Production of Phytotoxic Sirodesmins by Aggressive Strains of Leptosphaeria maculans Differing in Interactions with Oil Seed Rape Genotypes

Sirodesmins were extracted from culture filtrates of 3 aggressive strains of Leptosphaeria maculans which showed different specific interactions with the oilseed rape cultivars Quinta and Jet Neuf. Up to 8 compounds were detectable showing reactions on thin layer plates which are characteristic for sirodesmins. Sirodesmin PL, sirodesmin C, deacetylsirodesmin PL and deacetyl-sirodesmin C were identified on the basis of their UV-spectra, by various conversion reactions and by cochromatography with known standards. Two further compounds were tentatively identified as sirodesmin B and deacetylsirodesmin B on the basis of conversion reactions only. In the cultures of all strains sirodesmin PL was the main component, followed by deacetylsirodesmin PL, sirodesmin C, and deacetylsirodesmin C. These 4 metabolities were mainly responsible for the phytotoxic activity of all culture filtrates. They were systemically transported to leaves of root treated rape seedlings. No correlation was found between the production of the 4 main sirodesmins and the differential host parasite interactions of the 3 aggressive strains of Leptosphaeria maculans.     

Stress-driven discovery of metabolites from the phytopathogenic fungus Leptosphaeria maculans: structure and activity of leptomaculins A-E

A search for stress inducing metabolites produced by the plant pathogenic fungus Leptosphaeria maculans led to the isolation and structure elucidation of eight new metabolites, the leptomaculins and deacetylleptomaculins A-E. The chemical structures and absolute stereochemistry of the new metabolites were deduced by detailed analysis of 1D and 2D NMR spectroscopic data and chemical degradation of the toxin sirodesmin PL. Leptomaculins A and B are the first examples of naturally occurring 2,3-oxopiperazinethione and 2,3-dioxopiperazine, respectively. Stress inducing activity was found in the fungal phytotoxins sirodesmin PL and deacetylsirodesmin PL but not in any of the new leptomaculins, phomalide or phomamide. A metabolic pathway for biosynthesis of the first 2,3-(di)oxopiperazine(thione) from sirodesmin PL is proposed.     

Pathogenicity of Sirodesmin-deficient Mutants of Phoma lingam

Sirodesmin-deficient mutants were produced from aggressive isolates of Phoma lingam by UV-mutagenesis. The mutation rates were 7.1 × 10^-3 for mutants expressing a 100-fold reduction of sirodesmin PL in liquid cultures and 1.8 × 10^-3 for mutants with a 1000-fold reduction. The low ability of the mutants to produce sirodesmins was also apparent in planta after infection of cotyledons or the stem basis of Brassica napus. Growth rates under in vitro conditions, colony morphology, mating types and the formation of pycnidia and asci were not affected by the mutation. The mutants had the same ability as the wild types to infect cotyledons of the susceptible B. napus cv. Lirabon and to cause a typical grey-green tissue collapse. On the stem basis of the cv. Cobra, however, mutants caused significantly smaller lesions. These results indicate that phytotoxic sirodesmins produced by P. lingam have no basic role in pathogenesis and apparently are not involved in the virulence on cotyledons. However, it cannot be excluded that the compounds act as virulence factors on the stem base of B. napus.     

Differences in response to the toxin sirodesmin PL produced by Phoma lingam (Tode ex fr.) Desm. on protoplasts,cell aggregates and intact plants of resistant and susceptible Brassica accessions

Summary The selective property of sirodesmin PL, a toxin produced by Phoma lingam, was studied on protoplasts, cell aggregates, leaves and roots. Directly after isolation, protoplasts from all the different Brassica accessions were sensitive when treated with toxin in a concentration higher than 1 M. When more differentiated plant tissue. i.e. cell aggregates, leaves or roots, were investigated, insensitivity to the toxin was found in the plant material resistant to P. lingam, while the plant material susceptible to P. lingam was sensitive. The results reveal that a clear correlation between resistance to P. lingam and insensitivity to sirodesmin PL is present, and that the toxin can be used to distinguish resistant plant material from susceptible both in vivo and in vitro.     

