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PubMed2002

Pathogen challenge, salicylic acid, and jasmonic acid regulate expression of chitinase gene homologs in pine.

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable beta-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.

Amino Acid Sequence↗
PubMed1991

Regulation of chitinase synthesis in Trichoderma harzianum.

The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.

Acetylglucosamine↗
PubMed2004

Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae.

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families. To test this hypothesis, the chitin synthase and chitinase activities of cell-free extracts were measured, as well as the chitin content of cell walls isolated from isogenic mutant strains that contained single or multiple knock-outs in members of these two gene families, in both Candida albicans and Saccharomyces cerevisiae. However, deletion of chitinase genes did not markedly affect specific chitin synthase activity, and deletion of single CHS genes had little effect on in vitro specific chitinase activity in either fungus. Chitin synthesis and chitinase production was, however, regulated in C. albicans during yeast-hypha morphogenesis. In C. albicans, the total specific activities of both chitin synthase and chitinase were higher in the hyphal form, which was attributable mainly to the activities of Chs2 and Cht3, respectively. It appeared, therefore, that chitin synthesis and hydrolysis were not coupled, but that both were regulated during yeast-hypha morphogenesis in C. albicans.

Benzenesulfonates↗
PubMed1997

Differential effect of purified spruce chitinases and beta-1,3-glucanases on the activity of elicitors from ectomycorrhizal fungi.

Two chitinases (EC 3.2.1.14) and two beta-1,3-glucanases (EC 3.2.1.39) were purified from the culture medium of spruce (Picea abines [L.] Karst.) cells to study their role in modifying elicitors, cell walls, growth, and hyphal morphology of ectomycorrhizal fungi. The 36-kD class I chitinase (isoelectric point [pl] 8.0) and the 28-kD chitinase (pl 8.7) decreased the activity of elicitor preparations from Hebeloma crustuliniforme (Bull. ex Fries.) Quél., Amanita muscaria (L.) Pers., and Suillus variegatus (Sw.: Fr.) O.K., as demonstrated by using the elicitor-induced extracellular alkalinization in spruce cells as a test system. In addition, chitinases released monomeric products from the walls of these ectomycorrhizal fungi. The beta-1,3-glucanases (35 kD, pl 3.7 and 3.9), in contrast, had little influence on the activity of the fungal elicitors and released only from walls of A. muscaria some polymeric products. Furthermore, chitinases alone and in combination with beta-1,3-glucanases had no effect on the growth and morphology of the hyphae. Thus, it is suggested that apoplastic chitinases in the root cortex destroy elicitors from the ectomycorrhizal fungi without damaging the fungus. By this mechanism the host plant could attenuate the elicitor signal and adjust its own defense reactions to a level allowing symbiotic interaction.

Amanita↗
PubMed1996

Potential chitinase activating factor from yeast cells of Candida albicans.

Microsomal chitinase from yeast and hyphal cells of Candida albicans was activated endogenously by incubation at 30 degrees C and exogenously by trypsin. The putative activating factor of yeast cells was separated from chitinase activity by fractionation of lysed protoplasts on an Iodixanol density gradient. The vacuole fraction contained no significant chitinase activity, but was enriched in chitinase activating factor. Activity of microsomal chitinase increased upon incubation with this, but no other gradient factor. Results suggest that the regulatory system governing microsomal chitinase activity, like that governing chitin synthase, involves a 'vacuolar' activating factor in Candida albicans.

Candida albicans↗
PubMed2005

Enzymatic properties of wild-type and active site mutants of chitinase A from Vibrio carchariae, as revealed by HPLC-MS.

