A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed.     

Induction of chitinases and of translatable mRNA for these enzymes in melon plants infected with Collectotrichum lagenarium

In melon plants infected with Colletotrichum langenarium, there is a strong increase in the activity of chitinase, an enzyme with a potential defensive function against pathogens. In order to investigate the molecular mechanisms involved in the regulation of chitinase gene expression, antisera have been raised against two purified chitinases (I and II) from infected melon plants. Changes induced by infection in the rate of synthesis of chitinases were determined using direct immunoprecipitation of enzymes labelled in vivo with [³⁵S]methionine. A large but transient increase in the rate of chitinase synthesis occurred 5 days after inoculation. The in vitro synthesis of chitinases was then studied in healthy and infected melon plants. Poly(A)RNA was fractionated by sucrose gradient density centrifugation, and translated in a rabbit reticulocyte lysate. The transition products were then separated by fast protein liquid chromatography (FPLC) and further analysed by SDS-polyacrylamide gel electrophoresis. The identification of in vitro synthesized chitinases was performed by immunoprecipitation. The obtained results indicated the presence of chitinases in in vitro translation products of mRNA from infected, but not from healthy melon seedlings. It is concluded therefore, that infection of melon seedlings by a pathogen caused an increase in the translatable mRNAs for host chitinases.     

Characterzation of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variantts11

To characterize the acidic endochitinase EP3, able to rescue somatic embryos of the carrot cell linets11, 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 ofts11 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-stagets11 embryos. The class II endochitinase and a heterologous class IV chitinase from sugar-beet were not active onts11. This suggests that there are differences in the specificity of chitinases in terms of their effect on plant somatic embryos.     

Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.     

A tobacco gene encoding a novel basic class II chitinase: a putative ancestor of basic class I and acidic class II chitinase genes

Various chitinases have been identified in plants and categorized into several groups based on the analysis of their sequences and domains. We have isolated a tobacco gene that encodes a predicted polypeptide consisting of a 20-amino acid N-terminal signal peptide, followed by a 245-amino acid chitinolytic domain. Although the predicted mature protein is basic and shows greater sequence identity to basic class I chitinases (75%) than to acidic class II chitinases (67%), it lacks the N-terminal cysteine-rich domain and the C-terminal vacuolar targeting signal that is diagnostic for class I chitinases. Therefore, this gene appears to encode a novel, basic, class II chitinase, which we have designated NtChia2;B1. Accumulation of Chia2;B1 mRNA was induced in leaves in association with the local-lesion response to tobacco mosaic virus (TMV) infection, and in response to treatment with salicylic acid, but was only slightly induced by treatment with ethephon. Little or no Chia2;B1 mRNA was detected in roots, flowers, and cell-suspension cultures, in which class I chitinase mRNAs accumulate to high concentrations. Sequence comparisons of Chia2;B1 with known tobacco class I and class II chitinase genes suggest that Chia2;B1 might encode an ancestral prototype of the present-day class I and class II isoforms. Possible mechanisms for chitinase gene evolution are discussed. Received: 25 May 1998 / Accepted: 29 June 1998     

Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.     

Entamoeba invadens: Cloning and molecular characterization of chitinases

Entamoeba histolytica, the causative agent of amebiasis infects through its cyst form and this transmission may be blocked using encystation specific protein as drug target. In this study, we have characterized the enzyme chitinase which express specifically during encystation. The reptilian parasite Entamoeba invadens, used as a model for encystation study contain three chitinases. We report the molecular cloning, over-expression and biochemical characterization of all three E. invadens chitinase. Cloned chitinases were over-expressed in bacterial system and purified by affinity chromatography. Their enzymatic profiles and substrate cleaving patterns were characterized. All of them showed binding affinity towards insoluble chitin though two of them lack the chitin binding domain. All the chitinases cleaved and released dimmers from the insoluble substrate and act as an exochitinase. Homology modeling was also done to understand the substrate binding and cleavage pattern.     