The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans

Sirodesmin PL is a phytotoxin produced by the fungus Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus). This phytotoxin belongs to the epipolythiodioxopiperazine (ETP) class of toxins produced by fungi including mammalian and plant pathogens. We report the cloning of a cluster of genes with predicted roles in the biosynthesis of sirodesmin PL and show via gene disruption that one of these genes (encoding a two-module non-ribosomal peptide synthetase) is essential for sirodesmin PL biosynthesis. Of the nine genes in the cluster tested, all are co-regulated with the production of sirodesmin PL in culture. A similar cluster is present in the genome of the opportunistic human pathogen Aspergillus fumigatus and is most likely responsible for the production of gliotoxin, which is also an ETP. Homologues of the genes in the cluster were also identified in expressed sequence tags of the ETP producing fungus Chaetomium globosum. Two other fungi with publicly available genome sequences, Magnaporthe grisea and Fusarium graminearum, had similar gene clusters. A comparative analysis of all four clusters is presented. This is the first report of the genes responsible for the biosynthesis of an ETP.     

Transfer of resistance against Phoma lingam to Brassica napus by asymmetric somatic hybridization combined with toxin selection

Summary Irradiated mesophyll protoplasts from nine different accessions of B. juncea, B. nigra and B. carinata, all resistant to Phoma lingam, were used as gene donors in fusion experiments with hypocotyl protoplasts isolated from B. napus as the recipient. A toxin, sirodesmin PL, was used to select those fusion products in which the resistant gene(s) was present. In the fusion experiments different gene donors, various irradiation dosages and toxin treatments were combined. Symmetric and asymmetric hybrid plants were obtained from the cell cultures with and without toxin selection. Isozymes were used to verify hybrid characters in the symmetric hybrids, whereas two DNA probes were used to identify donor-DNA in the asymmetric hybrids. Resistance to P. lingam was expressed in all symmetric hybrids, and in 19 of 24 toxin-selected asymmetric hybrids, while all the unselected asymmetric hybrids were susceptible.     

Antibacterial Activity of Sirodesmin PL Phytotoxin: Application to the Selection of Phytotoxin-Deficient Mutants

Sirodesmin PL, a phytotoxin and mycotoxin produced by Leptosphaeria maculans, the causal agent of stem-canker disease of crucifers, exhibited antibacterial activity against gram-positive bacteria and particularly Bacillus subtilis. The importance of the disulfide bridge of the molecule in antibacterial activity was demonstrated. A simple and reliable bioassay based on the antibacterial activity of the toxin was performed for screening sirodesmin PL-deficient mutants when grown on solid culture medium. A mutant was selected and found to produce 3,700-fold less toxin than did the wild-type strain. A sensitive procedure for quantification of the toxin by high-pressure liquid chromatography was developed. Levels of product as low as 100 ng could be detected by this procedure.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Biosynthetic gene clusters for epipolythiodioxopiperazines in filamentous fungi

Epipolythiodioxopiperazines (ETPs) are toxic secondary metabolites made only by fungi. Recent research efforts have provided insight into the molecular mechanisms of the toxicity of these compounds. The availability of complete genome sequences for many fungi has also facilitated the identification of putative ETP biosynthetic gene clusters. Expression and mutational analyses have confirmed the role of such gene clusters in the biosynthesis of two ETPs, sirodesmin PL and gliotoxin. The creation of mutants unable to produce these ETPs has facilitated the assessment of these molecules as virulence factors in interactions between pathogenic fungi and their hosts.     

Strain typing of polish Leptosphaeria maculans isolates supports at the genomic level the multi-species concept of aggressive and non-aggressive strains