The enzymatic properties of chitinase A from Vibrio carchariae have been studied in detail by using combined HPLC and electrospray MS. This approach allowed the separation of alpha and beta anomers and the simultaneous monitoring of chitooligosaccharide products down to picomole levels. Chitinase A primarily generated beta-anomeric products, indicating that it catalyzed hydrolysis through a retaining mechanism. The enzyme exhibited endo characteristics, requiring a minimum of two glycosidic bonds for hydrolysis. The kinetics of hydrolysis revealed that chitinase A had greater affinity towards higher Mr chitooligomers, in the order of (GlcNAc)6 > (GlcNAc)4 > (GlcNAc)3, and showed no activity towards (GlcNAc)2 and pNP-GlcNAc. This suggested that the binding site of chitinase A was probably composed of an array of six binding subsites. Point mutations were introduced into two active site residues - Glu315 and Asp392 - by site-directed mutagenesis. The D392N mutant retained significant chitinase activity in the gel activity assay and showed approximately 20% residual activity towards chitooligosaccharides and colloidal chitin in HPLC-MS measurements. The complete loss of substrate utilization with the E315M and E315Q mutants suggested that Glu315 is an essential residue in enzyme catalysis. The recombinant wild-type enzyme acted on chitooligosaccharides, releasing higher quantities of small oligomers, while the D392N mutant favored the formation of transient intermediates. Under standard hydrolytic conditions, all chitinases also exhibited transglycosylation activity towards chitooligosaccharides and pNP-glycosides, yielding picomole quantities of synthesized chitooligomers. The D392N mutant displayed strikingly greater efficiency in oligosaccharide synthesis than the wild-type enzyme.

Binding Sites↗
PubMed2004

Temporal and spatial profiles of chitinase expression by norway spruce in response to bark colonization by Heterobasidion annosum.

Pathogen colonization and transcript levels of three host chitinases, putatively representing classes I, II, and IV, were monitored with real-time PCR after wounding and bark infection by Heterobasidion annosum in 32-year-old trees of Norway spruce (Picea abies) with low (clone 409) or high (clone 589) resistance to this pathogen. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. At 14 days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for clone 589 but had progressed further into the host tissue in clone 409. Transcript levels of the class II and IV chitinases increased after wounding or inoculation, but the transcript level of the class I chitinase declined after these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in clone 589 than in similar sites in clone 409 3 days after inoculation. This difference was even more pronounced 2 to 6 mm away from the inoculation point, where no infection was yet established, and suggests that the clones differ in the rate of chitinase-related signal perception or transduction. At 14 days after inoculation, these transcript levels were higher in clone 409 than in clone 589, suggesting that the massive upregulation of class II and IV chitinases after the establishment of infection comes too late to reduce or prevent pathogen colonization.

Agaricales↗
PubMed2005

Cloning of Beauveria bassiana chitinase gene Bbchit1 and its application to improve fungal strain virulence.

Entomopathogenic fungi can produce a series of chitinases, some of which act synergistically with proteases to degrade insect cuticle. However, chitinase involvement in insect fungus pathogenesis has not been fully characterized. In this paper, an endochitinase, Bbchit1, was purified to homogeneity from liquid cultures of Beauveria bassiana grown in a medium containing colloidal chitin. Bbchit1 had a molecular mass of about 33 kDa and pI of 5.4. Based on the N-terminal amino acid sequence, the chitinase gene, Bbchit1, and its upstream regulatory sequence were cloned. Bbchit1 was intronless, and there was a single copy in B. bassiana. Its regulatory sequence contained putative CreA/Crel carbon catabolic repressor binding domains, which was consistent with glucose suppression of Bbchit1. At the amino acid level, Bbchit1 showed significant similarity to a Streptomyces avermitilis putative endochitinase, a Streptomyces coelicolor putative chitinase, and Trichoderma harzianum endochitinase Chit36Y. However, Bbchit1 had very low levels of identity to other chitinase genes previously isolated from entomopathogenic fungi, indicating that Bbchit1 was a novel chitinase gene from an insect-pathogenic fungus. A gpd-Bbchit1 construct, in which Bbchit1 was driven by the Aspergiullus nidulans constitutive promoter, was transformed into the genome of B. bassiana, and three transformants that overproduced Bbchit1 were obtained. Insect bioassays revealed that overproduction of Bbchit1 enhanced the virulence of B. bassiana for aphids, as indicated by significantly lower 50% lethal concentrations and 50% lethal times of the transformants compared to the values for the wild-type strain.

