Frontal cortex chitinase and pentraxin neuroinflammatory alterations during the progression of Alzheimer's disease.

BACKGROUND: Chitinase 3-like 1 (CHI3L1), chitinase 3-like 2 (CHI3L2), and neuronal pentraxin II (NPTX2) are inflammatory biomarkers of Alzheimer's disease (AD). Although studies have demonstrated that cerebrospinal fluid levels of these proteins are changed in AD, no studies have undertaken a detailed examination of alterations in protein levels, cellular expression, and interaction with amyloid in the brain during the progression of AD. METHODS: The study evaluated levels of both CHI3L1 and CHI3L2, NPTX2, ionized calcium-binding adapter molecule 1 (Iba1), complement component 1q (C1q), glial fibrillary acidic protein (GFAP), and CD44, in the frontal cortex of people who died with an antemortem clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), mild/moderate AD (mAD), and severe AD (sAD) using immunoblot and immunohistochemical techniques. RESULTS: CHI3L1-immunoreactive (-ir) astrocyte numbers were increased in the frontal cortex and white matter in sAD compared to NCI. On the other hand, increases in GFAP and Iba1-ir cell numbers were observed in MCI compared to NCI but only in white matter. Western blot analyses revealed significantly lower frontal cortex CHI3L2 levels, whereas CD44 levels were increased in sAD. No significant differences for CHI3L1, GFAP, C1q, and NPTX2 protein levels were detected between clinical groups. Strong significant correlations were found between frontal cortex CHI3L1 and Iba1-ir cell numbers in white matter and CHI3L1 and C1q protein levels in the early stages of the disease. C1q and Iba1, CD44 with CHI3L2, and GFAP protein levels were associated during disease progression. CHI3L1 and Iba1 cell numbers in white matter showed a significant associations with episodic memory and perceptual speed. CONCLUSIONS: White matter CHI3L1 inflammatory response is associated with cognitive impairment early in the onset of AD.

Chitinases and thaumatin-like proteins in Sauvignon Blanc and Chardonnay musts during alcoholic fermentation.

Protein precipitation, also referred to as protein instability, may lead to haziness in bottled wines and result in significant commercial losses. To avoid problems of this nature, fining finished wines with clay (bentonite) is the most commonly applied methodology. However, bentonite fining reduces yield and may affect wine quality. Protein haze has been primarily linked to grape pathogenesis-related proteins, in particular chitinases and thaumatin-like proteins. To better understand the persistence of these proteins during fermentation, reverse phase chromatography was used to monitor the evolution of total grape proteins as well as of chitinases and thaumatin-like proteins during alcoholic fermentation. The data confirm a previously reported significant decrease in total protein content during fermentation. This reduction in total protein levels was observed throughout fermentation, and was affected by factors such as fermentation temperature, yeast strain or grape cultivar. However, significant changes in the concentration of free chitinases were observed in a yeast strain-dependent manner. The data thus confirm the correlation between the levels of yeast cell wall chitin and changes in chitinase concentration, and suggest that it is primarily the amount of lateral chitin, and not the chitin in bud scars, that is responsible for this activity.

Type IV chitinase quantification in four different grape cultivars (Vitis vinifera) in northeast of Iran by an indirect sandwich enzyme-linked immunosorbent assay.

The incidence of grape (Vitis vinifera) allergy in the northeast of Iran is second to melon allergy. Type IV chitinase is one of the major grape allergens. The current study investigates the level of type IV chitinase in four grape variants for the first time in Khorasan Razavi Province using a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA). This assay was developed using a polyclonal antibody as a capture antibody and monoclonal antibody as a secondary one. Finally, the amount of type IV chitinase was measured by the validated ELISA test. The sensitivity of the developed sandwich ELISA is 16 ± 0.05 ng/ml, and its mean coefficients of intraday and interday variations are <5% and <15%, respectively. The recovery of the designed ELISA is 64 ± 0.9 %. The assessments showed that the highest level of type IV chitinase was 39.7 ± 2.3 µg/g in Peykani grape, whereas in the Sultana cultivar, it was 1.76 ± 0.1 µg/g. According to the data, the level of type IV chitinase is variable in different cultivars, and hence, it will be helpful for clinicians to recommend a less allergenic variety to the patient.

