Molecular cloning and functional expression of chitinase-encoding cDNA from the cabbage moth, Mamestra brassicae

Chitinase is a rate-limiting and endo-splitting enzyme involved in the bio-degradation of chitin, an important component of the cuticular exoskeleton and peritrophic matrix in insects. We isolated a cDNA-encoding chitinase from the last larval integument of the cabbage moth, Mamestra brassicae (Lepidoptera; Noctuidae), cloned the ORF cDNA into E. coli to confirm its functionality, and analyzed the deduced amino acid sequence in comparison with previously described lepidopteran chitinases. M. brassicae chitinase expressed in the transformed E. coli cells with the chitinase-encoding cDNA enhanced cell proliferation to about 1.6 times of the untransformed wild type strain in a colloidal chitin-including medium with only a very limited amount of other nutrients. Compared with the wild type strain, the intracellular levels of chitin degradation derivatives, glucosamine and N-acetylglucosamine were about 7.2 and 2.3 times higher, respectively, while the extracellular chitinase activity was about 2.2 times higher in the transformed strain. The ORF of M. brassicae chitinaseencoding cDNA consisted of 1686 nucleotides (562 amino acid residues) except for the stop codon, and its deduced amino acid composition revealed a calculated molecular weight of 62.7 and theoretical pI of 5.3. The ORF was composed of N-terminal leading signal peptide (AA 1-20), catalytic domain (AA 21-392), linker region (AA 393-498), and C-terminal chitin-binding domain (AA 499-562) showing its characteristic structure as a molting fluid chitinase. In phylogenetic analysis, the enzymes from 6 noctuid species were grouped together, separately from a group of 3 bombycid and 1 tortricid enzymes, corresponding to their taxonomic relationships at both the family and genus levels.     

Expression and characterization of the chitinases from Serratia marcescens GEI strain for the control of Varroa destructor, a honey bee parasite

Serratia marcescens GEI strain was isolated from the gut of the workers of Chinese honey bee Apis cerana and evaluated in the laboratory for the control of Varroa destructor, a parasite of western honey bee A. mellifera. The supernatant and the collected proteins by ammonium sulfate from the bacterial cultures showed a strong miticidal effect on the female mites, with 100% mite mortality in 5 days. Heat (100 °C for 10 min) and proteinase K treatment of the collected proteins destroyed the miticidal activity. The improved miticial activity of this bacterial strain on chitin medium indicated the involvement of chitinases. The expressed chitinases ChiA, ChiB and ChiC1 from S. marcescens GEI by recombinant Escherichia coli showed pathogenicity against the mites in the laboratory. These chitinases were active in a broad pH range (5-9) and the optimum temperatures were between 60 and 75 °C. Synergistic effects of ChiA and ChiB on the miticidal activity against V. destructor were observed. The workers of both honey bee species were not sensitive to the spraying and feeding chitinases. These results provided alternative control strategies for Varroa mites, by formulating chitinase agents and by constructing transgenetic honey bees.     

Cloning and Expression of a Chitinase Gene from Sanguibacter sp. C4

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank dadabase. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.     

Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): cloning and effects of eyestalk ablation on gene expression

