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ABSTRACT: Subtilisin-like serine proteases play an important role in pathogenic fungi during the penetration and colonization of their hosts. In this study, we perform an evolutionary analysis of the subtilisin-like serine protease genes of subphylum Pezizomycotina to find if there are similar pathogenic mechanisms among the pathogenic fungi with different life styles, which utilize subtilisin-like serine proteases as virulence factors. Within Pezizomycotina, nematode-trapping fungi are unique because they capture soil nematodes using specialized trapping devices. Increasing evidence suggests subtilisin-like serine proteases from nematode-trapping fungi are involved in the penetration and digestion of nematode cuticles. Here we also conduct positive selection analysis on the subtilisin-like serine protease genes from nematode-trapping fungi.
Phylogenetic analysis of 189 subtilisin-like serine protease genes from Pezizomycotina suggests five strongly-supported monophyletic clades. The subtilisin-like serine protease genes previously identified or presumed as endocellular proteases were clustered into one clade and diverged the earliest in the phylogeny. In addition, the cuticle-degrading protease genes from entomopathogenic and nematode-parasitic fungi were clustered together, indicating that they might have overlapping pathogenic mechanisms against insects and nematodes. Our experimental bioassays supported this conclusion. Interestingly, although they both function as cuticle-degrading proteases, the subtilisin-like serine protease genes from nematode-trapping fungi and nematode-parasitic fungi were not grouped together in the phylogenetic tree. Our evolutionary analysis revealed evidence for positive selection on the subtilisin-like serine protease genes of the nematode-trapping fungi.
Our study provides new insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. Pezizomycotina subtilisins most likely evolved from endocellular to extracellular proteases. The entomopathogenic and nematode-parasitic fungi likely share similar properties in parasitism. In addition, our data provided better understanding about the duplications and subsequent functional divergence of subtilisin-like serine protease genes in Pezizomycotina. The evidence of positive selection detected in the subtilisin-like serine protease genes of nematode-trapping fungi in the present study suggests that the subtilisin-like serine proteases may have played important roles during the evolution of pathogenicity of nematode-trapping fungi against nematodes.
BMC Evolutionary Biology 03/2010; 10:68. · 3.52 Impact Factor
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ABSTRACT: In this study, the mature peptide sequence of a pectin lyase gene A was amplified from Aspergillus niger strain EIM-6 by using RT-PCR reverse transcription technique. The cloned gene was then inserted into a Pichia pastoris expression vector pPIC9k to produce the recombinant expression plasmid pPIC9K-pelA. By using electric shocks, we successfully transformed the recombinant pPIC9K-pelA into Pichia pastoris GS115. The activity of the engineered strain reached to 2.3 U/mL after induction with the final concentration of 1.5% methanol. SDS-PAGE analysis revealed that the pPIC9K-pelA transformant had an additional protein band of approximately 38 kD, which was not present in the control. There were no significant differences between the recombinant and native pectin lyase with regard to their hydrolysis activities.
Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2009; 25(12):1962-8.
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ABSTRACT: In order to facilitate the understanding of the actual process of enzyme-based degradation of nematodes, we visualized the localization of BLG4, a cuticle-degrading protease from the nematophagous bacterium Brevibacillus laterosporus G4, on nematode cuticle by using an improved immuno-labeled fluorescent method. Live nematodes, heat-killed nematodes and extracted nematode cuticles were exposed to the protease, and the localization of the protease and the resulting tissue degradation and destruction were observed microscopically. The bioassay findings showed that live nematodes were significantly more resistant to the protease than the dead nematodes and the extracted cuticles were. The observation of the immuno-labeling fluorescence for BLG4 revealed that the protease localized first in the tail region of the live target; and then spread over the entire target and ultimately destroyed it, including the cuticle. The results indicated the resistance of nematode cuticles to enzymatic attacks and the differences in protease susceptibilities at different regions on the nematode cuticles.
