Cloning,DNA sequence,and expression of Aeromonas caviae WS7b chitinase gene

A chitinase-producing bacterium, designated WS7b, was isolated from a soil sample obtained from a black-pepper plantation on Bangka Island, Indonesia. Fatty-acid methyl-ester analysis indicated that the isolate was Aeromonas caviae. A chitinase gene from WS7b was cloned in a pUC19-based plasmid vector, but without its natural promoter. The complete nucleotide sequence of the gene was determined, and the structural gene consisted of a 2748-bp region encoding 864 amino acids. DNA sequence analysis indicated that the gene had been cloned without its promoter, and this was confirmed by chitinase-plate assay of the truncated version of the gene in Escherichia coli. The chitinase gene product showed amino-acid sequence similarity to chiA from A. caviae. Chitinase enzyme activity was determined spectrophotometrically, using colloidal chitin azure as substrate for extracellular and intracellular fractions. The ability of the chitinase cloned in E. coli to hydrolyze chitin was less than that of the enzyme in its indigenous host.     

Purification and properties of a chitinase from Penicillium sp. LYG 0704

The chitinase producing Penicillium sp. LYG 0704 was procured from soil of the Chonnam National University crop field. The chitinase activity was detected after the first day which increased gradually and reached its maximum after 3 days of cultivation. The chitinase was purified from a culture medium by precipitation with isopropanol and column chromatography with Mono Q and Butyl-Sepharose. The molecular mass of chitinase was estimated to be 47 kDa by SDS–PAGE. Optimal pH and temperature were 5.0 and 40 °C, respectively. The N-terminal amino acid sequence of the enzyme was determined to be ¹AGSYRSVAYFVDWAI¹⁵. The fully cloned gene, 1287 bp in size, encoded a single peptide of 429 amino acids. BLAST search of the chitinase gene sequence showed similarity with chitinase of Aspergillus fumigatus Af293 chitinase gene (58%) and A. fumigatus class V chitinase ChiB1 gene (56%).     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro

Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. ‘Sponsor’ were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants.     

Molecular cloning and characterization of 58 kDa chitinase gene fromSerratia marcescens KCTC 2172

A chitinase gene (pCHi58) encoding a 58 kDa chitinase was isolated from theSerratia marcescens KCTC 2172 cosmid library. The chitinase gene consisted of a 1686 bp open reading frame that encoded 562 amino acids.Escherichia coil harboring the pChi58 gene secreted a 58 kDa chitinase into the culture supernatant. The 58 kDa chitinase was purified using a chitin affinity column and mono-S column. A nucleotide andN-terminal amino acid sequence analysis showed that the 58 kDa chitinase had a leader peptide consisting of 23 amino acids which was cleaved prior to the 24th alanine. The 58 KDa chitinase exhibited a 98% similarity to that ofS. marcescens QMB 1466 in its nuclotide sequence. The chitinolytic patterns of the 58 kDa chitinase released N,N′-diacetyl chitobiose (NAG2) as the major hydrolysis end-product with a trace amount ofN-acetylglucosamine. When a 4-methylumbellyferyl-N-acetylglucosamin monomer, dimmer, and tetramer were used as substrates, the 58 kDa chitinase did not digest the 4-Mu-NAG monomer (analogue of NAG₂), thereby indicating that the 58 kDa chitinase was likely an endochitinase. The optimum reaction temperature and pH of the enzyme were 50°C and 5.0, respectively.     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding,fungal infection and the elicitor chitosan

The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chil. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco

The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F₁ progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities.     

Characterization of Antifungal Chitinase from Marine Streptomyces sp. DA11 Associated with South China Sea Sponge Craniella Australiensis

