Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1

We describe the overexpression and characterization of a new 30 kDa family 18 chitinase (Ech30) from Trichoderma atroviride strain P1. Sequence alignments indicate that the active site architecture of Ech30 resembles that of endochitinases such as hevamine from the rubber tree (Hevea brasiliensis). The ech30 gene was overexpressed in Escherichia coli without its signal peptide and with an N-terminal His-tag. The enzyme was produced as inclusion bodies, from which active chitinase could be recovered using a simple refolding procedure. The enzyme displayed an acidic pH-optimum (pH 4.5-5.0), probably due to the presence of a conserved Asn residue near the catalytic glutamate, which is characteristic for acidic family 18 chitinases. Studies with oligomers of N-acetylglucosamine [(GlcNAc)(n)], 4-methylumbelliferyl (4-MU) labelled GlcNAc oligomers and beta-chitin reveal enzymatic properties typical of an endochitinase: 1) low activity towards short substrates (kinetic parameters for the hydrolysis of 4-MU-(GlcNAc)2 were K(m), 149+/-29 microM and k(cat), 0.0048+/-0.0005 s(-1)), and 2) production of relatively large amounts of trimers and tetramers during degradation of beta-chitin. Detailed studies with GlcNAc oligomers indicated that Ech30 has as many as seven subsites for sugar binding. As expected for a family 18 chitinase, catalysis proceeded with retention of the beta-anomeric configuration.     

Cloning and phylogenetic analysis of the chitinase gene from the facultative pathogen Paecilomyces lilacinus

AIMS: To PCR-amplify the full-length genomic-encoding sequence for one chitinase from the facultative fungal pathogen Paecilomyces lilacinus, analyse the DNA and deduced amino acid sequences and compare the amino acid sequence with chitinases reported from mycopathogens, entomopathogens and nematopathogens. METHODS AND RESULTS: The encoding gene (designated as PLC) was isolated using the degenerate PCR primers and the DNA-Walking method. The gene is 1458 bp in length and contains three putative introns. A number of sequence motifs that might play a role in its regulation and function had also been found. Alignment of the translation product (designated as Plc, molecular mass of 45.783 kDa and pI of 5.65) with homologous sequences from other species showed that Plc belongs to Class V chitinase within the glycosyl hydrolase family 18. The phylogenetic and molecular evolutionary analysis using mega (Molecular Evolutionary Genetics Analysis) indicated that these chitinases from mycopathogens, entomopathogens and nematopathogens, the majority of which belong to glycosyl hydrolase family 18, were clustered into two well-supported subgroups corresponding to ascomycetes fungal and nonfungal chitinases (bacteria, baculoviruses). CONCLUSIONS: Our study showed that chitinases from mycoparasitic, entomopathogenic and nematophagous fungi are closely related to each other and reaffirmed the hypothesis that baculovirus chitinase is most likely to be of a bacterial origin - acquired by gene transfer. Bacterial and baculoviral chitinases in our study are potential pathogenicity factors; however, we still cannot ascribe any specific function to those chitinases from the fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report describing the chitinase gene and its translation product from Paecilomyces lilacinus, which constitutes the largest number of formulated biological nematicides reported so far, this is also the first study to analyse and resolve the phylogenetic and molecular evolutionary relationships among the chitinases produced by mycopathogens, entomopathogens and nematopathogens.     

High-multiplicity of chitinase genes in Streptomyces coelicolor A3(2).

Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp. as probes, and cloned. The genes were sequenced and analyzed. The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification. The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains. The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70% identity with the catalytic domain of ChiC of Streptomyces griseus, the sole gene for family 19 chitinase so far found in an organism other than higher plants.     

Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases

The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.     

Role of the Trichoderma harzianum endochitinase gene, ech42, in mycoparasitism

The role of the Trichoderma harzianum endochitinase (Ech42) in mycoparasitism was studied by genetically manipulating the gene that encodes Ech42, ech42. We constructed several transgenic T. harzianum strains carrying multiple copies of ech42 and the corresponding gene disruptants. The level of extracellular endochitinase activity when T. harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains as compared with the wild type, whereas gene disruptants exhibited practically no activity. The densities of chitin labeling of Rhizoctonia solani cell walls, after interactions with gene disruptants were not statistically significantly different than the density of chitin labeling after interactions with the wild type. Finally, no major differences in the efficacies of the strains generated as biocontrol agents against R. solani or Sclerotium rolfsii were observed in greenhouse experiments.     