Genome Analysis of Fimbriiglobus ruber SP5T, a Planctomycete with Confirmed Chitinolytic Capability.

Members of the bacterial order Planctomycetales have often been observed in associations with Crustacea. The ability to degrade chitin, however, has never been reported for any of the cultured planctomycetes although utilization of N-acetylglucosamine (GlcNAc) as a sole carbon and nitrogen source is well recognized for these bacteria. Here, we demonstrate the chitinolytic capability of a member of the family Gemmataceae, Fimbriiglobus ruber SP5T, which was isolated from a peat bog. As revealed by metatranscriptomic analysis of chitin-amended peat, the pool of 16S rRNA reads from F. ruber increased in response to chitin availability. Strain SP5T displayed only weak growth on amorphous chitin as a sole source of carbon but grew well with chitin as a source of nitrogen. The genome of F. ruber SP5T is 12.364 Mb in size and is the largest among all currently determined planctomycete genomes. It encodes several enzymes putatively involved in chitin degradation, including two chitinases affiliated with the glycoside hydrolase (GH) family GH18, GH20 family ß-N-acetylglucosaminidase, and the complete set of enzymes required for utilization of GlcNAc. The gene encoding one of the predicted chitinases was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The genome also contains genes required for the assembly of type IV pili, which may be used to adhere to chitin and possibly other biopolymers. The ability to use chitin as a source of nitrogen is of special importance for planctomycetes that inhabit N-depleted ombrotrophic wetlands.IMPORTANCE Planctomycetes represent an important part of the microbial community in Sphagnum-dominated peatlands, but their potential functions in these ecosystems remain poorly understood. This study reports the presence of chitinolytic potential in one of the recently described peat-inhabiting members of the family Gemmataceae, Fimbriiglobus ruber SP5T This planctomycete uses chitin, a major constituent of fungal cell walls and exoskeletons of peat-inhabiting arthropods, as a source of nitrogen in N-depleted ombrotrophic Sphagnum-dominated peatlands. This study reports the chitin-degrading capability of representatives of the order Planctomycetales.

Analysis of variation in virulence of Beauveria bassiana against insect pests of pigeonpea using qPCR.

Beauveria bassiana is a broad spectrum microbial bioagent used for the control of agriculturally important insect pests. However, in our experiments, two virulent isolates of B. bassiana (B2 and B10) showed specific preference toward Maruca vitrata and Helicoverpa armigera of pigeonpea. To better understand this feature, we developed a qPCR assay to quantify the chitinase (virulence factor) of B. bassiana during the infection process. Isolates of B. bassiana were grown on insect cuticle amended medium and minimal medium (without insect cuticle) to assess the induction of chitinase. Our results revealed a positive correlation between expression of chitinase by B. bassiana and the substrates (with or without cuticles of M. vitrata and H. armigera) used. This study showcases the methodology to quantify the chitinase and analysis of variation in virulence of B. bassiana (B2 and B10) against M. vitrata and H. armigera.

Structural basis for carbohydrate binding properties of a plant chitinase-like agglutinin with conserved catalytic machinery.

A new chitinase-like agglutinin, RobpsCRA, related to family GH18 chitinases, has previously been identified in black locust (Robinia pseudoacacia) bark. The crystal structure of RobpsCRA at 1.85Å resolution reveals unusual molecular determinants responsible for the lack of its ancestral chitinase activity. Unlike other chitinase-like proteins, which lack chitinase catalytic residues, RobpsCRA has conserved its catalytic machinery. However, concerted rearrangements of loop regions coupled to non-conservative substitutions of aromatic residues central to the chitin-binding groove explain the lack of hydrolytic activity against chitin and the switch toward recognition of high-mannose type N-glycans. Identification of close homologs in flowering plants with conservation of sequence motifs associated to the structural adaptations seen in RobpsCRA defines an emerging class of agglutinins, as emphasized by a phylogenetic analysis, that are likely to share a similar carbohydrate binding specificity for high-mannose type N-glycans. This study illustrates the recent evolution and molecular adaptation of a versatile TIM-barrel scaffold within the ancestral GH18 family.

Interindividual variation, correlations, and sex-related differences in the salivary biochemistry of young healthy adults.