The gene cloning, purification, properties, kinetics, and antifungal activity of chitinase from marine Streptomyces sp. DA11 associated with South China sponge Craniella australiensis were investigated. Alignment analysis of the amino acid sequence deduced from the cloned conserved 451 bp DNA sequence shows the chitinase belongs to ChiC type with 80% similarity to chitinase C precursor from Streptomyces peucetius. Through purification by 80% ammonium sulfate, affinity binding to chitin and diethylaminoethyl-cellulose anion-exchange chromatography, 6.15-fold total purification with a specific activity of 2.95 Umg⁻¹ was achieved. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular weight of approximately 34 kDa and antifungal activities were observed against Aspergillus niger and Candida albicans. The optimal pH, temperature, and salinity for chitinase activity were 8.0, 50°C, and 45 g‰ psu, respectively, which may contribute to special application of this marine microbe-derived chitinase compared with terrestrial chitinases. The chitinase activity was increased by Mn²⁺, Cu²⁺, and Mg²⁺, while strongly inhibited by Fe²⁺ and Ba²⁺. Meanwhile, SDS, ethyleneglycoltetraacetic acid, urea, and ethylenediaminetetraacetic acid were found to have significantly inhibitory effect on chitinase activity. With colloidal chitin as substrates instead of powder chitin, higher V _max (0.82 mg product/min·mg protein) and lower K _m (0.019 mg/ml) values were achieved. The sponge’s microbial symbiont with chitinase activity may contribute to chitin degradation and antifungal defense. To our knowledge, it was the first time to study sponge-associated microbial chitinase.     

Mechanisms of biocontrol of soil-borne plant pathogens by Rhizobacteria

Bacterial antagonism, responsible for biological control, may operate by antiobiosis, competition or parasitism. Parasitism relies on lytic enzymes for the degradation of cell walls of pathogenic fungi. Serratia marcescens was found to be an efficient biocontrol agent of Sclerotium rolfsii and Rhizoctonia solani under greenhouse conditions. Populations of 10⁵ or 10⁶ colony forming units g^-1 soil were the most effective. Drench and drip application of S. marcescens suspension were more effective in controlling S. rolfsii than spraying, mixing in soil or seed coating. The highest population density of the bacteria in the rhizosphere was found on the proximal portion of the root, decreasing significantly until the tips, where it increased again. The isolated Serratia, found to possess chitinolytic activity, was able to release N-acetyl D-glucosamine from cell walls of S. rolfsii. The gene coding for chitinase was cloned into Escherichia coli and the enzyme was uniquely excreted from the bacterium into its growth medium. When S. rolfsii was sprayed by partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was effective in reducing disease incidence caused by S. rolfsii in beans and R. solani in cotton, under greenhouse conditions. A similar effect was obtained when a viable E. coli cell, containing the plasmid with the chitinase gene (pLCHIA), was applied. It appears that genetic engineering of the lytic enzymes, such as chitinase which play an important role in plant disease control, may improve the efficacy of biocontrol agents.     

Polyclonal antibody against <Emphasis Type="Italic">Manduca sexta</Emphasis> chitinase and detection of chitinase expressed in transgenic cotton

The chitinase gene of Manduca sexta was cloned into the expression vector, pET-28a, and expressed in Escherichia coli BL21 (DE3) host cells. The protein product was expressed in inclusion bodies. After denaturation and renaturation procedures using a Ni²⁺-NTA affinity chromatography column, soluble chitinase was obtained. The authenticity of the renatured protein was confirmed by Western blotting. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed chitinase and was used to quantify its presence in transgenic cotton being developed to resist attack by various insects.Revisions requested 24 September 2004; Revisions received 18 November 2004     

Novel insect resistance in Brassica napus developed by transformation of chitinase and scorpion toxin genes

Transgenic plants with introduced pest-resistant gene offer an efficient alternative insect control. The novel insect-resistant gene combination, chitinase(chi) and BmkIT(Bmk), containing an insect-specific chitinase gene and a scorpion insect toxin gene was introduced into Brassica napus cultivar via Agrobacterium-mediated transformation. Fifty-seven regenerated plantlets with kanamycin-resistance were obtained. Transgenic plants were verified by Southern blot analysis. Enzyme-linked immunosorbent assay (ELISA) and bioassay of artificial inoculation with diamondback moth (Plutella maculipenis) (DBM) larvae indicated that some of the transgenic plants were high-level expression for both chitinase and scorpion toxin proteins and performed high resistance against the tested pest infestation. The genetic analysis of T₁ progeny confirmed that the inheritance of introduced genes followed the Mendelian rules.     

Elicitor-specific induction of one member of the chitinase gene family in Arachis hypogaea

Summary Chitinases are believed to play an important role in plant defence against bacterial and fungal attack. In peanut (Arachis hypogaea) chitinase genes form a small multigene family. Four chitinase cDNAs (chit 1–4) were isolated from cultured peanut cells. Expression of individual chit genes was assayed by the polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphisms (RFLP). UV irradiation, dilution of cell cultures and treatment with Phytophthora megasperma (Pmg) elicitor or yeast extract were used to induce expression of chit genes. The chit 3 gene is constitutively expressed at a low level in untreated as well as in treated cultures; the expression of chit 4 gene is induced by each of the stimuli tested, whereas the chit 1 gene is activated by cell culture dilution and by yeast extract treatment. The chit 2 gene is strongly activated by treatment with cell wall components from the fungus Phytophthora megasperma but not by the other stimuli. These results indicate that chit 2 gene expression may be controlled by pathogen-specific regulatory elements.     