Journal of Invertebrate Pathology 06/2009; 101(2):143-6. · 2.06 Impact Factor
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ABSTRACT: To investigate docosahexaenoic acid (DHA, C22:6n-3) biosynthesis pathway in marine fungus Schizochytrium sp. FJU-512, a cDNA library of the fungus was constructed and analyzed. The titers of primary library were up to 5.0 x 10(6). A total of 4005 ESTs were assembled into 1947 unigenes. Sequences annotation and function analysis were carried out by using Blast, GO and KEGG programs. Compared with other eukaryote genomes, Schizochytrium sp. FJU-512 ESTs shared at least 26.6% genes with Arabidopsis thaliana (E < or = 10(-10)). The cDNA (Contig46, assembled by EH401977 and EH404532) and EH40321 were found to encode serine/threonine protein phosphatase type 1 and cell division control protein 2 which were involved in successive binary cell division. Notably, the key enzymes involved in biosynthesis of fatty acid via polyketide synthases (PKS) such as beta-ketoacyl synthase, beta-ketoacyl reductase, hydroxyacyl dehydrogenase, enoyl-CoA hydratase/isomerase, and enoyl reductase were found in the cDNA library. The results indicated that DHA synthesis in Schizochytrium sp. FJU-512 had undergone PKS pathway.
Journal of Biotechnology 09/2008; 138(1-2):9-16. · 3.05 Impact Factor
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ABSTRACT: Docosahexaenoic acid (DHA C22:6n-3), a typical long chain polyunsaturated fatty acids (PUFAs) has many positive effects on diseases such as artherosclerosis, hypertriglyceridemia, hypertension and cancers. Marine fungi, especially Thraustochytrium spp. producing much DHA can serve as model organisms for explaining the mechanism on the biosynthesis of PUFA. We described two elongase genes (TFD6 and TFD5) involved in the biosynthesis of DHA in Thraustochytrium sp. FJN-10 was cloned by using reverse transcription PCR and rapid amplification of cDNA ends. TFD6 cDNA was 816 bp in length and encoded a protein of 271 amino acids. TFD5 cDNA was 831 bp in length and encoded a protein of 276 amino acids. Transmembrane analysis revealed that TFD6 contained five transmembrane domains while TFD5 contained seven. Tertiary structures of TFD6, TFD5 elongases were predicted by HHMMSTR (Hidden markov model for local sequence-structure) model and Rosetta program. Alignment of TFD6, TFD5 with other elongases showed that both of them shared an HXXHH conserved histidine-rich motif. Phylogenetic analysis showed that TFD6 was the closest to Thraustochytrium 66 elongase, while TFD5 was the closest to Thraustochytrium sp. delta5 elongase. TFD6 and TFD5 were subcloned into the Hind III/Xba I restriction site of pYES2 vector respectively. Recombined plasmids were transformed into Saccharomyces cerevisiae using lithium acetate method. Gas chromatography analysis showed that TFD6 could elongate C18:3n-3 to C20:3n-3 while TFD5 could elongate C20:5n-3 to C22:5n-3.
ACTA MICROBIOLOGICA SINICA 03/2008; 48(2):176-83.
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ABSTRACT: As a group of important natural enemies of nematode pests, nematophagous bacteria exhibit diverse modes of action: these include parasitizing; producing toxins, antibiotics, or enzymes; competing for nutrients; inducing systemic resistance of plants; and promoting plant health. They act synergistically on nematodes through the direct suppression of nematodes, promoting plant growth, and facilitating the rhizosphere colonization and activity of microbial antagonists. This review details the nematophagous bacteria known to date, including parasitic bacteria, opportunistic parasitic bacteria, rhizobacteria, Cry protein-forming bacteria, endophytic bacteria and symbiotic bacteria. We focus on recent research developments concerning their pathogenic mechanisms at the biochemical and molecular levels. Increased understanding of the molecular basis of the various pathogenic mechanisms of the nematophagous bacteria could potentially enhance their value as effective biological control agents. We also review a number of molecular biological approaches currently used in the study of bacterial pathogenesis in nematodes. We discuss their merits, limitations and potential uses.