A cross-sectional observational study was conducted to evaluate interindividual biochemical variation in unstimulated whole saliva in a population of 268 systemically healthy young students, 18-30 yr of age, with no apparent caries lesions or periodontal disease. Salivary flow rate, protein content, pH, buffering capacity, mucins MUC5B and MUC7, albumin, secretory IgA, cystatin S, lactoferrin, chitinase, amylase, lysozyme, and proteases were measured using ELISAs and enzymatic activity assays. Significant differences were found between male and female subjects. Salivary pH, buffering capacity, protein content, MUC5B, secretory IgA, and chitinase activity were all lower in female subjects compared with male subjects, whereas MUC7 and lysozyme activity were higher in female subjects. There was no significant difference between sexes in salivary flow rate, albumin, cystatin S, amylase, and protease activity. Principal component analysis (PCA) and spectral clustering (SC) were used to assess intervariable relationships within the data set and to identify subgroups. Spectral clustering identified two clusters of participants, which were subsequently described. This study provides a comprehensive overview of the distribution and inter-relations of a set of important salivary biochemical variables in a systemically healthy young adult population, free of apparent caries lesions and periodontal disease. It highlights significant gender differences in salivary biochemistry.

Isolation, characterization, kinetics, and enzymatic and nonenzymatic microbicidal activities of a novel c-type lysozyme from plasma of Schistocerca gregaria (Orthoptera: Acrididae).

A protein, designated as Sgl, showing a muramidase lytic activity to the cell wall of the Gram-positive bacterium Micrococcus lysodeikticus was isolated for the first time from plasma of Escherichia coli-immunized fifth instar Schistocerca gregaria. The isolated Sgl was detected as a single protein band, on both native- and SDS-PAGE, has a molecular weight of ∼15.7 kDa and an isoelectric point (pI) of ca 9.3 and its antiserum has specifically recognized its isolated form. Fifty-nine percentage of Sgl lytic activity was recovered in the isolated fractions and yielded ca 126-fold increase in specific activity than that of the crude. The partial N-terminal amino acid sequence of the Sgl has 55 and 40% maximum identity with Bombyx mori and Gallus gallus c-type lysozymes, respectively. The antibacterial activity against the Gram-positive and the Gram-negative bacteria were comparatively stronger than that of the hen egg white lysozyme (HEWL). The detected Sgl poration to the inner membrane that reach a maximum ability after 3 h was suggested to operate as a nonenzymatic mechanism for Gram-negative bacterial cell lysis, as tested in a permease-deficient E. coli, ML-35 strain. Sgl showed a maximal muramidase activity at pH 6.2, 30-50°C, and 0.05 M Ca(2+) or Mg(2+); and has a Km of 0.5 µg/ml and a Vmax of 0.518 with M. lysodeikticus as a substrate. The Sgl displayed a chitinase activity against chitin with a Km of 0.93 mg/ml and a Vmax of 1.63.

Characterization and expression analysis of six chitinase genes from the desert beetle Microdera punctipennis in response to low temperature.

BACKGROUND: Chitinase is responsible for chitin metabolism in a wide range of organisms. However, current knowledge on insect chitinase and their possible functions in relation to low temperature stress is very limited. OBJECTIVE: Six chitinase genes from cold treated desert beetle Microdera punctipennis obtained by RNA-seq technology were characterized, and their expression patterns in different tissues and in response to cold were investigated. METHODS: Multiple sequence alignment was carried out using ClustalW1.81 and Phylogenetic trees were generated by MEGA5. The expression patterns were studied by quantitative real-time PCR. RESULTS: These genes were belong to three different chitinase groups. Almost all of them were highly expressed in midgut, and some are expressed in fat body or hindgut. Subzero-4 degree C had stronger effect than 4 degree C in inducing chitinase expression. CONCLUSION: The tissue specific and cold inducible expressions suggest that the chitinases may have diverse functions and play roles in insect cold adaptation.

Molecular and immunohistochemical characterization of the chitinase gene from Pieris rapae granulovirus.