Sequence variation, differential expression and chromosomal location of rice chitinase genes

Rice chitinases are encoded by a small multigene family. To clarify the overall organization of rice chitinase genes, we have isolated and characterized the genes Cht-1, Cht-2 and Cht-3. Although all the three genes encode class I chitinase, the nucleotide sequences of the coding regions of Cht-1 and Cht-3 are very similar (90%), while that of Cht-2 is clearly more divergent (78%). Only Cht-2 has a 130 by intron and encodes a C-terminal peptide sequence similar to that known to function as a vacuolar targeting signal. In 5 flanking regions of Cht-1 and Cht-3, but not of Cht-2, conserved sequences (GGCCGGCYGCCCYAG) were found. Related sequences were found also in the 5 flanking regions of another chitinase gene and a -glucanase gene which has also been reported to be stress-induced in rice. RNA blot hybridization analysis demonstrated that the stress-induced expression patterns of the Cht-1 and Cht-3 genes are similar, but quite different from that of Cht-2. However, all three genes are active in unstressed roots. By restriction fragment length polymorphism (RFLP) linkage analysis, Cht-1 and Cht-3 were mapped onto chromosome 6 and shown to be closely linked (0.8 cM). Cht-2 was mapped onto chromosome 5. All these features suggest that the expression patterns of rice class I chitinase genes may be correlated with their levels of sequence divergence and their chromosomal location.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Introduction and constitutive expression of a tobacco hornworm (<Emphasis Type="Italic">Manduca sexta</Emphasis>) chitinase gene in soybean

Summary Embryogenic soybean [Glycine max (L.) Merrill] cultures were transformed with a Manduca sexta chitinase (msc) gene using microprojectile bombardment. A 1.7 kb DNA fragment encoding a tobacco hornworm chitinase was cloned into the rice transformation vector pGL2, under the control of the maize ubiquitin promoter and linked to the hpt gene as a selectable marker. After bombardment, hygromycin-resistant tissues were isolated and cultured to give rise to clones of transgenic material. Four hygromycin-resistant clones were converted into plants. Two clones were positive for the msc gene via polymerase chain reaction (PCR) and Southern blot analysis. The integration inheritance, and expression of transgenes were confirmed by molecular analysis of transgenic soybean plants. Progeny analysis showed that the introduced genes were inherited and segregated in a 3:1 Mendelian fashion. DNA blot experiments and progeny inheritance analysis indicated that the plants contained several copies of the msc gene and that the insertion occurred at a single locus. Northern blotting analysis confirmed the expression of the transgenes. Western blot analysis of transgenic plants and their progeny revealed the presence of a protein with a molecular weight of 48kDa that reacted with the Manduca sexta antibody. Progeny from the chitinase-positive plants were tested for their resistance to the soybean cyst nematode. Plants expressing the insect chitinase did not manifest enhanced resistance to the soybean cyst nematode.     

Functional expression in Beauveria bassiana of a chitinase gene from Ophiocordyceps unilateralis,an ant-pathogenic fungus

A gene encoding chitinase was cloned from Ophiocordyceps unilateralis, a Formamidae-specific fungus, collected from Sirindhorn Peat Swamp Forest, Thailand. The O. unilateralis chitinase (OuChi) full-length gene (1311 bp) encodes 436 amino acids with the first 20 amino acids as a putative signal peptide. The gene showed highest identity (78%) to Isaria farinose endochitinase. To investigate if cross-species chitinase expression also enhances fungal toxicity, the mature OuChi gene was subcloned into an Agrobacterium binary vector pPZP-bar and then transformed into Beauveria bassiana strain BCC2659. Chitinase activity was detected using 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside. The fungal transformant expressing O. unilateralis chitinase showed higher toxicity against Spodoptera exigua. These results support the hypothesis that chitinolytic enzymes are one of several ‘virulence’ factors produced by entomopathogenic fungi during host encounter.     

Directed evolution for increased chitinase activity

Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.