47 Polish isolates of the blackleg fungus Leptosphaeria maculans (Phoma lingam) were compared with eight well-defined reference strains from Germany, France, Denmark, Australia and one Polish isolate of Phoma nigrificans. The isolates were tested (i) for growth characteristics, (ii) for their ability to form sirodesmins, (iii) for cellulolytic enzymes, and (iv) for pathotype-differentiating molecular markers generated by RAPD-PCR, PCR analysis with pathotype-specific primer pairs and PFGE. With two exceptions all Polish isolates do not form sirodesmins. grow rapidly without penetrating into the substrate and form in most cases yellow or brown pigments in Czapek-Dox liquid cultures. With respect to cellulase secretion and molecular fingerprinting Polish A strains (aggressive) fit into the general picture of the aggressive pathotype group, whereas the NA isolates (non-aggressive) display a higher degree of heterogeneity. This matches with inoculation tests on rape seedlings, which revealed a considerable number of isolates ranging in aggressivity between the conventional A and NA pathotype group. Molecular fingerprinting techniques unequivocally sorted intermediately aggressive isolates into the NA pathotype group. Isolate Ph Bial, which produces sirodesmin but groups within NA isolates according to molecular and physiological markers, may represent a novel third group besides A and NA strains with intermediate aggressivity (IA). We hybridized Southern blots of electrophoretically separated chromosomes with radioactively labelled PCR fragments used for differentiation between A and NA isolates. The specificity of diagnostic PCR amplicons is reflected at the genomic level. The A probe reveals a single hybridizing chromosome exclusively in A strains. The NA probe reveals several chromosomes and is specific for the NA pathotype group. Chromosomes from intermediately aggressive strains are equally well recognized by the NA probe as are Polish isolates with low aggressivity and give no signal with the A probe. Both diagnostic DNA sequences are highly specific for the pathotype group they were derived from. The lack of correspondence of both genetic elements between A and NA strains strongly supports the idea of ascribing the pathotype groups to different species. Whereas the A pathotype group is genetically homogeneous and congruent with the species Leptosphaeria maculans, the NA group needs to be revised taxonomically. NA isolates will presumably have to be split into several independent species.     

Purification and Characterization of Thermostable Pectate Lyase with Protopectinase Activity from Thermophilic Bacillus sp. TS 47

A strain of thermophilic bacterium, Bacillus sp., with pectolytic activity has been isolated. It produced an extracellular endo-polygalacturonate trans-eliminase (PL, EC 4.2.2.1) when grown at 60°C on a medium containing polygalacturonate (PGA). The PL was purified by hydrophobic, cation exchange, and size exclusion column chromatographies. The molecular mass of the enzyme was 50 kDa by SDS-PAGE. The isoelectric point of the enzyme was pH 5.3. The enzyme had a half-life of 13 and 1 h at 65 and 70°C, respectively, and showed optimal activity around at 70°C and pH 8.0. It had protopectinase activity, besides PL activity, on lemon protopectin and cotton fibers. The first 20 amino acids sequence of the enzyme had significant similarity with that of PL from methophilic Bacillus subtilis, with 50% identity.     

Phospholipase and Proteinase Activities of Clinical Isolates of Candida from Immunocompromised Patients

Sixty-one isolates of Candida recovered from HIV seropositive and cancer patients were studied for elaboration of putative virulence determinants – phospholipase (PL) and secreted aspartyl proteinase (Sap). Forty two (68.85%) isolates examined were PL producers and 51 (83.6%) were positive for Sap. 57.37% (35/61) isolates produced both enzymes. Enzymatic activity was more pronounced in Candida albicans with 100% PL and 94.1% Sap activity. In contrast, non-C. albicans species demonstrated only 29.6% PL and 70.3% Sap activity, indicating interplay of other virulence determinants in these yeasts in colonization and disease.     

Effects of Feeding Pigs Increasing Levels of C 18:1 Trans Fatty Acids on Fatty Acid Composition of Backfat and Intramuscular Fat as well as Backfat Firmness

Forty Large White pigs were fed from 30kg to 103kg body mass on diets supplemented with 6% of pure high-oleic sunflower oil (HO) or HO plus increasing amounts of partially hydrogenated rape seed oil (HR; 1.85%, 3.70%, 5.55%), containing high levels of j 6 to j 11 C 18:1 trans fatty acid isomers. Increasing dietary C 18: trans fatty acids resulted in a linear increase in C 18:1 trans fatty acids and conjugated linoleic acid (cis-9, trans-11 CLA) in backfat (BF) as well as in neutral lipids (NL) and phospholipids (PL) of M. long. dorsi. Thus, the rate of bioconversion of trans vaccenic acid (TVA) into CLA and incorporation of C 18:1 trans and CLA into pig adipose tissue was not limited up to 25g total C 18:1 trans fatty acids including 3.3g of TVA perkg feed. BF was higher in C 18:1 trans fatty acids and CLA than M. long. dorsi NL and PL. In BF and NL the sum of saturated fatty acids (SFA) increased with increasing dietary amounts of HR, while in PL SFA were reduced. Thus, according to their physical properties, C 18:1 trans fatty acids partly replaced SFA in PL. Firmness of backfat was also significantly increased (P<0.05) with increasing amounts of HR in feed.     