The chitinase gene of baculoviruses is expressed in the late phase of virus replication in insects and possesses high exo- and endochitinase activity, which can hydrolyze chitin in the body of the insect, thus promoting terminal host liquefaction. Alphabaculovirus viral chitinases (vChitA) have been well analyzed, but information regarding viral chitinases from betabaculoviruses is limited. Whole-genome sequencing of a Korean isolate of Pieris rapae GV (PiraGV-K) predicted a putative chitinase gene corresponding to ORF10. The PiraGV-K chitinase gene had a coding sequence of 1,761 bp, encoding a protein of 586 amino acid (aa) residues, including an 18-aa putative signal peptide. Time course induction pattern observed by SDS-PAGE and subsequent Western blot with anti-PiraGV-K chitinase antibody revealed the cleavage of the signal peptide from the intact chitinase. Edman sequencing analysis was further conducted to confirm the exact nature of the mature chitinase, and the N-terminal amino acid sequence (KPGAP) exactly matched the sequence following the signal peptide sequence. The transcriptomics of PiraGV-K chitinase in infected P. rapae larvae, examined by real-time PCR, revealed a significant 75-fold increase after four days of feeding with PiraGV-K-treated leaves, with a subsequent decline at the later stages of infection. Confocal microscopic analysis showed that PiraGV-K chitinase possibly exists as a secreted protein, with strong chitinase-specific signals in fat body cells and integument at four days postinfection. Furthermore, immunogold labeling and electron microscopy studies localized the PiraGV-K chitinase in the cytoplasm and sparsely within vacuolar structures in the fat body apart from the extensive aggregation in the cuticular lining of the integument.

Modular structure of HEL protein from Arabidopsis reveals new potential functions for PR-4 proteins.

Plants possess an innate immune system enabling them to defend themselves against pathogen attack.The accumulation of newly synthesized pathogenesis related proteins (PRs) is one of the most studied inducible plant defence response. In this paper, we report on the characterization of a class I PR4 vacuolar protein from Arabidopsis, named At HEL. The protein has a modular structure consisting of an N-terminal hevein-like domain(CB-HEL) and a C-terminal domain (CD-HEL) that are posttranslationally processed. Both domains show a strong antifungal activity, but they do not have chitinolitic properties.CD-HEL was found to be endowed with RNase, but not DNase activity. Molecular modeling carried out on both domains revealed that CB-HEL possesses a chitin binding site strictly conserved between hevein-type peptides and that the cavity involved in substrate interaction of CD-HEL do not show any residue substitution with respect to the orthologous wheatwin1 from wheat. Using a fishing for partners approach, CB-HEL was found to interact with a fungal fruiting body lectin. According to literature, we can hypothesize that CB-HEL could cross the pathogen hyphal membrane and that its interaction with a fungal lectin could knock out one of the weapons that the fungus uses.

Characterization of novel Trichoderma spp. isolates as a search for effective biocontrollers of fungal diseases of economically important crops in Argentina.

Monoconidial cultures of 33 isolates of Trichoderma from Buenos Aires Province, Argentina were characterized on the basis of twenty eight morphological, physiological and biochemical features. All of them were screened for proteinase, endochitinase and ß-1,3 glucanase activity. Universally primed PCR (UP-PCR) and inter-simple sequence repeat (ISSR) techniques were used to examine the genetic variability among isolates, which resulted in 127 bands for the total number of isolates. These results were subjected to numerical analysis revealing 20 haplotypes grouped in five clusters. The ability of Trichoderma isolates to antogonize soil-borne fungal plant pathogens using a dual culture assay was done against five fungal species: Alternaria sp., Bipolaris sorokiniana, Fusarium graminearum, F. solani, and Pyricularia oryzae. The highest inhibition values (85% RI) were obtained against B. sorokiniana and P. oryzae. Three isolates of T. harzianum named as FCCT2, FCCT3 and FCCT9 were capable of causing a high growth inhibition on four of the fungal species assayed, which was in agreement with their higher extracellular hydrolytic activity. Our results suggest that these isolates have the potential to be effective agents for biocontrol of cereal and tomato fungal pathogens.

Chemical and Molecular Composition of the Chrysalis Reveals Common Chitin-Rich Structural Framework for Monarchs and Swallowtails.

