Bacterial Chitinases Research Articles

Analysis of a chitinase from EpapGV, a fast killing betabaculovirus

The main function of baculoviral chitinase protein (V-CHIA) is to promote the final liquefaction of infected host larvae, facilitating the dispersion of occlusion bodies (OBs) in the environment. In this study, a v-chiA from Epinotia aporema Granulovirus (EpapGV) was identified and characterized. The 1,713 base pairs long open reading frame encodes a protein of 570 amino acids with a predicted molecular weight of 63 kDa. EpapGV V-CHIA sequence alignment resulted 62 % identical to Pieris rapae GV and Blastp search revealed a high conservation among all baculovirus chitinases. Amino acid sequence analysis indicated that the C-terminal KDEL present in most alphabaculovirus chitinases is absent in EpapGV V-CHIA, as well as in the rest of the betabaculoviruses. Phylogenetic analysis was performed with bacterial, lepidopteran, and baculoviral chitinase sequences available in databases. Using an AcMNPV bacmid (bApGOZA) a recombinant Ac-chiAEpapGV was obtained in order to overexpress EpapGV V-CHIA in cell culture. The presence of chitinase was detected in purified AcMNPV-chiAEpapGV OBs. Peritrophic membranes of Anticarsia gemmatalis larvae fed with recombinant OBs showed an altered structure. The results presented in this study show that EpapGV chitinase overexpression in recombinant baculovirus can cause association of this protein with OBs, and suggest that this could be used to evaluate the protein role in early stages of baculoviral infections.

Peculiarities of the Brevundimonas diminuta Gl7ACA-acylase quaternary structure formation and obtaining stable enzyme analogues

The physicochemical and enzymatic properties of hybrid analogues of the Brevundimonas diminuta Gl7ACA-acylase (BrdGIA), containing the N-terminal chitin-binding domain of the bacterial chitinase (BrdG1A/NmChBD) or the C-terminal oligohistidine sequence (BrdGIA/H), were studied. An enhanced thermostability level of BrdG1A/NmChBD could suggest the stabilizing effect of the chitin-binding domain. An analysis of pH profiles of the enzymatic activity of recombinat BrdGIA analogues did not reveal significant differences: the catalytic activity of both variants changed slightly in the.interval ofpH values from 6.0 to 9.0 but drastically decreased at lower pH values. Both analogues demonstrated similar sensitivity towards denaturing agents: addition of 2.0 M ofguanidine chloride resulted in the complete inactivation of both enzymes. A scheme was developed for obtaining isolated recombinant alpha- and beta-subunits of BrdGLA. In vitro enzyme reconstructions indicated that the alpha-subunit was necessary for the formation of a correct spatial structure of the beta-subunit and for the formation of a functionally active enzyme.

Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics

Plant disease caused by fungal pathogens results in vast crop damage globally. Microbial communities of soil that is suppressive to fungal crop disease provide a source for the identification of novel enzymes functioning as bioshields against plant pathogens. In this study, we targeted chitin-degrading enzymes of the uncultured bacterial community through a functional metagenomics approach, using a fosmid library of a suppressive soil metagenome. We identified a novel bacterial chitinase, Chi18H8, with antifungal activity against several important crop pathogens. Sequence analyses show that the chi18H8 gene encodes a 425-amino acid protein of 46 kDa with an N-terminal signal peptide, a catalytic domain with the conserved active site F175DGIDIDWE183, and a chitinase insertion domain. Chi18H8 was expressed (pGEX-6P-3 vector) in Escherichia coli and purified. Enzyme characterization shows that Chi18H8 has a prevalent chitobiosidase activity with a maximum activity at 35 °C at pH lower than 6, suggesting a role as exochitinase on native chitin. To our knowledge, Chi18H8 is the first chitinase isolated from a metagenome library obtained in pure form and which has the potential to be used as a candidate agent for controlling fungal crop diseases. Furthermore, Chi18H8 may also answer to the demand for novel chitin-degrading enzymes for a broad range of other industrial processes and medical purposes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-013-5287-x) contains supplementary material, which is available to authorized users.

Purification and Characterization of an Antifungal Chitinase from Citrobacter freundii str. nov. haritD11

The purpose of the research was to study the purification and partial characterization of antifungal alkaline chitinase from a newly isolated Citrobacter freundii haritD11. The enzyme was purified in a three-step procedure involving ammonium sulfate precipitation, dialysis, and Sephadex G-100 gel filtration chromatography. The enzyme was shown to have a relative high molecular weight of 64 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was purified 7.3-fold with a yield of 18.8 %. It was most active at 35 °C, pH 8.0, with colloid chitin as substrate and was very stable at alkaline pH contradicting the characteristic that most of the bacterial chitinases are active at acidic pH. Further, the purified chitinase exhibited remarkable antifungal activity against pathogenic fungi Aspergillus flavus MTCC 2798 and Aspergillus niger MTCC 9652 showing diametric inhibition zones of 27 mm and 21 mm, respectively.

