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PubMed2006

Novel extracellular chitinases rapidly and specifically induced by general bacterial elicitors and suppressed by virulent bacteria as a marker of early basal resistance in tobacco.

Early basal resistance (EBR, formerly known as early induced resistance) is triggered by general bacterial elicitors. EBR has been suggested to inhibit or retard expression of the type III secretion system of pathogenic bacteria and may also prevent nonpathogenic bacteria from colonizing the plant tissue. The quickness of EBR here plays a crucial role, compensating for a low bactericidal efficacy. This inhibitory activity should take place in the cell wall, as bacteria do not enter living plant cells. We found several soluble proteins in the intercellular fluid of tobacco leaf parenchyma that coincided with EBR under different environmental (light and temperature) conditions known to affect EBR. The two most prominent proteins proved to be novel chitinases (EC 3.2.1.14) that were transcriptionally induced before and during EBR development. Their expression in the apoplast was fast and not stress-regulated as opposed to many pathogenesis-related proteins. Nonpathogenic, saprophytic, and avirulent bacteria all induced EBR and the chitinases. Studies using these chitinases as EBR markers revealed that the virulent Pseudomonas syringae pv. tabaci, being sensitive to EBR, must suppress it while suppressing the chitinases. EBR, the chitinases, as well as their suppression are quantitatively related, implying a delicate balance determining the outcome of an infection.

Amino Acid Sequence↗
PubMed1999

Expression of a Serratia marcescens chitinase gene in Sinorhizobium fredii USDA191 and Sinorhizobium meliloti RCR2011 impedes soybean and alfalfa nodulation.

A gene encoding chitinase from Serratia marcescens BJL200 was cloned into a broad-host-range vector (pRK415) and mobilized into Sinorhizobium fredii USDA191. Chitinolytic activity was detected in S. fredii USDA191 transconjugants that carried the S. marcescens chiB gene. Chitinase-producing S. fredii USDA191 formed nodules on soybean cultivar McCall. However, there was a delay in nodule formation and a marked decrease in the total number of nodules formed by the chitinase-producing S. fredii in comparison with the wild-type strain. Expression of chitinase in S. meliloti RCR2011 also impeded alfalfa nodulation. Thin-layer chromatography of 14C-labeled Nod factors from chitinase-producing S. fredii USDA191 revealed hydrolysis of lipochitooligosaccharides.

Chitinases↗
PubMed2002

Partial redistribution of the Autographa californica nucleopolyhedrovirus chitinase in virus-infected cells accompanies mutation of the carboxy-terminal KDEL ER-retention motif.

During virus infection of insect cells, the Autographa californica nucleopolyhedrovirus chitinase is localized primarily within the endoplasmic reticulum (ER), which is consistent with the presence of a carboxy-terminal ER retention motif (KDEL). Release of chitinase into the extracellular medium appears to be concomitant with terminal cell lysis, rather than by active secretion. In this study, we have shown that mutation of the KDEL motif induces a partial redistribution of the chitinase at both early and late times post-infection. Deletion of the KDEL motif or substitution with glycine residues allowed chitinase to move through the secretory pathway, accumulating to detectable levels in the extracellular medium by 24 h post-infection; more than 48 h prior to cell lysis. Deletion of the KDEL motif did not compromise enzyme activity, with the modified enzyme exhibiting characteristic endo- and exo-chitinolytic activity. Trichoplusia ni larvae infected with the modified virus were found to liquefy approximately 24 h earlier than larvae infected with a control virus in which the chitinase KDEL motif had not been deleted.

Amino Acid Motifs↗
PubMed1989

Purification and some properties of two forms of chitinase from mycelial cells of Mucor rouxii.

Chitinase activity was measured in extracts of mycelial cells of Mucor rouxii as a function of the culture age. There was a peak of specific activity at the mid-exponential phase of growth (10 h), which paralleled chitin synthase activity. An additional peak of chitinase with higher specific activity was detected in 4 h cultures, which coincided with the onset of germination. Purification of chitinase activities from the cytoplasm revealed two enzymes, I and II, with different molecular mass and ionic charge. Antibodies induced with chitinase I did not cross-react with chitinase II. Both enzymes digested nascent chitin preferentially over preformed chitin, yielding diacetylchitobiose as the sole product of hydrolysis.