Amino Acid Sequence↗
PubMed2005

Roles of four chitinases (chia, chib, chic, and chid) in the chitin degradation system of marine bacterium Alteromonas sp. strain O-7.

Alteromonas sp. strain O-7 secretes four chitinases (ChiA, ChiB, ChiC, and ChiD) in the presence of chitin. To elucidate why the strain produces multiple chitinases, we studied the expression levels of the four genes and proteins, their enzymatic properties, and their synergistic effects on chitin degradation. Among the four chitinases, ChiA was produced in the largest quantities, followed by ChiD, and the production of ChiB and ChiC changed at lower levels than those of ChiA and ChiD. The expression of the chiA, chiB, chiC, and chiD genes was investigated at the transcriptional level. The RNA transcript of chiA was most strongly induced in the presence of chitin, the expression of chiD followed, and the RNA transcripts of chiB and chiC changed at low levels. The hydrolyzing activities of the four chitinases against various substrates were examined. ChiA was the most active enzyme against powdered chitin, whereas ChiC was the most active against soluble chitin among the four chitinases. ChiD had activities closer to those of ChiA than to those of ChiB and ChiC. ChiB showed no distinctive feature against the chitinous substrates tested. When powdered chitin was treated with the proper combination of four chitinases, an approximately 2.0-fold increase in the hydrolytic activity was observed. These results, together with the results described above, indicate that ChiA plays a central role in chitin degradation for this strain.

Alteromonas↗
PubMed1994

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.

Amino Acid Sequence↗
PubMed2006

Distribution, structure, organ-specific expression, and phylogenic analysis of the pathogenesis-related protein-3 chitinase gene family in rice (Oryza sativa L.).

Rice (Oryza sativa L.) pathogenesis-related (PR)-3 chitinases, like other PR proteins, are each coded by one of the genes of a multigene family in the plant genome. We assembled the database information about rice PR-3 chitinase sequences. A total of 12 PR-3 chitinase loci (Cht1 to Cht12) were found deployed in the rice genome. Some of the loci were occupied by 2 or more alleles. For all the loci expect Cht4, Cht5, Cht6, and Cht11, the amino acid sequence was polymorphic between japonica and indica varieties of rice, but glutamic acid acting as a catalytic residue was completely conserved in all the loci expect Cht7. All the genes except Cht7, which was not tested in this study, were transcripted in some organs (leaf, sheath, root, and meristem) of rice plants. These results suggest that chitinase proteins encoded by the genes at these loci have important biological effects, at least antifungal activities, on rice plants. We also proposed a new classification of rice PR-3 chitinases based on their domain structures. This classification was consistent with the results of phylogenetic analysis of rice chitinases.

Alleles↗
PubMed2003

Actinobacterial chitinase-like enzymes: profiles of rhizosphere versus non-rhizosphere isolates.

The objective of this study was to determine if antifungal actinomycetes isolated from rhizosphere and non-rhizosphere soils exhibit different chitinase-like production and (or) induction patterns. Selected isolates from both habitats were compared. Chitinase-like levels and isoform characteristic patterns were evaluated over time in culture fluids of isolates grown on media containing different combinations of colloidal chitin and fungal cell wall (FCW) preparation. Supernatants were also subjected to native and non-native polyacrylamide gel electrophoresis (PAGE), using glycol chitin amended gels. For non-native PAGE, protein samples were denatured by two different approaches. Multiple active bands, ranging from 20 to 53 kDa and present in varying amounts, were detected in gels for most strains. Different substrate preferences were observed among strains, and different chitinase-like enzymes were produced, depending upon the substrate combinations used. The presence of FCW in the medium induced specific chitinase-like enzymes not observed otherwise. Enzymatic activities and profiles of the isolates, however, were strain and substrate specific rather than habitat specific. However, a sagebrush rhizosphere soil had a larger actinomycete community with higher chitinolytic activities than the nearby bulk soil. The use of PAGE to compare chitinase-like proteins induced in media with and without FCW was useful for identifying chitinase-like enzymes potentially involved in antifungal activity.