FEMS Microbiology Ecology 09/2007; 61(2):197-213. · 3.41 Impact Factor
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ABSTRACT: An extracellular serine protease (Ac1) with a molecular mass of 35 kDa was purified from the nematode-trapping fungus Arthrobotrys conoides. The optimum activity of Ac1 is at pH 7.0 and 53.2 degrees C (over 20 min). Ac1 can degrade a broad range of substrates including casein, gelatin, bovine serum albumin, collagen, and nematode cuticles. Moreover, the enzyme can immobilize the free-living nematode Panagrellus redivivus and the pine wood nematode Bursaphelenchus xylophilus, indicating Ac1 may be involved in infection against nematodes. The encoding gene of Ac1 contains one intron of 60-bp and two exons encoding a polypeptide of 411 amino acid residues. The deduced polypeptide sequence of Ac1 showed a high degree of similarity to two previously reported serine proteases PII and Mlx from other nematode-trapping fungi (81% aa sequence identity). However, three proteases Ac1, Aoz1 and Mlx showed optimum temperatures at 53.2, 45 and 65 degrees C, respectively. Compared to PII, Ac1 appears to have a significantly higher activity against gelatin, bovine serum albumin, and non-denatured collagen. Moreover, our bioassay experiments showed that Ac1 is more effective at immobilizing P. redivivus than B. xylophilus.
Archives of Microbiology 08/2007; 188(2):167-74. · 1.43 Impact Factor
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ABSTRACT: Nematophagous fungi are an important group of soil microorganisms that can suppress the populations of plant-parasitic nematodes. The pathogenic mechanisms of nematophagous fungi are diverse: They can be parasitical-mechanical through producing specialized capturing devices, or toxin-dependent. During infections, a variety of virulence factors may be involved against nematodes by nematophagous fungi. In this review, we present up-to-date information on the modes of infection by nematophagous fungi. The roles of extracellular hydrolytic enzymes and other virulence factors involved in infection against nematodes were summarized. The biochemical properties and peptide sequences of a special group of enzymes, the serine proteases, were compared, and their implications in infections were discussed. We also discussed the impact of emerging new techniques on our understanding of this unique group of fungi.
Applied Microbiology and Biotechnology 06/2007; 75(1):21-31. · 3.42 Impact Factor
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ABSTRACT: Proteases have been proposed as virulence factors in microbial pathogenicity against nematodes. However, what kinds of extracellular proteases from these pathogens and how they contribute to the pathogenesis of infections against nematode in vivo remain largely unknown. A previous analysis using a strain with a deletion in an extracellular alkaline protease BLG4 gene from Brevibacillus laterosporus demonstrated that BLG4 was responsible for the majority of nematicidal activity by destroying host's cuticle. In recent studies, a neutral protease NPE-4, purified from the mutant BLG4-6, was found to be responsible for the majority of the remaining EDTA-inhibited protease activity. However, the purified NPE-4 and recombinant NPE-4 in a related species Bacillus subtilis showed little nematicidal activity in vitro and were unable to degrade the intact cuticle of the host. It is interesting to note that the addition of NPE-4 improved the pathogenicity of crude enzyme extract from wild-type B. laterosporus but had no effect on the BLG4-deficient mutant. This result suggests that NPE-4 functions in the presence of protease BLG4. Moreover, NPE-4 could degrade proteins from the inner layer of purified cuticles from nematode Panagrellus redivivus in vitro. These results indicated that the two different bacterial extracellular proteases might play differential roles at different stages of infection or a synthetic role in penetration of nematode cuticle in B. laterosporus. This is among the first reports to systematically evaluate and define the roles of different bacterial extracellular proteases in infection against nematodes.
