Showing papers on "Chitinase published in 1996"

Comparative studies of chitinases A and B from Serratia marcescens.

Abstract: Serratia marcescens produces several chitinolytic enzymes, including chitinase A (ChiA) and chitinase B (ChiB). In this study, ChiB was purified to homogeneity using a newly developed protocol based on hydrophobic interaction chromatography. Subsequently, characteristics of ChiB and of the hitherto only partly characterized ChiA were determined and compared. Pure ChiA and ChiB shared several characteristics such as a broad pH optimum around pH 5.0-6.0, and a temperature optimum between 50 and 60 degrees C. Both enzymes were fairly stable, with half-lives of more than 10 d at 37 degrees C, pH 6.1. Analyses of the degradation of various N-acetylglucosamine oligomers, fluorogenic substrates and colloidal chitin showed that both enzymes cleave chitobiose [(GlcNAc)2] from (GlcNAc)n and thus possess an exo-N,N'-diacetylchitobiohydrolase activity. Both enzymes were also capable of producing monomers from longer (GlcNAc)n substrates, indicating that they also have an endochitinase (ChiA) or exo-N,N',N"-triacetylchitotriohydrolase (ChiB) activity. Kinetic analyses with 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside, an analogue of (GlcNAc)3, showed cooperative kinetics for ChiA, whereas for ChiB normal hyperbolic kinetics were observed. ChiA had a higher specific activity towards chitin than ChiB and synergistic effects on the chitin degradation rate were observed upon combining the two enzymes. These results, together with the results of sequence comparisons and previous studies of the cellular localization of the two chitinases in S. marcescens indicate possible roles for ChiA and ChiB in chitin breakdown.

A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037.

Abstract: The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the beta-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases. In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases. The previously purified chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases. This is the first report of a family 19 chitinase found in an organism other than higher plants.

Characterization and Ultrastructural Localization of Chitinases From Metarhizium Anisopliae, M. Flavoviride, and Beauveria Bassiana During Fungal Invasion of Host (Manduca Sexta) Cuticle

Abstract: Extracellular chitinases have been suggested to be virulence factors in fungal entomopathogenicity. We employed isoelectric focusing and a set of three fluorescent substrates to investigate the numbers and types of chitinolytic enzymes produced by the entomopathogenic fungi Metarhizium anisopliae, Metarhizium flavoviride, and Beauveria bassiana. Each species produced a variety of N-acetyl-(beta)-d-glucosaminidases and endochitinases during growth in media containing insect cuticle. M. flavoviride also produced 1,4-(beta)-chitobiosidases. The endochitinases could be divided according to whether they had basic or acidic isoelectric points. In contrast to those of the other two species, the predominant endochitinases of M. anisopliae were acidic, with isoelectric points of about 4.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the acidic chitinases of M. anisopliae into two major bands (43.5 and 45 kDa) with identical N-terminal sequences (AGGYVNAVYFY TNGLYLSNYQPA) similar to an endochitinase from the mycoparasite Trichoderma harzianum. Use of polyclonal antibodies to the 45-kDa isoform and ultrastructural immunocytochemistry enabled us to visualize chitinase production during penetration of the host (Manduca sexta) cuticle. Chitinase was produced at very low levels by infection structures on the cuticle surface and during the initial penetration of the cuticle, but much greater levels of chitinase accumulated in zones of proteolytic degradation, which suggests that the release of the chitinase is dependent on the accessibility of its substrate.

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis

Abstract: The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

Response of transgenic cucumber and carrot plants expressing different chitinase enzymes to inoculation with fungal pathogens.