Cloning and characterization of a small family 19 chitinase from moss (Bryum coronatum)

Chitinase-A (BcChi-A) was purified from a moss, Bryum coronatum, by several steps of column chromatography. The purified BcChi-A was found to be a molecular mass of 25 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.5. A cDNA encoding BcChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1012 nucleotides and encoded an open reading frame of 228 amino acid residues. The predicted mature BcChi-A consists of 205 amino acid residues and has a molecular weight of 22,654. Sequence analysis indicated that BcChi-A is glycoside hydrolase family-19 (GH19) chitinase lacking loops I, II, IV and V, and a C-terminal loop, which are present in the catalytic domain of plant class I and II chitinases. BcChi-A is a compact chitinase that has the fewest loop regions of the GH19 chitinases. Enzymatic experiments using chitooligosaccharides showed that BcChi-A has higher activity toward shorter substrates than class II enzymes. This characteristic is likely due to the loss of the loop regions that are located at the end of the substrate-binding cleft and would be involved in substrate binding of class II enzymes. This is the first report of a chitinase from mosses, nonvascular plants.     

Salmonella enterica serovar Typhimurium chitinases modulate the intestinal glycome and promote small intestinal invasion

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the leading causes of food-borne illnesses worldwide. To colonize the gastrointestinal tract, S. Typhimurium produces multiple virulence factors that facilitate cellular invasion. Chitinases have been recently emerging as virulence factors for various pathogenic bacterial species, and the S. Typhimurium genome contains two annotated chitinases: STM0018 (chiA) and STM0233. However, the role of these chitinases during S. Typhimurium pathogenesis is unknown. The putative chitinase STM0233 has not been studied previously, and only limited data exists on ChiA. Chitinases typically hydrolyze chitin polymers, which are absent in vertebrates. However, chiA expression was detected in infection models and purified ChiA cleaved carbohydrate subunits present on mammalian surface glycoproteins, indicating a role during pathogenesis. Here, we demonstrate that expression of chiA and STM0233 is upregulated in the mouse gut and that both chitinases facilitate epithelial cell adhesion and invasion. S. Typhimurium lacking both chitinases showed a 70% reduction in invasion of small intestinal epithelial cells in vitro. In a gastroenteritis mouse model, chitinase-deficient S. Typhimurium strains were also significantly attenuated in the invasion of small intestinal tissue. This reduced invasion resulted in significantly delayed S. Typhimurium dissemination to the spleen and the liver, but chitinases were not required for systemic survival. The invasion defect of the chitinase-deficient strain was rescued by the presence of wild-type S. Typhimurium, suggesting that chitinases are secreted. By analyzing N-linked glycans of small intestinal cells, we identified specific N-acetylglucosamine-containing glycans as potential extracellular targets of S. Typhimurium chitinases. This analysis also revealed a differential abundance of Lewis X/A-containing glycans that is likely a result of host cell modulation due to the detection of S. Typhimurium chitinases. Similar glycomic changes elicited by chitinase deficient strains indicate functional redundancy of the chitinases. Overall, our results demonstrate that S. Typhimurium chitinases contribute to intestinal adhesion and invasion through modulation of the host glycome.     

Two sequence-specific endonucleases from Moraxella bovis

Two new sequence-specific endodeoxyribonucleases have been partially purified from Moraxella bovis. These restriction-like enzymes, MboI and MboII, each cleave bacteriophage lambda DNA and adenovirus-2 DNA at more than 50 sites. MboI recognizes the sequence 5′ ↓ G-A-T-C 3′ 3′ C-T-A-G ↑ 5′ and cleaves at the sites indicated by the arrows. A specific endonuclease, MosI, has also been purified from Moraxella osloenis and recognizes the same sequence as MboI.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Properties of purified folate-binding proteins from chronic myelogenous leukemia cells

Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate.Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid would be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8 and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydroflate, lower affinity of N⁵-methyl-tetrahydrofolate, and no apparent affinity for N⁵-formytetrahydrofolate or methotrexate.     