Applied Microbiology and Biotechnology 03/2007; 74(2):372-80. · 3.42 Impact Factor
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ABSTRACT: Brevibacillus laterosporus G4, which was isolated from soil sample, kills free-living nematodes (Panagrellus redivius) and plant-parasite nematodes (Bursaphelenchus xylophilus) and degrades their cuticle in previous bioassay. Our works for B. laterosporus G4 had demonstrated that an extracellular alkaline protease BLG4 played a key role as a pathogenic factor in infection against nematode. In this study, the nematicidal activity of BLG4 was further verified by an in vitro assay with purified recombinant BLG4. The encoding gene of BLG4 was cloned and showed high degree of homology with the subtilisin subclass of serine protease gene and another reported cuticle-degrading protease gene from nematophagous bacterium Bacillus sp. B16. Deletion of BLG4 by homologous recombinant had a significant effect on the pathogenicity of B. laterosporus. In infection assays the BLG4-deficient strain (BLG4-6) lost about 50% of its nematocidal activity and in toxicity tests the mortality rate of nematodes decreased with approximately 56% in comparison to wild-type strain. This is the first report analyzing the function of a subtilisin enzyme involved in bacterium against nematode at the molecular level, and it is possible to use B. laterosporus as a model to study host-parasite interaction and to gain detailed knowledge of the infection process.
Archives of Microbiology 11/2006; 186(4):297-305. · 1.43 Impact Factor
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ABSTRACT: The gene encoding a cuticle-degrading serine protease was cloned from three isolates of Lecanicillium psalliotae (syn. Verticillium psalliotae) by 3' and 5' RACE (rapid amplification of cDNA ends) method. The gene encodes for 382 amino acids and the protein shares conserved motifs with subtilisin N and peptidase S8. Comparison of translated cDNA sequences of three isolates revealed one amino acid polymorphism at position 230. The deduced protease sequence shared high degree of similarities to other cuticle-degrading proteases from other nematophagous fungi.
Biotechnology Letters 10/2005; 27(17):1329-34. · 1.68 Impact Factor
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ABSTRACT: Lecanicillium psalliotae produced an extracellular protease (Ver112) which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 32 kDa. The optimum activity of Ver112 was at pH 10 and 70 degrees C (over 5 min). The purified protease degraded a broad range of substrates including casein, gelatin, and nematode cuticle with 81% of a nematode (Panagrellus redivivus) being degraded after treating with Ver112 for 12 h. The protease was highly sensitive to PMSF (1 mM) indicating it to be a serine protease. The N-terminal amino acid residues of Ver112 shared a high degree of similarity with other cuticle-degrading proteases from nematophagous fungi which suggests a role in nematode infection.
Biotechnology Letters 09/2005; 27(15):1123-8. · 1.68 Impact Factor
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ABSTRACT: Brevibacillus laterosporus is an aerobic spore-forming bacterium with the ability to produce canoe-shaped lamellar parasporal inclusions adjacent to spores. An isolate named G4 was identified as a B. laterosporus which does not produce parasporal crystals and shows significant toxic activity toward nematodes. Crude extracellular protein extract from culture supernatant of B. laterosporus G4 killed the nematodes within 12 h and finally destroyed the targets within 24 h, which suggested possible proteinaceous pathogeny. A homogeneous extracellular protease with nematicidal activities, purified by chromatography, confirmed the hypothesis that it might serve as a pathogenic factor during infection of the G4 strain. Characterization of the purified protease revealed a molecular mass of 30 kDa and optimum activity at pH 10 and 50 degrees C. The protease hydrolyzed relatively broad substrates including collagen and the cuticle of nematodes, and histopathological observations demonstrated the resulting destroyed nematode cuticle upon treatment by purified protease. Our present study reveals that extracellular protease, but not previously reported parasporal crystals, can be employed in infection against invertebrates by the B. laterosporus G4 strain.
Research in Microbiology 156(5-6):719-27. · 2.76 Impact Factor