Cloning and expression of a Bacillus circulans KA-304 gene encoding chitinase I, which participates in protoplast formation of Schizophyllum commune

KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune, has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, isolated from KA-prep, brings about the protoplast-forming activity. The gene of chitinase I was cloned from B. circulans KA-304 into pGEM-T Easy vector. The gene consists of 1,239 nucleotides, which encodes 413 amino acids including a putative signal peptide (24 amino acid residues). The molecular weight of 40,510, calculated depending on the open reading frame without the putative signal peptide, coincided with the apparent molecular weight of 41,000 of purified chitinase I estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal domain of the deduced amino acid sequence showed high similarity to that of family 19 chitinases of actinomycetes and other organisms, indicating that chitinase I is the first example of family 19 chitinase in Bacillus species. Recombinant chitinase I without the putative signal peptide was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the purified recombinant enzyme were almost the same as those of chitinase I purified from KA-prep, and showed the protoplast-forming activity when it was combined with alpha-1,3-glucanase from KA-prep. Recombinant chitinase I as well as the native enzyme inhibited hyphal extension of Trichoderma reesei.     

Cloning, sequence, and expression of a chitinase gene from a marine bacterium, Altermonas sp. strain O-7.

The gene encoding an extracellular chitinase from marine Alteromonas sp. strain O-7 was cloned in Escherichia coli JM109 by using pUC18. The chitinase produced was not secreted into the growth medium but accumulated in the periplasmic space. A chitinase-positive clone of E. coli produced two chitinases with different molecular weights from a single chitinase gene. These proteins showed almost the same enzymatic properties as the native chitinase of Alteromonas sp. strain O-7. The N-terminal sequences of the two enzymes were identical. The nucleotide sequence of the 3,394-bp SphI-HindIII fragment that included the chitinase gene was determined. A single open reading frame was found to encode a protein consisting of 820 amino acids with a molecular weight of 87,341. A putative ribosome-binding site, promoter, and signal sequence were identified. The deduced amino acid sequence of the cloned chitinase showed sequence homology with chitinases A (33.4%) and B (15.3%) from Serratia marcescens. Regardless of origin, the enzymes of the two bacteria isolated from marine and terrestrial environments had high homology, suggesting that these organisms evolved from a common ancestor.     

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.     

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.     

Characterization of two antifungal endochitinases from barley grain

A basic chitinase (chitinase T, EC 3.2.1.14, molecular mass 33 kDa, pI 9.8) was isolated and compared with a previously described chitinase (chitinase C, molecular mass 28 kDa, pI 9.7). The two chitinases were isolated in homogeneous form from barley ( Hordeum vulgare L.) Bomi mutant 1508 grains either by two cation exchange steps or by one affinity step followed by cation exchange. Both chitinases are endochitinases with specific activities of 168 and 54 nkat (mg protein)⁻¹ for chitinase T and chitinase C, respectively. Both inhibit the growth of Trichoderma viride efficiently. The lysozyme activity of both chitinases is 10⁴ times lower than that of hen egg-white lysozyme as measured by lysis of cell walls of Micrococcus lysodeikticus . The amino acid composition and two partial amino acid sequences of chitinase T were determined. A 23 residue sequence of the N-terminal domain of chitinase T, which was not present in chitinase C, showed 73% identity with domain B of wheat germ lectin and 65% identity with the N-terminal domain of an endochitinase from bean leaves (deduced from cDNA). A 9 amino acid sequence of a cyanogen bromide fragment of chitinase T was identical with a cDNA deduced sequence of a barley aleurone endochitinase but differed in one residue from chitinase C. Generally, the two grain chitinases have physico-chemical and enzymatic properties similar to the plant leaf chitinases characterized. Both chitinases are localized in the aleurone layer and starchy endosperm of developing and germinating grain, but not in the embryo. The appearance of chitinases T and C at a late state of grain development suggests a role for these enzymes as a defense against fungi in the quiescent and germinating grain.     

Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria

In organisms other than higher plants, family 19 chitinase was first discovered in Streptomyces griseus HUT6037, and later, the general occurrence of this enzyme in Streptomyces species was demonstrated. In the present study, the distribution of family 19 chitinases in the class Actinobacteria and the phylogenetic relationship of Actinobacteria family 19 chitinases with family 19 chitinases of other organisms were investigated. Forty-nine strains were chosen to cover almost all the suborders of the class Actinobacteria, and chitinase production was examined. Of the 49 strains, 22 formed cleared zones on agar plates containing colloidal chitin and thus appeared to produce chitinases. These 22 chitinase-positive strains were subjected to Southern hybridization analysis by using a labeled DNA fragment corresponding to the catalytic domain of ChiC, and the presence of genes similar to chiC of S. griseus HUT6037 in at least 13 strains was suggested by the results. PCR amplification and sequencing of the DNA fragments corresponding to the major part of the catalytic domains of the family 19 chitinase genes confirmed the presence of family 19 chitinase genes in these 13 strains. The strains possessing family 19 chitinase genes belong to 6 of the 10 suborders in the order Actinomycetales, which account for the greatest part of the Actinobacteria: Phylogenetic analysis suggested that there is a close evolutionary relationship between family 19 chitinases found in Actinobacteria and plant class IV chitinases. The general occurrence of family 19 chitinase genes in Streptomycineae and the high sequence similarity among the genes found in Actinobacteria suggest that the family 19 chitinase gene was first acquired by an ancestor of the Streptomycineae and spread among the Actinobacteria through horizontal gene transfer.     

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.     

Garlic (Allium sativum) chitinases: characterization and molecular cloning

Leaves and bulbs of garlic ( Allium sativum L.) contain a chitinase which can be separated into three different isoforms with similar molecular structure and N- terminal amino acid sequence. SDS-PAGE of the alkylated chitinase revealed two distinct polypeptides of 32 and 33 kDa. Induction studies of the chitinase in leaves of garlic plants indicated that not only treatment with ethephon or salicylate and wounding but also a temperature shock strongly increased the enzyme level.
cDNA libraries constructed from poly(A)-rich RNA isolated from young garlic shoots and bulbs were screened for chitinase clones using the cDNA clone CCH4 encoding a basic potato chitinase as a probe. Two different cDNA clones (designated CHITAS 1 and CHITAS 2)of ca 1 000 bp were isolated and their sequences analyzed. The amino acid sequences deduced from both cDNA clones were homologous though not identical to the N-terminal sequences of the mature chitinases. Although both clones encode highly homologous chitinases their sequences definitely differ in that they have different signal peptides and one of them contains a glycine-rich domain. The garlic chitinases are apparently translated from an mRNA of 1200 nucleotides which encodes a proprotein of approximately 32 or 33 kDa for CHITAS 1 and CHITAS 2, respectively. Co-translational removal of the signal peptide will result in a 30 (for CHITAS 1) or 31 kDa (for CHITAS 2) protein with an isoelectric point of 4. 94 (for CHITAS 1) or 6. 12 (for CHITAS 2). Garlic chitinases are encoded by a small gene family as shown by Southern blot analysis of genomic DNA isolated from garlic.
The garlic chitinases show a high degree of sequence homology to the previously isolated chitinases from dicotyledonous as well as monocotyledonous species, indicating that these proteins have been conserved from an evolutionary point of view.     

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

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

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.     

Characterization of a chitinolytic enzyme from Serratia sp. KCK isolated from kimchi juice

The novel chitinolytic bacterium Serratia sp. KCK, which was isolated from kimchi juice, produced chitinase A. The gene coding for the chitinolytic enzyme was cloned on the basis of sequencing of internal peptides, homology search, and design of degenerated primers. The cloned open reading frame of chiA encodes for deduced polypeptide of 563 amino acid residues with a calculated molecular mass of 61 kDa and appears to correspond to a molecular mass of about 57 kDa, which excluded the signal sequence. The deduced amino acid sequence showed high similarity to those of bacterial chitinases classified as family 18 of glycosyl hydrolases. The chitinase A is an exochitinase and exhibits a greater pH range (5.0-10.0), thermostability with a temperature optimum of 40 degrees C, and substrate range other than Serratia chitinases thus far described. These results suggested that Serratia sp. KCK chitinase A can be used for biotechnological applications with good potential.     

A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.

A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.     

Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S. coelicolor A3(2)

Streptomyces coelicolor A3(2) has 13 chitinase genes encoding 11 family 18 and two family 19 chitinases. To compare enzymatic properties of family 19 chitinase and family 18 chitinases produced by the same organism, the four chitinases (Chi18bA, Chi18aC, Chi18aD, and Chi19F), whose genes are expressed at high levels in the presence of chitin, were produced in Escherichia coli and purified. The effect of pH on the hydrolytic activity was very different not only among the four chitinases but also among the substrates. The hydrolytic activity of Chi19F, family 19 chitinase, against soluble substrates was remarkably high as compared with three family 18 chitinases, but was the lowest against crystalline substrates among the four chitinases. On the contrary, Chi18aC, a family 18-subfamily A chitinase, showed highest activity against crystalline substrates. Only Chi19F exhibited significant antifungal activity. Based on these observations, the roles of family 19 chitinases are discussed.