Isolation and Characterization of a Chitinase and its cDNA Clone from Rice

A 35 kD chitinase has been purified to apparent homogeneity from extracts of rice bran of cv New Bonnet by ammonium sulfate fractionation, chitin affinity chromatography, cation exchange chromatography on carboxymethyl cellulose and gel filtration. The purified enzyme has an isoelectric point of 8.8. The enzyme inhibited the growth of Rhizoctonia solani (the sheath blight pathogen), Trichoderma viride, T. harzianum, Fusarium graminaerum and F. culmorum in vitro. A cDNA clone for chitinase was isolated from a developing rice seed cDNA library by probing with a barley chitinase cDNA probe. The nucleotide sequence of this 654 bp clone was determined, it contains an open reading frame of 519 nucleotides. The protein product encoded by this clone is homologous to chitinases from tobacco, bean and barley. Southern blot analysis of rice genomic DNA with this probe revealed that chitinases are encoded by a small multi-gene family in the rice genome.     

Purification and biochemical characterization of glucose–cellobiose-tolerant cellulases from Scytalidium thermophilum

Two cellulases from Scytalidium thermophilum were purified and characterized, exhibiting tolerance to glucose and cellobiose. Characterization of purified cellulases I and II by mass spectrometry revealed primary structure similarities with an exoglucanase and an endoglucanase, respectively. Molecular masses were 51.2 and 45.6 kDa for cellulases I and II, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cellulases I and II exhibited isoelectric points of 6.2 and 6.9 and saccharide contents of 11 and 93 %, respectively. Optima of temperature and pH were 60–65 °C and 4.0 for purified cellulase I and 65 °C and 6.5 for purified cellulase II. Both cellulases maintained total CMCase activity after 60 min at 60 °C. Cysteine, Mn²⁺, dithiotreitol and ß-mercaptoethanol-stimulated cellulases I and II. The tolerance to cellulose hydrolysis products and the high thermal stabilities of Scytalidium cellulases suggest good potential for industrial applications.     

A comparative proteomic approach to analyse structure, function and evolution of rice chitinases: a step towards increasing plant fungal resistance

Glycoside hydrolase family 19 chitinases (EC 3.2.1.14) widely distributed in plants, bacteria and viruses catalyse the hydrolysis of chitin and play a major role in plant defense mechanisms and development. Rice possesses several classes of chitinase, out of which a single structure of class I has been reported in PDB to date. In the present study an attempt was made to gain more insight into the structure, function and evolution of class I, II and IV chitinases of GH family 19 from rice. The three-dimensional structures of chitinases were modelled and validated based on available X-ray crystal structures. The structural study revealed that they are highly α-helical and bilobed in nature. These enzymes are single or multi domain and multi-functional in which chitin-binding domain (CBD) and catalytic domain (CatD) are present in class I and IV whereas class II lacks CBD. The CatD possesses a catalytic triad which is thought to be involved in catalytic process. Loop III, which is common in all three classes of chitinases, reflects that it may play a significant role in their function. Our study also confirms that the absence and presence of different loops in GH family 19 of rice may be responsible for various sized products. Molecular phylogeny revealed chitinases in monocotyledons and dicotyledons differed from each other forming two different clusters and may have evolved differentially. More structural study of this enzyme from different plants is required to enhance the knowledge of catalytic mechanism and substrate binding.     

A new class of tobacco chitinases homologous to bacterial exo-chitinases displays antifungal activity

A novel chitinase gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I–IV and are designated as class V chitinases. Comparison of the chitinase class V peptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratia marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation. Two related chitinase class V proteins of 41 and 43 kDa were purified from Samsun NN tobacco leaves inoculated with tobacco mosaic virus. The proteins were purified by Chelating Superose chromatography and gel filtration. In vitro assays demonstrated that class V chitinases have endo-chitinase activity and exhibit antifungal activity toward Trichoderma viride and Alternaria radicina. In addition, it was shown that class V chitinase acts synergistically with tobacco class I β-1,3-glucanase against Fusarium solani germlings.     

Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity

Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.