Abstract: Three lines of cucumber cv. Endeavor, each transformed with a chitinase gene originating from petunia (acidic), tobacco (basic), or bean (basic) using Agrobacterium tumefaciens, were compared with nontransgenic plants for response to inoculation with Alternaria cucumerina, Botrytis cinerea, Colletotrichum lagenarium, and Rhizoctonia solani. In both growth chamber studies using whole plants and in vitro inoculations conducted with detached leaves, no differences in disease development (rate and final levels) were detected between transgenic and nontransgenic plants. Carrot cvs. Nanco and Golden State transformed with two chitinase genes (from petunia and tobacco) were also evaluated for response to inoculation with the pathogens Alternaria radicini, B. cinerea, R. solani, Sclerotium rolfsii, and Thielaviopsis basicola. A detached petiole inoculation method was used to compare nontransgenic and transgenic plants. The rate and final extent of lesion development after 7 days were significantly (P = 0.01) lower in the transgenic plants expressing the tobacco (basic) chitinase gene upon inoculation with B. cinerea, R. solani, and S. rolfsii, but not in plants expressing the petunia (acidic) chitinase gene. There were no detectable differences with A. radicini or T. basicola in either group of transgenic punts. These results demonstrate the in planta efficacy of a basic chitinase protein in providing enhanced tolerance of carrot to three fungal pathogens; however, the efficacy of chitinase gene transformation as a strategy for enhancing disease tolerance in plants can be influenced by the plant species used, the type of chitinase protein expressed, and the characteristics of the fungal pathogen.

Constitutive expression of an inducible β-1,3-glucanase in alfalfa reduces disease severity caused by the oomycete pathogen Phytophthora megasperma f. sp medicaginis , but does not reduce disease severity of chitin-containing fungi

Abstract: cDNA sequences coding for an acidic glucanase (Aglul) that is expressed in elicited alfalfa cell suspension cultures, and a rice basic chitinase (RCH10) that is induced by elicitor and wounding, were placed into constitutive expression cassettes under control of the cauliflower mosaic virus 35S promoter or 35S enhancer sequences, and introduced in alfalfa plants of the regenerable cultivar Regen SY byAgrobacterium-mediated transformation. Southern and northern blot analysis confirmed stable incorporation and transcription, respectively, of the chimaeric genes in the transgenic plants. Active rice chitinase was expressed in alfalfa leaves, and leaves of plants transformed with theAglul sequence exhibited increased glucanase activity and the appearance of an additional glucanase band on activity gels. A glucanase of similar native electrophoretic mobility was constitutively present in root extracts of non-transformed alfalfa plants, and was induced in pathogen-infected leaves, presumably reflecting the expression pattern of the endogenousAglul gene. Thus, expression of the chimaericAglul transgene increased the amount, and broadened the tissue-type constitutive expression, of theAglul protein compared to control plants. Transgenic alfalfa plants containing a binary vector with both chimaeric genes in tandem expressed each gene to a much lesser extent than transgenic plants containing a single chimaeric gene. Expression of RCH10 in transgenic alfalfa did not appear to affect negatively theRhizobium/alfalfa interaction. Analysis of primary transformants for response to several fungal pathogens of alfalfa indicated statistically significant symptom reduction only in the case ofPhytophthora megasperma f. sp.medicaginis (Pmm), and only in plants overexpressingAglul. Resistance against Pmm segregated with glucanase expression in a cross between transgenic Regen SY and the commercial alfalfa cultivar Apollo.

Regulation, expression and function of a new basic chitinase gene in rice (Oryza sativa L.)

Abstract: A new basic chitinase gene, designated RC24, was isolated from a rice genomic library. The predicted RC24 protein contains 322 amino acid residues and exhibits 68% to 95% amino acid identity with known class I rice chitinases. RC24 protein expressed in Escherichia coli exhibited chitinase activity and strongly inhibited bacterial growth. Two transcription start sites of the RC24 gene were mapped by primer extension analysis of both rice native RNA and in vitro transcribed RNA using a RC24 promoter/GUS (β-glucuronidase) gene fusion as a template. The 5′-franking region of RC24 contained several putative stress-responsive cis-acting elements. A basal level of RC24 transcripts was detected in rice root and stem tissues, but not in leaf tissues. RC24 transcripts rapidly accumulated within 1 h after fungal elicitor treatment of suspension-cultured cells, and the levels continued to increase for at least 9 h. RC24 transcript accumulation was also observed in intact leaf tissues upon wounding. Transgenic rice plants containing the RC24/GUS gene fusion further confirmed that the RC24 gene showed a tissue-specific expression pattern and that transcription of the RC24 promoter was sensitively and rapidly activated by wounding.

