Showing papers on "Chitinase published in 2000"

Regulation of soil phosphatase and chitinase activity by N and P availability

Abstract: Soil microorganisms and plants produce enzymes thatmineralize organically bound nutrients When nutrientavailability is low, the biota may be able to increase production ofthese enzymes to enhance the supply of inorganicnitrogen (N) and phosphorus (P) Regulation of enzyme productionmay be a point where N and P cyclesinteract We measured acid phosphatase and chitinase(N-acetyl s-D-glucosaminide) activity in soilacross a chronosequence in Hawaii where N and Pavailability varies substantially among sites and longterm fertilizer plots had been maintained for over 4years Phosphatase activity was high at all sites Chitinaseactivity decreased significantly as age and Navailability increased across the chronosequencePhosphorus addition suppressed phosphatase activity atall sites, while N addition increased phosphataseactivity at the young, N-limited site In contrast,N addition repressed chitinase activity only at the Nlimited young site, and P additions had no effect onchitinase activity These results suggest that theregulatory relationship between nutrient supply andnutrient mineralization are asymmetric for N and P,and that the differences could help to explaindifferences observed in patterns of N and Pavailability

Rapid evolution in plant chitinases: molecular targets of selection in plant-pathogen coevolution.

Abstract: Many pathogen recognition genes, such as plant R-genes, undergo rapid adaptive evolution, providing evidence that these genes play a critical role in plant-pathogen coevolution. Surprisingly, whether rapid adaptive evolution also occurs in genes encoding other kinds of plant defense proteins is unknown. Unlike recognition proteins, plant chitinases attack pathogens directly, conferring disease resistance by degrading chitin, a component of fungal cell walls. Here, we show that nonsynonymous substitution rates in plant class I chitinase often exceed synonymous rates in the plant genus Arabis (Cruciferae) and in other dicots, indicating a succession of adaptively driven amino acid replacements. We identify individual residues that are likely subject to positive selection by using codon substitution models and determine the location of these residues on the three-dimensional structure of class I chitinase. In contrast to primate lysozymes and plant class III chitinases, structural and functional relatives of class I chitinase, the adaptive replacements of class I chitinase occur disproportionately in the active site cleft. This highly unusual pattern of replacements suggests that fungi directly defend against chitinolytic activity through enzymatic inhibition or other forms of chemical resistance and identifies target residues for manipulating chitinolytic activity. These data also provide empirical evidence that plant defense proteins not involved in pathogen recognition also evolve in a manner consistent with rapid coevolutionary interactions.

Biological control of Sclerotinia minor using a chitinolytic bacterium and actinomycetes

Abstract: Isolates of 85 bacteria and 94 streptomycete and 35 nonstreptomycete actinomycetes were obtained from a lettuce-growing field in Al-Ain, United Arab Emirates, on colloidal chitin agar, and screened for their ability to produce chitinase. Twenty-three bacteria and 38 streptomycete and 15 nonstreptomycete actinomycete isolates produced high levels of chitinase and were examined in vitro for their ability to suppress the growth of Sclerotinia minor, a pathogen causing basal drop disease of lettuce. The three most suppressive isolates were examined further for their production of β-1,3-glucanase and antifungal activity as well as their ability to colonize the roots and rhizosphere of lettuce in vitro and in planta. The three isolates, Serratia marcescens, Streptomyces viridodiasticus and Micromonospora carbonacea, significantly reduced the growth of S. minor in vitro, and produced high levels of chitinase and β-1,3-glucanase. Streptomyces viridodiasticus also produced antifungal metabolite(s) that significantly reduced the growth of the pathogen in vitro. When the pathogen was presented as the sole carbon source, all three isolates caused extensive hyphal plasmolysis and cell wall lysis. Serratia marcescens and St. viridodiasticus were competent to varying degrees in colonizing the roots of lettuce seedlings after 8 days on agar plates and the rhizosphere within 14 days in pots, with their competency being superior to that of M. carbonacea. All three isolates, individually or in combination, were antagonistic to S. minor and significantly reduced incidence of disease under controlled glasshouse conditions.