Chitin Synthase↗
PubMed1995

Chitinase B from Serratia marcescens BJL200 is exported to the periplasm without processing.

A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55.5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of the chitinase was studied, using two types of cell fractionation methods of immunocytochemical techniques. These analyses showed that the chitinase is located in the cytoplasm in E. coli, whereas it is exported to the periplasm in S. marcescens. Analysis of chitinase isolated from periplasmic fractions of S. marcescens carrying the cloned gene showed that export of the enzyme is not accompanied by processing at the N-terminus. The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.

Amino Acid Sequence↗
PubMed1999

Distribution of chitinase in guinea pig tissues and increases in levels of this enzyme after systemic infection with Aspergillus fumigatus.

Intravenous infection of guinea pigs with the fungus Aspergillus fumigatus resulted in increased levels of chitinase in serum and tissues of the animals. The molecular properties of the enzyme were demonstrated to be different from those of the fungal chitinase, but also from guinea pig lysozyme and beta-N-acetylhexosaminidase. Bio-Gel P-100 gel filtration showed that in liver, spleen, heart and lung tissue of control animals there were two molecular mass forms present with apparent molecular masses of 35 kDa and 15 kDa. In brain and serum, only the 35 kDa form was detectable. Kidney showed only the 15 kDa form. Upon infection the 35 kDa form appeared in kidney and increased in the other tissues. When a less pathogenic form of the fungus was used the 35 kDa form remained absent in kidney. In contrast to human serum chitinase, the enzyme from guinea pig serum and tissues did bind to concanavalin A-Sepharose. This was the case for both molecular mass forms. The mode of cleavage of the substrate 4-methylumbelliferyl-tri-N-acetylchitotrioside (MU-[GlcNAc]3, where GlcNAc is N-acetylglucosamine) by the two forms of the enzyme was the same: both [GlcNAc]2 and [GlcNAc]3 were released. The chitinase activity levels in the control tissues showed a large variation in this order: spleen > lung, kidney > liver > heart > brain. The fact that spleen showed the highest chitinase level is in agreement with its major role as a lymphoid organ in cases of systemic infections. The relative increases upon infection were the highest for the tissues that showed low control values.

Acetylglucosamine↗
PubMed1997

A class IV chitinase is highly expressed in grape berries during ripening.

Chitinase activity increased markedly at the onset of ripening in grape (Vitis vinifera L.) berries and continued to increase throughout the sugar accumulation phase of berry development. In contrast, beta-1,3-glucanase activity was not detected in grape berries at any stage of development. Two closely related chitinase cDNAs (VvChi4A and VvChi4B) were cloned from grapes. Sequence and Southern analysis indicate that these two clones may represent alleles of the same gene. The predicted proteins are acidic and have a signal peptide followed by a cysteine-rich, chitin-binding domain and a catalytic region. An analysis of their sequences indicates that they are class IV chitinase. The deduced protein sequence of VvChi4A has a high level of identity with the 32- and 28-kD chitinases present as haze proteins in wine. Expression of VvChi4 was high in berries and low in flowers but was not detected in leaves, roots, or seeds. No expression was detected in berries 2 to 8 weeks postflowering, but expression was high 12 to 16 weeks postflowering, which coincided with sugar accumulation and an increase in chitinase activity. Constitutive expression of VvChi4 appears to be fruit-specific and induced at high levels in grapes during ripening.

Amino Acid Sequence↗
PubMed2001

Class I beta-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence.

beta-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. beta-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. beta-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of beta-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 microM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both beta-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.

Abscisic Acid↗
PubMed2002

Mutation of a chitinase-like gene causes ectopic deposition of lignin, aberrant cell shapes, and overproduction of ethylene.