Six cDNAs encoding chitinase proteins in Pandalopsis japonica were isolated by using polymerase chain reaction (PCR) cloning methods and bioinformatic analysis of expressed sequence tags (ESTs). The cDNAs, designated Pj-Cht1, 2, 3A, 3B, 3C, and 4, encoded proteins ranging from 388 to 607 amino acid residues in length (43.61-67.62 kDa) and displayed a common structural organization: an N-terminal catalytic domain, a Thr/Pro-rich linker region, and either 0 (Pj-Cht2, 3A), 1 (Pj-Cht1, 3B, and 3C), or 2 (Pj-Cht4) C-terminal chitin-binding domain(s) (CBD). Pj-Cht1 and 2 lacked the 5′ end of the open reading frame (ORF); the other Pj-Chts contained the complete ORF. All known decapod crustacean chitinases were segregated into at least four groups based on phylogenetic analysis and domain organization. Group 1 chitinases, represented by Pj-Cht1, were most closely related to insect group I chitinases and may function in the digestion of the peritrophic membrane. Group 2 chitinases including Pj-Cht2 show different domain organizations and pI value from other chitinases and appear to function in degradation of the old exoskeleton during the premolt period. Group 3 chitinases, represented by Pj-Cht3A, 3B, and 3C, may function in digestion of chitin-containing food and defense against pathogens. Group 4 chitinases, represented by Pj-Cht4, have two CBDs and their functions are unknown. Five Pj-Chts (Pj-Cht1, 3A, 3B, 3C, and 4) are expressed in the hepatopancreas and intestine, whereas Pj-Cht2 is expressed in epidermis and SG/XO complex suggesting crustacean chitinases can be classified into two groups (hepatopancreatic and epidermal) based on the expression profile. Eyestalk ablation (ESA) down-regulated the hepatopancreatic chitinase expression (Pj-Cht1, 3A, and 3C); Pj-Cht3B expression was not significantly affected by ESA. By contrast, mRNA levels of Pj-Cht2 were significantly upregulated in 7 days post-ESA. Pj-Cht4 mRNA levels were too low for measurement with quantitative polymerase chain reaction. ESA had no significant effect on chitinase expression in the intestine. These data indicate that Pj-Cht1, 3A, 3B, 3C, and 4 are hepatopancreatic chitinases that may function in the digestion of ingested chitin and the modification of peritrophic membrane in the intestine. By contrast, epidermal chitinase, Pj-Cht2 may play a role in chitin metabolism during molt cycle as shown in other crustacean group 2 chitinases.     

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 expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.     

A stress responsive alternative splicing mechanism in Citrus clementina leaves

Chitinases are often considered pathogenesis-related proteins since their activity can be induced by viral infections, fungal and bacterial cell wall components, and also by more general sources of stress such as wounding, salicylic acid, ethylene, auxins and cytokinins. In the present study, comparative proteomic analysis showed the defense-related acidic chitinase II to be specifically induced in Citrus clementina leaves infested by the two-spotted spider mite Tetranychus urticae or treated with MeJA. In parallel, changes in the mRNA profiles of two partially homologous chitinase forms were shown by RT-PCR. In particular, the appearance of an additional cDNA chitinase fragment in T. urticae-infested and MeJA-treated leaves was observed. This finding may indicate a specific regulatory mechanism of chitinase expression. We report evidence for alternative splicing in T. urticae-infested C. clementina, where a premature stop codon after the first 135 amino acids was introduced. We observed inducible chitinase activity after MeJA treatment, indicative of a rapid plant response to infestation. This work provides the first evidence of chitinase alternative splicing in C. clementina. In addition, the presence of the dual-band pattern for chitinase cDNA by RT-PCR may represent a suitable predictive marker for early diagnosis of plant biotic stress.     

Molecular Docking and Site-directed Mutagenesis of a Bacillus thuringiensis Chitinase to Improve Chitinolytic,Synergistic Lepidopteran-larvicidal and Nematicidal Activities

Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi9602_35-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi9602_35-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed.     

Differential induction of chitinase in Piper colubrinum in response to inoculation with Phytophthora capsici,the cause of foot rot in black pepper

Plant chitinases have been of particular interest since they are known to be induced upon pathogen invasion. Inoculation of Piper colubrinum leaves with the foot rot fungus, Phytophthora capsici leads to increase in chitinase activity. A marked increase in chitinase activity in the inoculated leaves was observed, with the maximum activity after 60 h of inoculation and gradually decreased thereafter. Older leaves showed more chitinase activity than young leaves. The level of chitinase in black pepper (Piper nigrum L.) upon inoculation was found to be substantially high when compared to P. colubrinum. RT–PCR using chitinase specific primers revealed differential accumulation of mRNA in P. colubrinum leaves inoculated with P. capsici. However, hyphal extension assays revealed no obvious differences in the ability of the protein extracts to inhibit growth of P. capsici in vitro.     

Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles

A serine protease and an enzyme preparation consisting of six chitinases, previously semi-purified from a liquid culture of Paecilomyces lilacinus strain 251, were applied to Meloidogyne javanica eggs to study the effect of the enzymes on eggshell structures. Transmission electron microscopic studies revealed that the protease and chitinases drastically altered the eggshell structures when applied individually or in combination. In the protease-treated eggs, the lipid layer disappeared and the chitin layer was thinner than in the control. The eggs treated with chitinases displayed large vacuoles in the chitin layer, and the vitelline layer was split and had lost its integrity. The major changes in the eggshell structures occurred by the combined effect of P. lilacinus protease and chitinases. The lipid layer was destroyed; the chitin layer hydrolyzed and the vitelline layer had lost integrity. The effect of P. lilacinus protease and chitinase enzymes on the hatching of M. javanica juveniles was also compared with a commercially available bacterial chitinase. The P. lilacinus protease and chitinase enzymes, either individually or in combination, reduced hatching of M. javanica juveniles whereas a commercial bacterial chitinase had an enhancing effect. Some juveniles hatched when the eggs were exposed to a fungal protease and chitinase mixture. We also established that P. lilacinus chitinases retained their activity in the presence of endogenous protease activity.     

Simultaneous cloning and expression of two cellulase genes from Bacillus subtilis newly isolated from Golden Takin (Budorcas taxicolor Bedfordi)

A bacterial strain with high cellulase activity was isolated of feces sample of Golden Takin (Budorcas taxicolor Bedfordi). The bacterium was classified and designated Bacillus subtilis LN by morphological and 16SrDNA gene sequence analysis. Two putative cellulase genes, CelL15 and CelL73, were simultaneously cloned from the isolated strain by PCR. The putative gene CelL15 consisted of an open reading frame (ORF) of 1470 nucleotides and encoded a protein of 490 amino acids with a molecular weight of 54 kDa. The CelL73 gene consisted of an open reading frame (ORF) of 741 nucleotides and encoded a protein of 247 amino acids with a molecular weight of 27 kDa. Both genes were purified and cloned into pET-28a for expression in Escherichia coli BL21 (DE3). The ability of E. coli to degrade cellulose was enhanced when the two recombinants were cultured together.     

The first chordate big defensin: identification, expression and bioactivity

Defensins are broadly present in plants, invertebrates and vertebrates, but little information is available about it in amphioxus, a protochordate holding a key phylogenetic position. In this study, a big defensin cDNA was identified from the amphioxus Branchiostoma japonicum (termed Bjbd). The cDNA contained an open reading frame (ORF) of 354 bp encoding a 117 amino acid protein, which had an N-terminal signal sequence followed by a propeptide and the mature big defensin. The mature peptide had the hydrophobic region GAAAVT(A)AA at N-terminus and the consensus pattern C-X6-C-X3-C-X13(14)-C-X4-C-C at C-terminus as well as four α-helices, four β-sheets, and three disulfide bridges (C1-C5, C2-C4 and C3-C6) in the predicted tertiary structure. This is the first big defensin gene ever identified in the phylum Chordata. Quantitative real-time PCR analysis revealed that Bjbd was constitutively expressed in most of the tissues examined, and its expression was remarkably up-regulated following the challenge with LPS, LTA, Aeromonas hydrophila and Staphylococcus aureus. Moreover, the recombinant BjBD was shown to be able to inhibit the growth of S. aureus, Escherichia coli and A. hydrophila. Taken together, these suggest that BjBD is a molecule involved in the removal of invading pathogens.     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

Electroantennogram (EAG) responses of Microplitis croceipes and Cotesia marginiventris and their lepidopteran hosts to a wide array of odor stimuli: Correlation between EAG response and degree of host specificity?

In order to test whether the electroantennogram (EAG) response spectrum of an insect correlates to its degree of host specificity, we recorded EAG responses of two parasitoid species with different degrees of host specificity, Microplitis croceipes (specialist) and Cotesia marginiventris (generalist), to a wide array of odor stimuli including compounds representing green leaf volatiles (GLVs), herbivore-induced plant volatiles (HIPV), ecologically irrelevant (not used by the parasitoid species and their hosts for host location) plant volatiles, and host-specific odor stimuli (host sex pheromones, and extracts of host caterpillar body and frass). We also tested the EAG responses of female moths of the caterpillar hosts of the parasitoids, Heliothis virescens and Spodoptera exigua, to some of the odor stimuli. We hypothesized that the specialist parasitoid will have a narrower EAG response spectrum than the generalist, and that the two lepidopteran species, which are similar in their host plant use, will show similar EAG response spectra to plant volatiles. As predicted, the specialist parasitoid showed greater EAG responses than the generalist to host-specific odor and one HIPV (cis-3-hexenyl butyrate), whereas the generalist showed relatively greater EAG responses to the GLVs and unrelated plant volatiles. We detected no differences in the EAG responses of H. virescens and S. exigua to any of the tested odor.     