Structure of a two-domain chitotriosidase from Serratia marcescens at 1.9-Å resolution

Abstract: In this paper, we describe the structure of chitinase B from Serratia marcescens, which consists of a catalytic domain with a TIM-barrel fold and a 49-residue C-terminal chitin-binding domain. This chitinase is the first structure of a bacterial exochitinase, and it represents one of only a few examples of a glycosyl hydrolase structure having interacting catalytic and substrate-binding domains. The chitin-binding domain has exposed aromatic residues that contribute to a 55-A long continuous aromatic stretch extending into the active site. Binding of chitin oligomers is blocked beyond the -3 subsite, which explains why the enzyme has chitotriosidase activity and degrades the chitin chain from the nonreducing end. Comparison of the chitinase B structure with that of chitinase A explains why these enzymes act synergistically in the degradation of chitin.

Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens.

Abstract: The rice chitinase gene (RCC2), classified as class I chitinase, was introduced into the somatic embryos of grapevine (Vitis vinifera L. cv. Neo Muscut) by Agrobacterium infection. After co-cultivation with Agrobacterium, somatic embryos were transferred onto Murashige and Skoog hormone-free medium supplemented with 50 mg/l kanamycin. Transformed secondary or tertiary embryos were selected, and then more than 20 transgenic plantlets were recovered. Two transformants showed enhanced resistance against powdery mildew caused by Uncinula necator. Few disease symptoms were observed on leaves of these transformants compared with those of the non-transformant, although browning and necrotic symptoms, which seemed to constitute a hypersensitive reaction, were observed. Scanning electron microscopic observation revealed that conidial germination, mycelial growth and conidial formation were suppressed on the leaf surface of the transformant. The transgenic grapevines obtained also exhibited slight resistance against Elisinoe ampelina inducing anthracnose, resulting in a reduction in disease lesions. The relationship between the expression of the foreign chitinase gene and the disease resistance is discussed.

Differential expression of eight chitinase genes in Medicago truncatula roots during mycorrhiza formation, nodulation, and pathogen infection.

Abstract: Expression of eight different chitinase genes, representing members of five chitinase classes, was studied in Medicago truncatula roots during formation of arbuscular mycorrhiza with Glomus intraradices, nodulation with Rhizobium meliloti, and pathogen attack by Phytophthora megasperma f. sp. medicaginis, Fusarium solani f. sp. phaseoli (compatible interactions with root rot symptoms), Ascochyta pisi (compatible, symptomless), and F. solani f. sp. pisi (incompatible, nonhost interaction). In the compatible plant-pathogen interactions, expression of class I, II, and IV chitinase genes was enhanced. The same genes were induced during nodulation. Transcripts of class I and II chitinase genes accumulated transiently during early stages of the interaction, and transcripts of the class IV chitinase gene accumulated in mature nodules. The pattern of chitinase gene expression in mycorrhizal roots was markedly different: Expression of class I, II, and IV chitinase genes was not enhanced, whereas expression of three class III chitinase genes, with almost no basal expression, was strongly induced. Two of these three (Mtchitinase III-2 and Mtchitinase III-3) were not induced at all in interactions with pathogens and rhizobia. Thus, the expression of two mycorrhiza-specific class III chitinase genes can be considered a hallmark for the establishment of arbuscular mycorrhiza in Medicago truncatula.

Chitinase genes responsive to cold encode antifreeze proteins in winter cereals

Abstract: Antifreeze proteins similar to two different chitinases accumulate during cold acclimation in winter rye (Secale cereale). To determine whether these cold-responsive chitinases require post-translational modification to bind to ice, cDNAs coding for two different full-length chitinases were isolated from a cDNA library produced from cold-acclimated winter rye leaves. CHT9 is a 1,193-bp clone that encodes a 31.7-kD class I chitinase and CHT46 is a 998-bp clone that codes for a 24.8-kD class II chitinase. Chitinase-antifreeze proteins purified from the plant were similar in mass to the predicted mature products of CHT9 and CHT46, thus indicating that there was little chemical modification of the amino acid sequences in planta. To confirm these results, the mature sequences of CHT9 and CHT46 were expressed in Escherichia coli and the products of both cDNAs modified the growth of ice. Transcripts of both genes accumulated late in cold acclimation in winter rye. Southern analysis of winter rye genomic DNA indicated the presence of a small gene family homologous to CHT46. In hexaploid wheat, CHT46 homologs mapped to the homeologous group 1 chromosomes and were expressed in response to cold and drought. We conclude that two novel cold-responsive genes encoding chitinases with ice-binding activity may have arisen in winter rye and other cereals through gene duplication.

Expression and characterization of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12.