Chitinase-like proteins have long been proposed to play roles in normal plant growth and development, but no mutations in chitinase-like genes have been obtained previously to support this hypothesis. In this study, we have shown that the gene responsible for the elp1 mutation in Arabidopsis encodes a chitinase-like protein (AtCTL1). Mutation of this chitinase-like gene caused ectopic deposition of lignin and aberrant shapes of cells with incomplete cell walls in the pith of inflorescence stems. The AtCTL1 gene was expressed in all organs during normal plant growth and development, but it was not induced by wounding, salicylic acid, pectin fragments, or ethylene. Consistent with its ubiquitous expression pattern, mutation of the AtCTL1 gene affected many aspects of plant growth and development, including exaggerated hook curvature, reduced length and increased diameter of hypocotyls in dark-grown seedlings, and reduced root length and increased number of root hairs in light-grown seedlings. The mutant phenotypes could be rescued partially by ethylene inhibitors, and ethylene production in the mutant was significantly greater than in the wild type. Together, these results suggest that AtCTL1, a chitinase-like gene, is essential for normal plant growth and development in Arabidopsis.

Amino Acid Sequence↗
PubMed2006

Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus.

The crystal structure of the catalytic domain of a chitinase from the hyperthermophilic archaeon Pyrococcus furiosus (AD2(PF-ChiA)) has been determined at 1.5 A resolution. This is the first structure of the catalytic domain of an archaeal chitinase. The overall structure of AD2(PF-ChiA) is a TIM-barrel fold with a tunnel-like active site that is a common feature of family 18 chitinases. Although the catalytic residues (Asp522, Asp524 and Glu526) are conserved, comparison of the conserved residues and structures with those of other homologous chitinases indicates that the catalytic mechanism of PF-ChiA is different from that of family 18 chitinases.

Amino Acid Sequence↗
PubMed2005

Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki.

AIMS: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. METHODS AND RESULTS: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. CONCLUSIONS: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase 'structure-function' relation.

Amino Acid Sequence↗
PubMed2001

Addition of substrate-binding domains increases substrate-binding capacity and specific activity of a chitinase from Trichoderma harzianum.

Chitinase Chit42 from Trichoderma harzianum CECT 2413 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin-binding domain (ChBD). We have produced hybrid chitinases with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Nicotiana tabacum ChiA chitinase and the cellulose-binding domain from cellobiohydrolase II of Trichoderma reesei. The chimeric chitinases had similar activities towards soluble substrate but higher hydrolytic activity than the native chitinase on high molecular mass insoluble substrates such as ground chitin or chitin-rich fungal cell walls.

Amino Acid Sequence↗
PubMed2006

Molecular cloning, characterization, and expression studies of a novel chitinase gene (ech30) from the mycoparasite Trichoderma atroviride strain P1.

We describe the cloning and characterization of a single copy gene from Trichoderma atroviride P1 encoding a novel 30 kDa chitinase, Ech30. Ech30 is a family 18 chitinase showing low sequence similarity to other Trichoderma chitinases. Real-time quantitative RT-PCR studies revealed that expression of the ech30 gene was induced by the presence of Botrytis cinerea in plate confrontation assays, but hardly by chitin in liquid cultures. Studies of Ech30 purified from an Escherichia coli strain overexpressing the ech30 gene devoid of the leader sequence and a predicted intron, showed that the gene encodes an active chitinase, which, as expected for family 18 chitinases, is inhibited by allosamidin.

5' Untranslated Regions↗
PubMed1981

Chitinase-overproducing mutant of Serratia marcescens.

Genetic modification of Serratia marcescens QMB1466 was undertaken to isolated mutants which produce increased levels of chitinolytic activity. After mutagenesis with ultraviolet light, ethyl methane sulfonate or N-methyl-N'-nitro-N-nitrosoguanidine, 19,940 colonies were screened for production of enlarged zones of clearing (indicative of chitinase activity) on chitin-containing agar plates. Forty-four chitinase high producers were tested further in shake flask cultures. Mutant IMR-1E1 was isolated which, depending on medium composition, produced two to three times more than the wild type of the other components of the chitinolytic enzyme system--a factor involved in the hydrolysis of crystalline chitin and chitobiase. After induction by chitin, endochitinase and chitobiase activity appeared at similar times for both IMR-1E1 and QMB1466, suggesting possible coordinate control of these enzymes. The results are consistent with IMR-1E1 containing a regulatory mutation which increased production of the components of the chitinolytic enzyme system and/or with IMR-1E1 containing a tandem duplication of the chitinase genes. The high rate of reversion of IMR-1E1 to decreased levels of chitinase production suggests that the overproduction of chitinase by IMR-1E1 is due to a tandem gene duplication.