Changes induced by Trichoderma harzianum in suppressive compost controlling Fusarium wilt

The addition of species of Trichoderma to compost is a widespread technique used to control different plant diseases. The biological control activity of these species is mainly attributable to a combination of several mechanisms of action, which may affect the microbiota involved in the suppressiveness of compost. This study was therefore performed to determine the effect of inoculation of Trichoderma harzianum (T. harzianum) on compost, focusing on bacterial community structure (16S rRNA) and chitinase gene diversity. In addition, the ability of vineyard pruning waste compost, amended (GCTh) or not (GC) with T. harzianum, to suppress Fusarium wilt was evaluated. The addition of T. harzianum resulted in a high relative abundance of certain chitinolytic bacteria as well as in remarkable protection against Fusarium oxysporum comparable to that induced by compost GC. Moreover, variations in the abiotic characteristics of the media, such as pH, C, N and iron levels, were observed. Despite the lower diversity of chitinolytic bacteria found in GCTh, the high relative abundance of Streptomyces spp. may be involved in the suppressiveness of this growing media. The higher degree of compost suppressiveness achieved after the addition of T. harzianum may be due not only to its biocontrol ability, but also to changes promoted in both abiotic and biotic characteristics of the growing media.

Bacterial chitinases and chitin-binding proteins as virulence factors

Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.

The Listeria monocytogenes ChiA Chitinase Enhances Virulence through Suppression of Host Innate Immunity

ABSTRACTEnvironmental pathogens survive and replicate within the outside environment while maintaining the capacity to infect mammalian hosts. For some microorganisms, mammalian infection may be a relatively rare event. Understanding how environmental pathogens retain their ability to cause disease may provide insight into environmental reservoirs of disease and emerging infections. Listeria monocytogenes survives as a saprophyte in soil but is capable of causing serious invasive disease in susceptible individuals. The bacterium secretes virulence factors that promote cell invasion, bacterial replication, and cell-to-cell spread. Recently, an L. monocytogenes chitinase (ChiA) was shown to enhance bacterial infection in mice. Given that mammals do not synthesize chitin, the function of ChiA within infected animals was not clear. Here we have demonstrated that ChiA enhances L. monocytogenes survival in vivo through the suppression of host innate immunity. L. monocytogenes ΔchiA mutants were fully capable of establishing bacterial replication within target organs during the first 48 h of infection. By 72 to 96 h postinfection, however, numbers of ΔchiA bacteria diminished, indicative of an effective immune response to contain infection. The ΔchiA-associated virulence defect could be complemented in trans by wild-type L. monocytogenes, suggesting that secreted ChiA altered a target that resulted in a more permissive host environment for bacterial replication. ChiA secretion resulted in a dramatic decrease in inducible nitric oxide synthase (iNOS) expression, and ΔchiA mutant virulence was restored in NOS2−/− mice lacking iNOS. This work is the first to demonstrate modulation of a specific host innate immune response by a bacterial chitinase.

Bacterial and fungal chitinase chiJ orthologs evolve under different selective constraints following horizontal gene transfer

BackgroundCertain bacteria from the genus Streptomyces are currently used as biological control agents against plant pathogenic fungi. Hydrolytic enzymes that degrade fungal cell wall components, such as chitinases, are suggested as one possible mechanism in biocontrol interactions. Adaptive evolution of chitinases are previously reported for plant chitinases involved in defence against fungal pathogens, and in fungal chitinases involved in fungal-fungal interactions. In this study we investigated the molecular evolution of chitinase chiJ in the bacterial genus Streptomyces. In addition, as chiJ orthologs are previously reported in certain fungal species as a result from horizontal gene transfer, we conducted a comparative study of differences in evolutionary patterns between bacterial and fungal taxa.FindingsChiJ contained three sites evolving under strong positive selection and four groups of co-evolving sites. Regions of high amino acid diversity were predicted to be surface-exposed and associated with coil regions that connect certain α-helices and β-strands in the family 18 chitinase TIM barrel structure, but not associated with the catalytic cleft. The comparative study with fungal ChiJ orthologs identified three regions that display signs of type 1 functional divergence, where unique adaptations in the bacterial and fungal taxa are driven by positive selection.ConclusionsThe identified surface-exposed regions of chitinase ChiJ where sequence diversification is driven by positive selection may putatively be related to functional divergence between bacterial and fungal orthologs. These results show that ChiJ orthologs have evolved under different selective constraints following the horizontal gene transfer event.