The metamorphosis of a caterpillar into a butterfly is an awe-inspiring example of how extraordinary functions are made possible through specific chemistry in nature's complex systems. The chrysalis exoskeleton is revealed and shed as a caterpillar transitions to butterfly form. We employed solid-state NMR to evaluate the chemical composition and types of biomolecules in the chrysalides from which Monarch and Swallowtail butterflies emerged. The chrysalis composition was remarkably similar between Monarch and Swallowtail. Chitin is the major polysaccharide component, present together with proteins and catechols or catechol-type linkages in each chrysalis. The high chitin content is comparable to the highest chitin-containing insect exoskeletons. Proteomics analyses indicated the presence of chitinases that could be involved in synthesis and remodeling of the chrysalis as well as cuticular proteins which play a role in the structural integrity of the chrysalis. The nearly identical 13C CPMAS NMR spectra of each chrysalis and similar structural proteins supports the presence of underlying design principles integrating chitin and protein partners to elaborate the chrysalis.

Estimating allergenicity of latex gloves using Hev b 1 and hevamine.

BACKGROUND: Latex allergy continues to be an increasingly serious occupational health problem in Taiwan, where it affects approximately 6.8% to 12% of health care workers. Contrasting with reports from western countries, Hev b 1 and hevamine, and not Hev b 3, 5 or 6.02, are the major latex allergens among health care workers in Taiwan. This study aimed at evaluating the allergenicity of 30 brands of commercially available medical latex gloves in Taiwan in 2007. METHODS: Residual Hev b 1 and hevamine from the gloves were measured by inhibition enzyme-linked immunosorbent assay using polyclonal antibodies against purified recombinant Hev b 1 and hevamine. The results were compared to those achieved with quantification of residual total extractable proteins and skin prick testing. RESULTS: The residual extractable protein levels in 30 medical gloves all conformed to United States Food and Drug Administration regulations. All the gloves except one yielded strong skin prick reactions in latex-allergic individuals. The only brand of gloves that consistently produced no skin prick reactions in latex-allergic individuals contained the lowest residual levels of Hev b 1 (0.60 microg/g) and hevamine (0.07 microg/g). CONCLUSIONS: Our results suggest that the measurement of residual extractable total proteins is not sufficient to assess the allergenicity of latex gloves and that Hev b 1 and hevamine may be used as indicator allergens in areas where they are major latex allergens, such as Taiwan.

Seed biopriming with antagonistic microbes and ascorbic acid induce resistance in tomato against Fusarium wilt.

Seed biopriming is an emerging technique to enhance seed germination under stress conditions. An integrated approach of tomato seed biopriming with ascorbic acid, Trichoderma asperellum BHU P-1 and Ochrobactrum sp. BHU PB-1 was applied to observe the response against wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici (FOL). Tomato seeds bioprimed with the aforementioned application expressed augmented seed germination and activated of defense response. Seed germination was recorded higher (80 %) at low concentration (1 pM) of ascorbic acid as compared to high concentration of 1 mM (41 %). Combination of both ascorbic acid and antagonistic microbe treatments (T5 & T6) significantly reduced disease incidence (up to 28 %) in tomato plants at 10 days. T5 and T6 treated plants exhibited higher accumulation of total phenol content and increased activity of Phenylammonia lyase (PAL), Peroxidase (PO), Chitinase (Chi) and Polyphenol oxidase (PPO) as compared to control (T1) plants. ROS formation in the form of H2O2 was also found to be reduced in combined treatment. Histochemical analysis revealed that phenylpropanoid pathway (lignin deposition) was more activated in combined priming treatment plants as compared to individual treatment upon challenge inoculation with FOL. Transcript expression analysis of defense genes confirmed the up-regulation of PAL (2.1 fold), Chi (0.92 fold), Pathogenesis related proteins (PR) (1.58 fold) and Lipoxygenase (Lox) (0.72 fold) in T6 treatment as compared to T1 treatment plants at 96 h. This study reveals that ascorbic acid treatment with antagonistic microbes through seed priming effectively induced seed germination and elicited defense mechanism to control wilt disease in tomato plants.

Characterisation of Plasmodium invasive organelles; an ookinete microneme proteome.

Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14-fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC-MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion.

The deletion of the AcMNPV ac124 gene resulted in a decrease in chitinase transcription.