Abstract: Chitinase A1 from Bacillus circulans WL-12 comprises an N-terminal catalytic domain, two fibronectin type III-like domains, and a C-terminal chitin-binding domain (ChBD). In order to study the biochemical properties and structure of the ChBD, ChBD(ChiA1) was produced in Escherichia coli using a pET expression system and purified by chitin affinity column chromatography. Purified ChBD(ChiA1) specifically bound to various forms of insoluble chitin but not to other polysaccharides, including chitosan, cellulose, and starch. Interaction of soluble chitinous substrates with ChBD(ChiA1) was not detected by means of nuclear magnetic resonance and isothermal titration calorimetry. In addition, the presence of soluble substrates did not interfere with the binding of ChBD(ChiA1) to regenerated chitin. These observations suggest that ChBD(ChiA1) recognizes a structure which is present in insoluble or crystalline chitin but not in chito-oligosaccharides or in soluble derivatives of chitin. ChBD(ChiA1) exhibited binding activity over a wide range of pHs, and the binding activity was enhanced at pHs near its pI and by the presence of NaCl, suggesting that the binding of ChBD(ChiA1) is mediated mainly by hydrophobic interactions. Hydrolysis of beta-chitin microcrystals by intact chitinase A1 and by a deletion derivative lacking the ChBD suggested that the ChBD is not absolutely required for hydrolysis of beta-chitin microcrystals but greatly enhances the efficiency of degradation.

Enzyme diffusion from Trichoderma atroviride (= T. harzianum P1) to Rhizoctonia solani is a prerequisite for triggering of Trichoderma ech42 gene expression before mycoparasitic contact.

Abstract: A plate confrontation experiment is commonly used to study the mechanism by which Trichoderma spp. antagonize and parasitize other fungi. Previous work with chitinase gene expression (ech42) during the precontact period of this process in which cellophane and dialysis membranes separated Trichoderma harzianum and its host Rhizoctonia solani resulted in essentially opposite results. Here, we show that cellophane membranes are permeable to proteins up to at least 90 kDa in size but that dialysis membranes are not. ech42 was expressed during the precontact stage of the confrontation between Trichoderma atroviride and its host only if the cellophane was placed between the two fungi. These results are consistent with enzyme diffusion from T. atroviride to R. solani generating the trigger of ech42 gene expression.

The Role of Chitinase Production by Stenotrophomonas maltophilia Strain C3 in Biological Control of Bipolaris sorokiniana.

Abstract: The role of chitinase production by Stenotrophomonas maltophilia strain C3 in biological control of leaf spot on tall fescue (Festuca arundinacea), caused by Bipolaris sorokiniana, was investigated in vitro and in vivo. The filtrate of a broth culture of C3, with chitin as the carbon source, was separated into fractions. A high molecular-weight fraction (>8 kDa) was chitinolytic and more inhibitory than a low-molecular-weight, nonchitinolytic fraction to conidial germination and hyphal growth by B. sorokiniana and to leaf spot development. A protein fraction derived by ammonium sulfate precipitation and a chitinase fraction purified by chitin affinity chromatography also were chitinolytic and highly antifungal. The chitinolytic fractions caused swelling and vacuolation of conidia and discoloration, malformation, and degradation of germ tubes. When boiled, the chitinolytic fractions lost chitinase activity along with most of the antifungal properties. Two chitinase-deficient and two chitinase-reduced mutants of C3 were compared with the wild-type strain for inhibition of germination of B. sorokiniana conidia on tall fescue leaves and for suppression of leaf spot development in vivo. The mutants exhibited reduced antifungal activity and biocontrol efficacy, but did not lose all biocontrol activity. An aqueous extract of leaves colonized by wild-type C3 had higher chitinase activity than that of noncolonized leaves and was inhibitory to conidial germination. The addition of chitin to leaves along with the wild-type strain increased both chitinase and antifungal activity. The chitinase activity level of extracts from leaves colonized by a chitinase-deficient mutant of C3, with and without added chitin, was no higher than the background, and the extracts lacked antifungal activity. Chitinolysis appears to be one mechanism of biological control by strain C3, and it functions in concert with other mechanisms.

Molecular detection of bacterial and streptomycete chitinases in the environment.