Citrate synthase and pyruvate kinase activities during early life stages of the shrimp Farfantepenaeus paulensis (Crustacea,Decapoda, Penaeidae): effects of development and temperature

Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 °C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII–XIV (PL XII–XIV). PK activity reached a pronounced peak in PL V–VI, followed by a further decrease in PL XII–XIV. Temperature reduction produced variation in oxygen consumption rates (QO₂), ammonia–N excretion and in enzyme activities. Ammonia–N excretion was higher at 20 °C in mysis III (M III), PL V–VI and PL XII–XIV, resulting in substantially lower O:N ratios in these stages. QO₂ was increased in protozoea II (PZ II) and mysis I (M I) at 26 °C, while no difference in QO₂ was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V–VI and PL XII–XIV at 20 °C compared with 26 °C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85–0.92 for CS and 1.1–1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 °C for both enzymes. Weight-specific CS activity was positively correlated with QO₂ at 20 and 26 °C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.     

Efficient secretory expression of an alkaline pectate lyase gene from Bacillus subtilis in E. coli and the purification and characterization of the protein

The gene encoding pectate lyase (PL) from Bacillus subtilis WSHB04-02 was amplified by PCR, fused with a periplasmic secretion signal peptide sequence, pelB, from pET22b(+), cloned and expressed in Escherichia coli cells using a temperature control vector, pHsh. The recombinant E. coil was grown in a 5 l fermentor. PL was secreted in broth at 22 U l⁻¹ after 20 h when temperature was increased from 30°C to 42°C. The recombinant enzyme was purified to homogeneity as judged by SDS-PAGE. It was optimally active at pH 9.4 and 50°C over 30 min. Analysis of polygalacturonic acid (PGA) degradation products by electrospray ionization (ESI)-mass spectrometry (MS) indicated that PL produced a mixture of unsaturated oligo-galacturonides including unsaturated tri-galacturonic acid and unsaturated bi-galacturonic acid but not unsaturated mono-galacturonic acid.     

Characterization of the binding of paylean and DNA by fluorescence,UV spectroscopy and molecular docking techniques

The interaction of paylean (PL) with calf thymus DNA (ctDNA) was investigated using fluorescence spectroscopy, UV absorption, melting studies, ionic strength, viscosity experiments and molecular docking under simulated physiological conditions. Values for the binding constant K_a between PL and DNA were 5.11 × 10³, 2.74 × 10³ and 1.74 × 10³ L mol^–1 at 19, 29 and 39°C respectively. DNA quenched the intrinsic fluorescence of PL via a static quenching procedure as shown from Stern–Volmer plots. The relative viscosity and the melting temperature of DNA were basically unchanged in the presence of PL. The fluorescence intensity of PL–DNA decreased with increasing ionic strength. The value of K_a for PL with double‐stranded DNA (dsDNA) was larger than that for PL with single‐stranded DNA (ssDNA). All the results revealed that the binding mode was groove binding, and molecular docking further indicated that PL was preferentially bonded to A–T‐rich regions of DNA. The values for ΔH, ΔS and ΔG suggested that van der Waals forces or hydrogen bonding might be the main acting forces between PL and DNA. The binding distance was determined to be 3.37 nm based on the theory of Förster energy transference, which indicated that a non‐radiation energy transfer process occurred. Copyright © 2015 John Wiley & Sons, Ltd.     

The Effect of Seasonal Shifts in Temperature on the Lipid Composition of Marine Macrophytes

The comparative study of lipid composition was carried out in four species of marine algae, Ahnfeltia tobuchiensis, Laminaria japonica, Sargassum pallidum, and Ulva fenestrata, as well as a higher plant grass wrack (Zostera marina). Plants were collected in the Japan Sea in spring at 2.9 and 5.5°C and in summer at 23°C. The main lipid components of membranes were determined, and the general patterns of the ratio of phospholipids (PL), glycolipids (GL), betaine (BL), and neutral (NL) lipids were discerned. The relative content of NL in all species (except A. tobuchiensis) was higher in summer. The level of triacylglycerols was as high as 18–37%. The content of individual classes of PL and GL varied between the spring and summer samples, the relative content of PL being higher in spring. In most species, the ratio of PL to GL decreased in summer. The content of free sterols did not depend on the season. The molar ratios of phosphatidylcholine and diacylglycerol-o-(hydroxymethyl)-(N,N,N-trimethyl)homoserine to free sterols varied from 0.9 to 1.7. The seasonal changes of lipid composition were apparently related to macrophyte adaptation to water temperature and to biology of their development.