Actinobacteria↗
PubMed2004

Transfer of a plant chitinase gene into a nitrogen-fixing Azospirillum and study of its expression.

Azospirillum is used extensively in rice and other cereal crops as a biofertilizer. There is a substantial opportunity to improve the efficiency of this bacterium through the transfer of genes of agricultural importance from other organisms. Chitinases are antifungal proteins, and expression of chitinase genes in Azospirillum would help to develop strains with potential antifungal activities. So far there are no reports about transfer of plant genes into Azospirillum and their expression. The present study was aimed at expressing an antifungal gene (a rice chitinase) of plant origin in Azospirillum brasilense. A rice chitinase cDNA (RC 7) that codes for a 35 kDa protein was subcloned into a broad host range plasmid pDSK519 under the control of LacZ promoter. The plasmid was mobilized into the nitrogen-fixing bacterium, Azospirillum brasilense strain SP51eFL1, through biparental mating. The conjugation frequency was in the range of 35-40 x 10(-6). The transconjugants grew in nitrogen-free media and fixed gaseous nitrogen in vitro. However, their growth and nitrogen-fixing ability were slightly less than those of the wild-type. Expression of the protein was demonstrated through western blotting of the total cell protein, which detected a 35 kDa band that was immuno-reactive to a barley chitinase antibody. The cell lysates also hydrolyzed various chitin substrates, which resulted in release of free sugars demonstrating the chitinase activity of transconjugants. The expressed protein also had antifungal activity as demonstrated by inhibition of growth of the plant pathogenic fungus, Rhizoctonia solani.

Antibiosis↗
PubMed2005

Isolation and characterization of chitinases from Verticillium lecanii.

Degenerate PCR primers corresponding to conserved domains of fungal chitinases were designed, and PCR was performed on genomic DNA of the entomogenous fungus Verticillium lecanii (Zimmermann) Viegas. Two distinct PCR fragments, chf1 and chf2, were isolated and used to identify two DNA contigs. Analyses of these two contigs revealed that we had obtained the full-length DNA sequence including the promoter, 5' untranslated region, open reading frame (ORF), and 3' untranslated regions for two distinct chitinase-like genes. These two genomic DNA sequences exhibited 51% identity at the amino acid (aa) level and were designed as acidic (chi1) and basic (chi2) chitinase-like genes. The isolated cDNA for chi1 gene is 1110 bp with a predicted protein of 370 aa and molecular mass of 40.93 kDa, and its ORF was uninterrupted in its corresponding genomic DNA sequence. The cDNA for the chi2 gene is 1269 bp, a predicted ORF of 423 aa and molecular mass of 45.95 kDa. In contrast, the ORF was interrupted by three introns in its corresponding genomic DNA. The basic chitinase gene (chi2) was successfully expressed in the Pichia pastoris system; optimum enzymatic activity was observed at 22 degrees C and at pH 7.5. CHI1 and CHI2 were clustered into two different phylogenetic groups according to their sequence alignments with 28 other fungal chitinases. A chitin-binding domain, comprising two sub-domains that exhibit similarities at the aa level to chitin binding domains in bacteria, was identified in 30 fungal chitinase sequences examined.

Amino Acid Sequence↗
PubMed1998

Complete amino acid sequence of chitinase-A from leaves of pokeweed (Phytolacca americana).