Effect of antifungal genes expressed in transgenic pea (Pisum sativum L.) on root colonization with Glomus intraradices

Pathogenic fungi have always been a major problem in agriculture. One of the effective methods for controlling pathogen fungi to date is the introduction of resistance genes into the genome of crops. It is interesting to find out whether the induced resistance in crops will have a negative effect on non-target organisms such as root colonization with the AM fungi. The objective of the present research was to study the influence of producing antifungal molecules by four transgenic pea (Pisum sativum L.) lines expressing PGIP gene from raspberry, VST-stilbene synthase from vine, a hybrid of PGIP/VST and bacterial Chitinase gene (Chit30) from Streptomyces olivaceoviridis respectively on the colonization potential of Glomus intraradices. Four different experiments were done in greenhouse and climate chamber, colonization was observed in all replications. The following parameters were used for evaluation: frequency of mycorrhization, the intensity of mycorrhization, the average presence of arbuscules within the colonized areas and the presence of arbuscules in the whole root system which showed insignificant difference between transgenic and non-transgenic plants. The root/shoot ratio exhibited different values according to the experiment condition. Compared with negative non-transgenic control all transgenic lines showed the ability to establish symbiosis and the different growth parameters had insignificant effect due to mycorrhization. The results of the present study proved that the introduced pathogen resistance genes did not affect the mycorrhization allocations in pea.

Characterization of a Chitinase (Chit62) from Serratia marcescens B4A and Its Efficacy as a Bioshield Against Plant Fungal Pathogens

Chitinases have been suggested to be involved in pathogen-antagonist interaction during biological control progress of plant pathogenic fungi. Here, a recombinant bacterial chitinase originally from Serratia marcescens B4A was produced, purified, and assayed biochemically to ascertain the activity and determine the kinetics parameters. Active enzyme was used to determine its biocontrol features against fungal phytopathogens. The results demonstrated that the optimum pH and temperature for the enzyme activity were 6.0 and 55 °C, respectively. The K(m) and V(max) values were 3.30 mg ml(-1) and 0.92 units, respectively. The recombinant chitinase was demonstrated to be highly active in controlling fungal pathogens.

FUNCTIONAL ANALYSIS OF TWO CHITINASE GENES DURING THE PUPATION AND ECLOSION STAGES OF THE BEET ARMYWORM Spodoptera exigua BY RNA INTERFERENCE

Insect chitinases are a multigene family that is encoded by a rather large and diverse group of genes. The main function of chitinases is to digest the chitin contained in tissues such as the cuticles and gut lining during molting. In this study, we examined the role of a chitinase (SeChi) and a bacterial type chitinase (SeChi-h) during the pupation and eclosion stages of Spodoptera exigua. First, efficient silencing of the SeChi and SeChi-h genes through specific double-stranded RNA (dsRNA) injection led to a significant reduction in the mRNA levels of SeChi and SeChi-h. Additionally, different phenotypic defects were observed at the pupal and adult stages after injection of the SeChi and SeChi-h dsRNAs. After injecting SeChi dsRNA in the pupal stage, the cuticle of the head split open and the pupal cuticle was visible under the old larval cuticle. However, after injecting the SeChi-h dsRNA, animals died without exhibiting any special phenotypes. At the adult death stage, animals injected with dsSeChi could not shed their pupal shell completely, and their old cuticles remained attached to their head or chest. However, the main lethal phenotype was that insects did not emerge after dsSeChi-h injection. Additionally, the average survival rates of S. exigua were 52.02% and 40.38% at the pupal and adult stages, respectively, after injection with SeChi dsRNA. For the insects injected with SeChi-h dsRNA, the survival rates were 72.38% and 48.52%, respectively. These results suggest that SeChi and SeChi-h may have different biologic functions during the pupal-adult molting.

Role of Chitotriosidase (Chitinase 1) Under Normal and Disease Conditions

Mammalian chitinases belong to the glycosyl hydrolase 18 family based on structural homology and the family includes a large number of bacterial and eukaryotic chitinases. Among the mammalian chitinases, chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase) are capable of hydrolyzing the β-(1, 4)-linkage between the adjacent N-acetyl glucosamine residues of chitin. CHIT1 is one of the most abundantly secreted proteins, being mainly produced by activated macrophages and epithelial cells. CHIT1 plays a pivotal role in the context of infectious disease including malaria and fungi infections as a host defense towards chitin in pathogen's cell structure and as a diagnostic marker of disease. In contrast, CHI1 released by activated Kupffer cells in liver could induce hepatic fibrosis and cirrhosis. Increased serum levels of CHIT1 were observed in patients with many disorders, including Gaucher's disease, bronchial asthma, and atherosclerosis. Therefore, CHIT1 seems to have dual (regulatory and pathogenic) roles depending on the disease and producing cell types during the inflammatory conditions.