Screening-based discovery of Aspergillus fumigatus plant-type chitinase inhibitors

A limited therapeutic arsenal against increasing clinical disease due to Aspergillus spp. necessitates urgent characterisation of new antifungal targets. Here we describe the discovery of novel, low micromolar chemical inhibitors of Aspergillus fumigatus family 18 plant-type chitinase A1 (AfChiA1) by high-throughput screening (HTS). Analysis of the binding mode by X-ray crystallography confirmed competitive inhibition and kinetic studies revealed two compounds with selectivity towards fungal plant-type chitinases. These inhibitors provide new chemical tools to probe the effects of chitinase inhibition on A. fumigatus growth and virulence, presenting attractive starting points for the development of further potent drug-like molecules.     

A simple bacterial transformation method using magnesium- and calcium-aminoclays

An efficient and user-friendly bacterial transformation method by simple spreading cells with aminoclays was demonstrated. Compared to the reported transformation approaches using DNA adsorption or wrapping onto (in)organic fibers, the spontaneously generated clay-coated DNA suprastructures by mixing DNA with aminoclay resulted in transformants in both Gram-negative (Escherichia coli) and Gram-positive cells (Streptococcus mutans). Notably, the wild type S. mutans showed comparable transformation efficiency to that of the E. coli host for recombinant DNA cloning. This is a potentially promising result because other trials such as heat-shock, electroporation, and treatment with sepiolite for introducing DNA into the wild type S. mutans failed. Under defined conditions, the transformation efficiency of E. coli XL1-Blue and S. mutans exhibited ~ 2 × 10⁵ and ~ 6 × 10³ CFU/μg of plasmid DNA using magnesium-aminoclay. In contrast, transformation efficiency was higher in S. mutans than that in E. coli XL1-Blue for calcium-aminoclay. It was also confirmed that each plasmid transformed into E. coli and S. mutans was stably maintained and that they expressed the inserted gene encoding the green fluorescent protein during prolonged growth of up to 80 generations.     

Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera)

We developed a protocol for obtaining high yields (10-15 mg per 1100 ml of culture supernatant) of highly purified (up to 95%) Vip3Aa protein from HD-1 cultures. The protocol is based on acetone precipitation of supernatant protein, followed by HPLC fractionation (DEAE-5PW column) and several concentration steps. Our protocol resulted in higher yields and purity of Vip3Aa than a previously published method [Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a 353 novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of 354 activities against lepidopteran insects. Proc. Nat. Acad. Sci. USA 93, 5389-5394.]. This was achieved by using acetone rather than ammonium sulfate for precipitation of proteins from culture supernatants, and a shallow rather than a steep NaCl gradient for elution of the toxin, and by conducting all the purification steps at low temperature to prevent toxin degradation. In bioassays of the purified protein, Choristoneura fumiferana and Lymantria dispar larvae were less susceptible than Spodopteraexigua (10- and ∼100-fold, respectively). A B. thuringiensis var. kurstaki strain HD-1 from which the vip3Aa gene had been deleted (EG12414) showed reduced toxicity to S. exigua relative to the unmodified parental strain (EG2001), but not to L. dispar or C. fumiferana. We interpret these results as indicating that the Vip3Aa toxin does not contribute measurably to pathogenicity of HD-1 in these species.     

Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae

Enterobactercloacae was isolated from the gut of the wood feeding termite, Heterotermesindicola, and a 2.25-kb fragment conferring cellulase activity was cloned in Escherichiacoli. The cloned fragment contained a 1083-bp ORF which could encode a protein belonging to glycosyl hydrolase family 8. The cellulase gene was introduced into Zymomonasmobilis strain Microbial Type Culture Collection centre (MTCC) on a plasmid and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. Using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (V/V) ethanol respectively, which was threefold higher than the amount obtained with the original E.cloacae isolate. The recombinant Z. mobilis strain could be improved further by simultaneous expression of cellulase cocktails before utilizing it for industrial level ethanol production.