The complete amino acid sequence of pokeweed leaf chitinase-A was determined. First all 11 tryptic peptides from the reduced and S-carboxymethylated form of the enzyme were sequenced. Then the same form of the enzyme was cleaved with cyanogen bromide, giving three fragments. The fragments were digested with chymotrypsin or Staphylococcus aureus V8 protease. Last, the 11 tryptic peptides were put in order. Of seven cysteine residues, six were linked by disulfide bonds (between Cys25 and Cys74, Cys89 and Cys98, and Cys195 and Cys208); Cys176 was free. The enzyme consisted of 208 amino acid residues and had a molecular weight of 22,391. It consisted of only one polypeptide chain without a chitin-binding domain. The length of the chain was almost the same as that of the catalytic domains of class IL chitinases. These findings suggested that this enzyme is a new kind of class IIL chitinase, although its sequence resembles that of catalytic domains of class IL chitinases more than that of the class IIL chitinases reported so far. Discussion on the involvement of specific tryptophan residue in the active site of PLC-A is also given based on the sequence similarity with rye seed chitinase-c.

Amino Acid Sequence↗
PubMed2001

Purification, characterization, and molecular cloning of a chitinase from the seeds of Benincasa hispida.

A chitinase was purified from the seeds of Benincasa hispida, a medicinal plant also called white gourd, and a member of the Cucurbitaceae family. Purification was done by using a procedure consisting of only two fractionation steps: an acid denaturation step followed by ion-exchange chromatography. The sequence of the N-terminal forty amino acid residues was analyzed and the sequence indicated that the enzyme is a class III chitinase. The enzyme, which is a basic chitinase, is one of at least five chitinases detected in the seed extract of B. hispida. Like other class III chitinases, this enzyme also has lysozyme activity. A genomic clone of the gene encoding the enzyme was isolated and sequenced. The gene has the potential to encode a protein of 301 amino acid residues. The deduced amino acid sequence of the protein, as expected from the N-terminal amino acid sequence, shares high degrees of similarity with other class III chitinases.

Amino Acid Sequence↗
PubMed2003

Nucleotide sequences of genes encoding allosamidin-sensitive and -insensitive chitinases produced by allosamidin-producing Streptomyces.

Allosamidin is a strong inhibitor of family 18 chitinases. We previously reported the presence of allosamidin-sensitive and -insensitive chitinases (chitinase S and IS) in the culture filtrate of the allosamidin-producing strain, Streptomyces sp. AJ9463. In this study, we cloned and sequenced the genes encoding the two chitinases, which clarified that chitinase S and IS belong to the family 18 and 19 chitinase, respectively.

Acetylglucosamine↗
PubMed2005

Microbial synthesis of chitinase in solid cultures and its potential as a biocontrol agent against phytopathogenic fungus Colletotrichum gloeosporioides.

Antifungal activity of chitinase can be effectively utilized in biologic pest control strategies. Because solid-state cultivation has been termed a cost-effective means for fungal growth and metabolite production, chitinase production by Trichoderma harzianum was studied using wheat bran-based solid medium containing 1% colloidal chitin. Chitinase synthesis was found to be growth associated because maximum enzyme (5.4 U/g of dry substrate) and biomass production occurred at 72 h. Substrate moisture had a critical impact on chitinase production; five grams of medium having an initial moisture content of 68.4% when incubated for 72 h increased the enzyme yield to 9.3 U/g of dry substrate. Optimization of colloidal chitin concentration showed that improvements in chitinase yield and maximum activity were attained with a 2% (w/w) concentration. Supplementation of additional nitrogen sources also influenced enzyme production, and the best yield was obtained with yeast extract. The effect of crude chitinase on hyphal morphology of the phytopathogenic fungus Colletotrichum gloeosporioides was swelling as well as lysis of hyphal wall, depending on the age of the mycelium. Studies of pH and thermal stability showed that crude culture filtrate was active over pH 4.0-6.0 and retained about 48.2% activity after 40 min of incubation at 40 degrees C.

Biomass↗