Acetylglucosaminidase↗
PubMed2001

Identification and characterization of a chitinase antigen from Pseudomonas aeruginosa strain 385.

A chitinase antigen has been identified in Pseudomonas aeruginosa strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH2-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the P. aeruginosa genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The Km and kcat for 4-nitrophenyl-beta-D-N,N'-diacetylchitobiose were 4.28 mM and 1.7 s(-1) respectively, and for 4-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, they were 0.48 mM and 0.16 s(-1) respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50 degrees C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.

Antibodies, Bacterial↗
PubMed2003

Characterization of chitinase genes from an alkaliphilic actinomycete, Nocardiopsis prasina OPC-131.

An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiB Delta, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiB Delta was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiB Delta were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiB Delta, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.

Actinomycetales↗
PubMed2003

Growth of hyperthermophilic archaeon Pyrococcus furiosus on chitin involves two family 18 chitinases.

Pyrococcus furiosus was found to grow on chitin, adding this polysacharide to the inventory of carbohydrates utilized by this hyperthermophilic archaeon. Accordingly, two open reading frames (chiA [Pf1234] and chiB [Pf1233]) were identified in the genome of P. furiosus, which encodes chitinases with sequence similarity to proteins from the glycosyl hydrolase family 18 in less-thermophilic organisms. Both enzymes contain multiple domains that consist of at least one binding domain and one catalytic domain. ChiA (ca. 39 kDa) contains a putative signal peptide, as well as a binding domain (ChiA(BD)), that is related to binding domains associated with several previously studied bacterial chitinases. chiB, separated by 37 nucleotides from chiA and in the same orientation, encodes a polypeptide with two different proline-threonine-rich linker regions (6 and 3 kDa) flanking a chitin-binding domain (ChiB(BD) [11 kDa]), followed by a catalytic domain (ChiB(cat) [35 kDa]). No apparent signal peptide is encoded within chiB. The two chitinases share little sequence homology to each other, except in the catalytic region, where both have the catalytic glutamic acid residue that is conserved in all family 18 bacterial chitinases. The genes encoding ChiA, without its signal peptide, and ChiB were cloned and expressed in Escherichia coli. ChiA exhibited no detectable activity toward chitooligomers smaller than chitotetraose, indicating that the enzyme is an endochitinase. Kinetic studies showed that ChiB followed Michaelis-Menten kinetics toward chitotriose, although substrate inhibition was observed for larger chitooligomers. Hydrolysis patterns on chitooligosaccharides indicated that ChiB is a chitobiosidase, processively cleaving off chitobiose from the nonreducing end of chitin or other chitooligomers. Synergistic activity was noted for the two chitinases on colloidal chitin, indicating that these two enzymes work together to recruit chitin-based substrates for P. furiosus growth. This was supported by the observed growth on chitin as the sole carbohydrate source in sulfur-free media.

Amino Acid Sequence↗
PubMed1995

Chitinase activity in human serum and leukocytes.

Using colloidal [3H] chitin as a substrate, we provide the first demonstration of a chitinase in human leukocytes; chitinolytic activity in whole and disrupted leukocyte preparations (approximately 0.6 and 5.5 nmol of N-acetylglucosamine [GlcNAc] released min-1 mg of protein-1, respectively) was partially inhibited by the specific chitinase inhibitor allosamidin (9 microM). Following fractionation of the leukocytes, much higher levels of chitinase activity were detected in granulocyte-rich homogenates (approximately 7.2 nmol of GlcNAc released min-1 mg of protein-1) than in lymphocyte- and monocyte-rich homogenates (approximately 0.22 and 0.26 nmol of GlcNAc released min-1 mg of protein-1, respectively). Low levels of chitinase activity were detected in human serum (approximately 4 pmol of GlcNAc released min-1 mg of protein-1). Chitinolytic activity in granulocyte-rich homogenates and serum was partially inhibited by allosamidin (9 microM). Proteins with chitinolytic activities (approximate molecular masses, 48 and 56 kDa) distinct from lysozyme (14.3 kDa) were detected on polyacrylamide gels following the electrophoresis of human granulocyte-rich preparations. Chitinase activity, detected consistently in serum and leukocytes from all human volunteers investigated, may contribute to the protection of the host by cleaving chitin in the cell walls of fungal pathogens.

Acetylglucosamine↗