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac124 gene has been previously characterized as a viral pathogenicity factor. In this study, an ac124-knockout virus (vAc124KO) was generated to examine the role of the ac124 gene in the context of the AcMNPV genome during infection. Our results showed that the absence of ac124 does not affect the production of budded virus (BV) and occlusion bodies (OBs) in infected Sf9 cells. Western blotting analysis showed that the deletion of ac124 does not affect the temporal expression and the relative levels of GP64, VP39, P6.9, and polyhedrin. qRT-PCR analysis showed that the transcription level of chitinase but not the adjacent cathepsin in vAc124KO infected cells was significantly lower than that of the vAcWT infected cells from 24 to 96 h p.i. Luciferase assays showed that the overexpression of Ac124 could significantly improve the ability of chitinase promoter to initiate reporter genes. Based on the above data, we hypothesize that Ac124 binds to the promoter of chitinase to regulate the expression of chitinase gene.

Floral nectar chitinase is a potential marker for monofloral honey botanical origin authentication: A case study from loquat (Eriobotrya japonica Lindl.).

Honey, as a commercial product, is a target of adulteration through inappropriate production practices and deliberate mislabelling of botanical origin. Floral nectar protein could be a good marker for determining the source flowers of honey, especially monofloral honeys. Here, nectar and monofloral honey from Eriobotrya japonica Lindl. (loquat) were systematically compared, especially regarding proteomic and enzymatic activity. Using two-dimensional electrophoresis and mass spectrometry, only bee-originated proteins were detected in loquat honey. Xylosidase, thaumatin, and two kinds of chitinases were detected in loquat floral nectar, and their activity in loquat nectar and honey were quantified. Following gel electrophoresis, loquat honey had similar chitinase activity profiles to loquat nectar, but both were clearly distinguishable from Camellia sinensis nectar and Brassica napus honey. To our knowledge, this is the first examination of nectar-origin enzyme activity in honey. Zymography of chitinases is a potential marker for determining or authenticating the botanical origin of honeys.

Chitinase is stored and secreted from the inner body of microfilariae and has a role in exsheathment in the parasitic nematode Brugia malayi.

Chitinase expression in microfilariae of the parasitic nematode Brugia malayi (B. malayi, Bm) is coincidental with the onset of their infectivity to mosquitoes. An antibody raised to Onchocerca volvulus (O. volvulus, Ov) infective-stage larval chitinase (Ov-CHI-1) was specifically reactive against B. malayi microfilarial chitinase and was used to study the localization of chitinase in B. malayi during microfilarial development and transmission to the insect vector. Immuno-electron microscopy (IEM) was used to demonstrate that the chitinase was confined to the inner body of the microfilariae and furthermore that chitinase was only present in sheathed microfilarial species, although the inner body is present in all species. Observation using the IEM implicates two distinct routes of chitinase secretion from the inner body, via either the pharyngeal thread, or during transmission of the microfilariae to the vector, contained in vesicle-like structures. Many morphological studies have described the structure of the inner body, but no function has been assigned to it as of yet. Although it has been commented that the cells surrounding the inner body and pharyngeal thread are those destined to become the intestine and pharynx and that the inner body represents a store of material. Our studies suggest that chitinase is one such product stored in the inner body and that it is secreted during the exsheathment of the microfilaria in the mosquito.

Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis.

AIMS: The present study focused on cloning and expression of chiA gene from a highly chitinolytic local isolate of Serratia marcescens in an anti-Coleopteran Bacillus thuringiensis and comparison of the characteristics of the native and recombinant ChiAs. METHODS AND RESULTS: chiA gene from Ser. marcescens was cloned, sequenced and compared with the previously cloned chiA genes. chiA gene was PCR cloned and expressed in anti-Coleopteran B. thuringiensis strain 3023 as verified by Western blot analysis. Specific ChiA activity of the recombinant B. thuringiensis (strain 3023-SCHI) reached its highest level at 21st hour of growth (16.93 U mg(-1)), which was 5.2- and 1.3-fold higher than that of its parental strain and Ser. marcescens, respectively. Temperature and pH effects on native and recombinant ChiAs were next determined. The recombinant plasmid was quite stable over 240 generations. CONCLUSIONS: Serratia marcescens ChiA was heterologously expressed in an anti-Coleopteran B. thuringiensis at levels even higher than that produced by the source organism. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thuringiensis 3023-SCHI co-expressing anti-Coleopteran Cry3Aa protein and Ser. marcescens chitinase offers a viable alternative to the use of chitinolytic microbes/enzymes in combination with entamopathogenic bacteria for an increased potency because of synergistic interaction between them.