Abstract: Sets of PCR primers were designed to amplify bacterial chitinases at different levels of specificity The bacterial chitinase group primers were successful in targeting enzymes classified within the group A glycosyl hydrolases of family 18 The widespread occurrence of group A bacterial chitinases in actinomycetes was demonstrated Streptomycete chitinase specific primers were designed and a collection of type strains of species changed in the genes Streptomyces were screened and shown to have at least one and usually multiple chitinase genes The presence of the gene for the chitin binding protein was also demonstrated within the streptomycete type strains These data indicate that streptomycetes are well equipped to degrade chitin The detection of group A chitinases in total community DNA is described and a sandy soil shown to contain more than 10 different genes using DGGE to indicate genetic diversity

Selected Chitinase Genes in Cultured and Uncultured Marine Bacteria in the α- and γ-Subclasses of the Proteobacteria

Abstract: PCR primers were patterned after chitinase genes in four γ-proteobacteria in the families Alteromonadaceae and Enterobacteriaceae (group I chitinases) and used to explore the occurrence and diversity of these chitinase genes in cultured and uncultured marine bacteria. The PCR results from 104 bacterial strains indicated that this type of chitinase gene occurs in two major groups of marine bacteria, α- and γ-proteobacteria, but not the Cytophaga-Flavobacter group. Group I chitinase genes also occur in some viruses infecting arthropods. Phylogenetic analysis indicated that similar group I chitinase genes occur in taxonomically related bacteria. However, the overall phylogeny of chitinase genes did not correspond to the phylogeny of 16S rRNA genes, possibly due to lateral transfer of chitinase genes between groups of bacteria, but other mechanisms, such as gene duplication, cannot be ruled out. Clone libraries of chitinase gene fragments amplified from coastal Pacific Ocean and estuarine Delaware Bay bacterioplankton revealed similarities and differences between cultured and uncultured bacteria. We had hypothesized that cultured and uncultured chitin-degrading bacteria would be very different, but in fact, clones having nucleotide sequences identical to those of chitinase genes of cultured α-proteobacteria dominated both libraries. The other clones were similar but not identical to genes in cultured γ-proteobacteria, including vibrios and alteromonads. Our results suggest that a closer examination of chitin degradation by α-proteobacteria will lead to a better understanding of chitin degradation in the ocean.

Argadin, a new chitinase inhibitor, produced by Clonostachys sp. FO-7314.

Abstract: A new chitinase inhibitor, designated as argadin (1), was isolated form the cultured broth of a fungal strain FO-7314. The strain was identified as Clonostachys sp. from the morphological characteristics. Argadin was purified from the cultured mycelium by a combination of cation exchange, adsorption and gel filtration chromatographic methods. The structure of argadin was elucidated as cyclo(Nω-acetyl-L-arginyl-D-prolyl-homoseryl-histidyl-L-2-aminoadipyl) in which homoseryl γ-methylene bonded to histidyl α-amino residue. The IC50 value of argadin against Lucilia cuprina (blowfly) chitinase was 150 nM at 37°C and 3.4 nM at 20°C. Argadin arrested the moult of cockroach larvae upon injection into the ventral abdominal part.

Introduction of the Serratia marcescens chiA gene into an endophytic Pseudomonas fluorescens for the biocontrol of phytopathogenic fungi.

Abstract: An endophytic strain of Pseudomonas fluorescens was isolated from micropropagated apple plantlets and introduced into beans (Phaseolus vulgaris) via their root tips. It was shown to be present as an endophyte in the roots at a level of 1.2 × 105 CFU/g fresh weight. The gene coding for the major chitinase of Serratia marcescens, chiA, was cloned under the control of the tac promoter into the broad-host-range plasmid pKT240 and the integration vector pJFF350. Pseudomonas fluorescens carrying tacchiA either on the plasmid or integrated into the chromosome is an effective biocontrol agent of the phytopathogenic fungus Rhizoctonia solani on bean seedlings under plant growth chamber conditions.Key words: endophyte, biological control, chitinase.

Interactions between a vesicular-arbuscular mycorrhizal fungus (Glomus intraradices) and Streptomyces coelicolor and their effects on sorghum plants grown in soil amended with chitin of brawn scales