Proteolytic Processing of Class IV Chitinase in the Compatible Interaction of Bean Roots with Fusarium solani

Abstract: Three chitinase isoenzymes, PvChiE, PvChiF, and PvChiG (molecular masses 29, 28, and 27 kD, respectively), were purified from bean (Phaseolus vulgaris L. cv Saxa) roots infected with the fungal pathogen Fusarium solani f. sp. phaseoli, and their amino acid sequence was partially determined. All sequences from all three isoenzymes exactly matched deduced amino acid sequences of the bean class IV chitinase PvChi4, formerly called PR4. The N terminus of PvChiF mapped to the hinge region, and the N terminus of PvChiG mapped to the catalytic domain of PvChi4. The N terminus of PvChiE was blocked. The appearance of PvChiE, PvChiF, and PvChiG correlated with an increase in protease activity in infected roots, and they could be generated in vitro by mixing extracts with high protease activity with extracts containing high amounts of PvChi4. Extracts from infected roots prepared in the presence of protease inhibitors also contained the processed forms of PvChi4, indicating that processing occurred in planta and not as an artifact of extraction. Processing of PvChi4 was not detected in incompatible interactions with a nonhost strain of F. solani and in symbiotic interactions with Glomus mosseae, and thus may be important only in compatible interactions with F. solani.

Characterization of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variant ts 11.

Abstract: To characterize the acidic endochitinase EP3, able to rescue somatic embryos of the carrot cell line ts11, the enzyme was purified from the medium of wild-type suspension cultures. Peptide sequences, deduced amino acid sequences of corresponding PCR-generated cDNA clones, serological relation and biochemical properties showed that there were at least five closely related chitinases, four of which could be identified as class IV EP3 chitinases with an apparent size of 30 kDa. Two other proteins were identified as a serologically related class I acidic chitinase (DcChitI) of 34 kDa, and a serologically unrelated 29 kDa class II acidic chitinase (DcChitII), respectively. Additional cDNA sequences, Western and Southern analysis showed the presence of a least two, but possibly more, highly homologous class IV EP3 genes in the carrot genome. Two class IV EP3 chitinases were tested and found to be able to increase the number of ts11 globular embryos formed under non-permissive conditions. One of the class IV EP3 chitinases as well as the class I chitinase DcChitI promoted the transition from globular to heart-stage ts11 embryos. The class II endochitinase and a heterologous class IV chitinase from sugar-beet were not active on ts11. This suggests that there are differences in the specificity of chitinases in terms of their effect on plant somatic embryos.

A novel chitinase inhibitor from a marine bacterium, Pseudomonas sp.

Abstract: A new chitinase inhibitor, CI-4, was isolated from the culture broth of a marine bacterium Pseudomonas sp. IZ208. The structure of CI-4 was determined to be cyclo(L-Arg-D-Pro) by spectral studies and comparison with a synthetic sample. CI-4 showed inhibitory activity against chitinase.

Chitinase levels in guinea pig blood are increased after systemic infection with Aspergillus fumigatus.

Abstract: The presence of chitinase activity in human serum has recently been described by us On that occasion we speculated on the possible role of mammalian chitinases as a defense mechanism against chitin-containing pathogens The results of the present study substantiate our hypothesis We demonstrate and partially characterize the chitinase activities that are present in plasma of guinea pigs and in homogenates of Afumigatus with the aid of the substrates MU-[GlcNAc]2,3 and also with glycol [3H]chitin Upon infection with Afumigatus the serum chitinase activity levels in the circulation of pathogen-free guinea pigs increased in a time-dependent manner The increase was also dependent on the size of the infecting fungal inoculum Antifungal treatment diminished the increases The increased chitinase activity was of guinea pig origin The activity of beta-hexosaminidase showed a very slight increase subsequent to the infection The activities of three other enzymes of lysosomal origin (alpha-mannosidase, beta-galactosidase and beta-glucosidase) did not increase

