomonas tolaasii by inducing the formation of a visible precipitate ("white line") in agar medium between both colonies. This phenomenon is attributed to the interaction of WLIP with a cyclic lipopeptide of a distinct structural group, the fungitoxic tolaasin, and has found application as a diagnostic tool to identify tolaasin-producing bacteria pathogenic to mushrooms. The genes encoding the WLIP nonribosomal peptide synthetases WlpA, WlpB, and WlpC were identified in two separate genomic clusters (wlpR-wlpA and wlpBC) with an operon organization similar to that of the viscosin, massetolide, and entolysin biosynthetic systems. Expression of wlpR is dependent on gacS, and the encoded regulator of the LuxR family (WlpR) activates transcription of the biosynthetic genes and the linked export genes, which is not controlled by the RW10S2 quorum-sensing system PmrR/PmrI. In addition to linking the known phenotypes of white line production and hemolytic activity of a WLIP producer with WLIP biosynthesis, additional properties of ecological relevance conferred by WLIP production were identified, namely, antagonism against Xanthomonas and involvement in swarming and biofilm formation.

The Solution Structure and Self‐Association Properties of the Cyclic Lipodepsipeptide Pseudodesmin A Support Its Pore‐Forming Potential

Pseudodesmin A is a cyclic lipodepsipeptide (CLP) of the viscosin group with a moderate in vitro biological activity. For several CLPs, including members of this group, this activity has been related to the ability to form ion pores in cellular membranes. As their size does not allow individual CLPs to span the membrane bilayer, individual monomers must somehow assemble into a larger structure. NMR spectroscopy has been used to demonstrate that in chloroform and other apolar organic solvents, pseudodesmin A monomers assemble into a supramolecular structure. These self-assembled structures can become sufficiently large to span the membrane bilayer as demonstrated with translational diffusion NMR spectroscopic measurements. With the aim to obtain more insight into the structural nature of this assembly, the solution conformation of pseudodesmin A was first determined by using ROESY (rOe) restraints measured in acetonitrile, in which no self-association occurs. The structure, which is found to be mostly similar to the previously described crystal structure, is shown to be retained within the supramolecular complex. Intermolecular rOe contacts obtained in chloroform together with chemical shift perturbation data provides structural insight into the organization of the self-associated complex. Based upon this analysis, a model for the organization of pseudodesmin A monomers in the supramolecular assembly is proposed, which is in agreement with the formation of bilayer spanning hydrophilic pores and provides the basis for a structure-function relationship for this type of CLPs. Finally, it is demonstrated that the differences previously reported between the crystal and solution conformation of the white line inducing principle (WLIP), a close analogue of pseudodesmin A, are the result of the use of dimethyl sulfoxide as solvent, whose strong hydrogen-bonding capacity induces conformational exchange.

Bioactive and structural metabolites of pseudomonas and burkholderia species causal agents of cultivated mushrooms diseases.

Pseudomonas tolaasii, P. reactans and Burkholderia gladioli pv. agaricicola, are responsible of diseases on some species of cultivated mushrooms. The main bioactive metabolites produced by both Pseudomonas strains are the lipodepsipeptides (LDPs) tolaasin I and II and the so called White Line Inducing Principle (WLIP), respectively, LDPs which have been extensively studied for their role in the disease process and for their biological properties. In particular, their antimicrobial activity and the alteration of biological and model membranes (red blood cell and liposomes) was established. In the case of tolaasin I interaction with membranes was also related to the tridimensional structure in solution as determined by NMR combined with molecular dynamic calculation techniques. Recently, five news minor tolaasins, tolaasins A-E, were isolated from the culture filtrates of P. tolaasii and their chemical structure was determined by extensive use of NMR and MS spectroscopy. Furthermore, their antimicrobial activity was evaluated on target micro-organisms (fungi-including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.-chromista, yeast and bacteria). The Gram positive bacteria resulted the most sensible and a significant structure-activity relationships was apparent. The isolation and structure determination of bioactive metabolites produced by B. gladioli pv. agaricicola are still in progress but preliminary results indicate their peptide nature. Furthermore, the exopolysaccharide (EPS) from the culture filtrates of B. gladioli pv. agaricicola, as well as the O-chain and lipid A, from the lipopolysaccharide (LPS) of the three bacteria, were isolated and the structures determined.

Bioactive and Structural Metabolites ofPseudomonasandBurkholderiaSpecies Causal Agents of Cultivated Mushrooms Diseases

Pseudomonas tolaasii, P. reactans and Burkholderia gladioli pv. agaricicola, are responsible of diseases on some species of cultivated mushrooms. The main bioactive metabolites produced by both Pseudomonas strains are the lipodepsipeptides (LDPs) tolaasin I and II and the so called White Line Inducing Principle (WLIP), respectively, LDPs which have been extensively studied for their role in the disease process and for their biological properties. In particular, their antimicrobial activity and the alteration of biological and model membranes (red blood cell and liposomes) was established. In the case of tolaasin I interaction with membranes was also related to the tridimensional structure in solution as determined by NMR combined with molecular dynamic calculation techniques. Recently, five news minor tolaasins, tolaasins A-E, were isolated from the culture filtrates of P. tolaasii and their chemical structure was determined by extensive use of NMR and MS spectroscopy. Furthermore, their antimicrobial activity was evaluated on target micro-organisms (fungi–-including the cultivated mushrooms Agaricus bisporus, Lentinus edodes, and Pleurotus spp.–-chromista, yeast and bacteria). The Gram positive bacteria resulted the most sensible and a significant structure-activity relationships was apparent. The isolation and structure determination of bioactive metabolites produced by B. gladioli pv. agaricicola are still in progress but preliminary results indicate their peptide nature. Furthermore, the exopolysaccharide (EPS) from the culture filtrates of B. gladioli pv. agaricicola, as well as the O-chain and lipid A, from the lipo-polysaccharide (LPS) of the three bacteria, were isolated and the structures determined.

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 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.

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.