Abstract: The effect of the interaction between a vesicular-arbuscular (VA)-mycorrhiza (Glomus intraradices no. LAP8) and Streptomyces coelicolor strain no. 2389 on the growth response, nutrition and metabolic activities of sorghum (Sorghum bicolor) plants grown in non-sterilized soil amended with chitin waste was studied in a greenhouse over 8 weeks. Chitin amendment resulted in an increase in the microbial population and chitinase activity in soils. Growth of mycorrhizal G. intraradices no. LAP8 and non-mycorrhizal sorghum plants increased as compared with other treatments either in the presence or absence of S. coelicolor strain 2389. VA-mycorrhizal inoculation significantly increased the growth, photosynthetic pigments, total soluble protein and nutrient contents of sorghum compared to non-mycorrhizal sorghum. Such increases were related to increased mycorrhizal colonization. Inoculation with S. coelicolor 2389 significantly increased the intensity of mycorrhizal root colonization and arbuscular formation, but the levels of mycorrhizal infection and their beneficial effects were significantly reduced with the addition of chitin waste to the soil. Analysis of the content of total amino acids and ammonia in leaves on the basis of dry matter production showed that, in most instances, total amino acids of mycorrhizal plants were significantly higher than those of non-inoculated plants. The microflora of the rhizosphere was highly affected by mycorrhizal inoculation. Quantitative changes in acid and alkaline phosphatase activities of the roots in response to the mycorrhizal inoculation are discussed.

Identification of a Chitin-Binding Protein Secreted by Pseudomonas aeruginosa

Abstract: One of the major proteins secreted by Pseudomonas aeruginosa is a 43-kDa protein, which is cleaved by elastase into smaller fragments, including a 30-kDa and a 23-kDa fragment. The N-terminal 23-kDa fragment was previously suggested as corresponding to a staphylolytic protease and was designated LasD (S. Park and D. R. Galloway, Mol. Microbiol. 16:263–270, 1995). However, the sequence of the gene encoding this 43-kDa protein revealed that the N-terminal half of the protein is homologous to the chitin-binding proteins CHB1 of Streptomyces olivaceoviridis and CBP21 of Serratia marcescens and to the cellulose-binding protein p40 ofStreptomyces halstedii. Furthermore, a short C-terminal fragment shows homology to a part of chitinase A of Vibrio harveyi. The full-length 43-kDa protein could bind chitin and was thereby protected against the proteolytic activity of elastase, whereas the degradation products did not bind chitin. The purified 43-kDa chitin-binding protein had no staphylolytic activity, and comparison of the enzymatic activities in the extracellular medium of a wild-type strain and a chitin-binding protein-deficient mutant indicated that the 43-kDa protein supports neither chitinolytic nor staphylolytic activity. We conclude that the 43-kDa protein, which was found to be produced by many clinical isolates of P. aeruginosa, is a chitin-binding protein, and we propose to name it CbpD (chitin-binding protein D).

Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol

Abstract: The crude enzyme extracts from five actinomycetes selected from a cerrado soil presented very good endochitinolytic activity when compared to a commercial chitinase. Exochitinase and chitobiase activities were also detected. They were identified as Streptomyces, but could not be characterized to species level, probably corresponding to new ones. The crude extracts, obtained from growth on fungal mycelium plus chitin of three of the strains, have shown a very pronounced activity against phytopathogenic fungi. In tests using growing cells, all five strains were active. These data suggest that these strains are potential biocontrol agents.

The most abundant glycoprotein of amebic cyst walls (Jacob) is a lectin with five Cys-rich, chitin-binding domains.

Abstract: The infectious stage of amebae is the chitin-walled cyst, which is resistant to stomach acids. In this study an extraordinarily abundant, encystation-specific glycoprotein (Jacob) was identified on two-dimensional protein gels of cyst walls purified from Entamoeba invadens. Jacob, which was acidic and had an apparent molecular mass of approximately 100 kDa, contained sugars that bound to concanavalin A and ricin. The jacob gene encoded a 45-kDa protein with a ladder-like series of five Cys-rich domains. These Cys-rich domains were reminiscent of but not homologous to the Cys-rich chitin-binding domains of insect chitinases and peritrophic matrix proteins that surround the food bolus in the insect gut. Jacob bound purified chitin and chitin remaining in sodium dodecyl sulfate-treated cyst walls. Conversely, the E. histolytica plasma membrane Gal/GalNAc lectin bound sugars of intact cyst walls and purified Jacob. In the presence of galactose, E. invadens formed wall-less cysts, which were quadranucleate and contained Jacob and chitinase (another encystation-specific protein) in secretory vesicles. A galactose lectin was found to be present on the surface of wall-less cysts, which phagocytosed bacteria and mucin-coated beads. These results suggest that the E. invadens cyst wall forms when the plasma membrane galactose lectin binds sugars on Jacob, which in turn binds chitin via its five chitin-binding domains.