Induction of chitinases and β‐l,3‐glucanases in Rhizoctonia solani‐infected rice plants: Isolation of an infection‐related chitinase cDNA clone

Abstract: Extracts from several rice cultivars (Oryza saliva L. cvs IR58, 74586 and SC33) infected with the sheath blight pathogen Rhizoctonia solani, were analyzed to determine the isozyme distribution of chitinases (EC 3.2.1.14) and β-1,3-glucanases (EC 3.2.1.39). Upon infection by the fungal pathogen, two chitinases of 28 and 35 kDa and two β-1,3-glucanases of 30 and 32 kDa were shown to be induced in all cultivars. They resolved into multiple isozymes under nondenaturing electrophoretic conditions. Wounding, but not bacterial infection, resulted in the induction of these hydrolytic enzymes. Even though fungal infection resulted in induction of chitinases and β-glucanases in all cultivars, some cultivars that were moderately resistant to R. solani appeared to have higher levels of specific isozymes. A chitinase cDNA clone was identified by screening a library, prepared from RNA isolated from R. solani-infected rice plants, with an antibody to a bean chitinase. This cDNA encoded a 35-kDa chitinase which was significantly different in amino acid sequence from other rice chitinases described so far. Northern blot analysis of RNA from infected rice plants indicated that transcripts corresponding to this chitinase gene were induced upon fungal infection.

Novel osmotically induced antifungal chitinases and bacterial expression of an active recombinant isoform.

Abstract: NaCl (428 mM)-adapted tobacco (Nicotiana tabacum L. var Wisconsin 38) cells accumulate and secrete several antifungal chitinases. The predominant protein secreted to the culture medium was a 29-kD peptide that, based on internal amino acid sequence, was determined to be a class II acidic chitinase with similarity to PR-Q. The four predominant chitinases (T1, T2, T3, and T4) that accumulated intracellularly in 428 mM NaCl-adapted cells were purified. Based on N-terminal sequence analyses, two of these were identified as class I chitinase isoforms, one similar to the N. tomentosiformis (H. Shinshi, J.M. Neuhaus, J. Ryals, F. Meins [1990] Plant Mol Biol 14:357-368) protein (T1) and the other homologous to the N. sylvestris (Y. Fukuda, M. Ohme, H. Shinshi [1991] Plant Mol Biol 16:1-10) protein (T2). The other two proteins (T3 and T4) were determined to be novel chitinases that have sequence similarity with class I chitinases, but each lacks a chitin-binding domain. All four chitinases inhibited Fusarium oxysporum f. sp. lycopersici and Trichoderma longibrachiatum hyphal growth in vitro, although the isoforms containing a chitin-binding domain were somewhat more active. Conditions were established for the successful expression of soluble and active bacterial recombinant T2. Expression of soluble recombinant T2 was achieved when isopropyl beta-D-thiogalactopyranoside induction occurred at 18 degrees C but not at 25 or 37 degrees C. The purified recombinant protein exhibited antifungal activity comparable to a class I chitinase purified from NaCl-adapted tobacco cells.

Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants

Abstract: Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.

Local and Systemic Induction of β‐1,3‐Glucanase and Chitinase in Coffee Leaves Protected Against Hemileia vastatrix by Bacillus thuringiensis

Abstract: β-1,3-glucanase and chitinase activities were induced locally and systemically 4-25 and 11-25 days, respectively, after spraying the surface of the third pair of coffee leaves from the apex of 8-month-old plants with a 50 mg/ml aqueous suspension of Bacillus thuringiensis in a commercial formulation (Thuricide HP-Sandoz). The treatment also induced local and systemic resistance against Hemileia vastatrix after the application of the inducer. Within 14-18 days of application of the Thuricide inducer, the β-1,3-glucanase activity in the locally-and systemically-protected unchallenged leaves reached maximum levels of 226% and 279% higher levels respectively, than in control plants. The chitinase activity reached maximum levels of 224% and 181% respectively, within 18-21 days after treatment with the inducer. Two β-1,3-glucanase bands were detected by native PAGE electrophoresis in extracts from locally-and systemically-protected unchallenged coffee leaves.

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens.

Abstract: Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.