Microbial diversity in the sediment of a crab pond in Nanjing, China

Chinese mitten crab (Eriocheir sinensis) is of great commercial significance and is bred extensively in Eastern and Northern China. Aquaculture is a complex ecosystem where microorganisms in the water, sediment and gastrointestinal tracts interact with each other to affect the health of aquatic animals (Cheng, Zhou, Xie, Ge, Zhu & Liu 2010). The microbial communities in crab pond water (Cheng et al. 2010) and in the gut of Chinese mitten crab (Li, Guan, Wei, Liu, Xu, Zhao & Zhang 2006) have been studied. However, there is no report on the microbial community in crab pond sediment. Generally a crab may require 12–16 moults to reach maturity (Zhang, Li & Cui 2001). Crab shell is rich in chitin, and the crab pond represents one of the most chitin-rich environments. Organisms produce chitinase for different physiological purposes. Bacterial chitinases play roles in nutrition and parasitism, and thus form one of the major sources of chitinases (Dahiya, Tewari & Hoondal 2006). Chitinase genes have been identified in a large pool of uncultured chitinolytic microorganisms in marine and soil environments and used as molecular markers (Cottrell, Wood, Yu & Kirchman 2000; Metcalfe, Krsek, Gooday, Prosser & Wellington 2002). Recently, chitinases of various aquatic habitats have been investigated (LeCleir, Buchan & Hollibaugh 2004; Hobel, Marteinsson, Hreggvidsson & Kristjansson 2005; Bhattacharya, Nagpure & Gupta 2007). Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rRNA gene fragments generated by PCR is a reliable, rapid and an easy method to evaluate microbial diversity (Liu, Zhou, Yao, Shi, He, Benjamisen Holvold & Ringo 2008; Zhou, Liu, Shi, He, Yao & Ringo 2009) and is capable to detect the dominant bacterial species in the environments (Muyzer, Waal & Uitterlinden 1993). We used PCR-DGGE and PCR screening technologies to investigate the microbial community in crab pond sediments. The sediment sample was collected from a pond rearing Chinese mitten crab near the city of Nanjing, Jiangsu province, China, in July 2008. The depth of the pond was 1.5 m, with temperature of 30°C, pH 7.5 and the culture area was approximately 100 acres. The crabs were fed the commercial diet (Crude protein 36% and crude lipid 6% without dietary antibiotics) manufactured by Jiangsu Danyang Lekaihuai Feed Co., Ltd (Zhenjiang City, Jiangsu Province, China). The earth pond has been used for the crab culture for about 3 years. The pH of the water 50 cm below the surface was measured at 11:00 hours on the sampling day. Due that dissolved oxygen level in the crab pond water was above 5.0 mg L 1 and the surface of the pond sediment was sampled, the sediment sampled was not regarded to be anoxic. The total genomic DNA of the crab pond sediment was extracted as described by Brady (2007) with some modifications. 10 g of sediment was suspended in a 50 mL centrifuge tube containing 20 mL of pre-heated (70°C) lysis buffer [100 mM Tris-HCl, 100 mM EDTA, 1.5 M NaCl, 1% (w/v)

Identification and structural studies of novel bacterial chitinases

Identification and structural studies of novel bacterial chitinases

Structure and function of chitinases from glycoside hydrolase family 19

AbstractGlycoside hydrolase family 19 chitinases are found in plants, bacteria and viruses, playing various roles. Plant chitinases are important for defence and development, whereas bacterial examples are useful for nutrition. The available structural studies show that family 19 enzymes are highly α‐helical bi‐lobed structures with a wide cleft lined by conserved residues important for catalysis and substrate binding. Class I and II plant chitinases possess loops which are absent in class IV and/or bacterial chitinases. One of the loops seems to retain a significant function. These inverting endochitinases demonstrate a possible opening–closing mechanism of the catalytic cleft during chitin hydrolysis due to loop movements. However, complex structures with inhibitors and/or substrate/product analogues are required for a deeper understanding of these enzymes. Copyright © 2011 Society of Chemical Industry

Production of N-Acetylglucosamine Using Recombinant Chitinolytic Enzymes

The pharmaceutically important compound N-acetylglucosamine (NAG), is used in various therapeutic formulations, skin care products and dietary supplements. Currently, NAG is being produced by an environment-unfriendly chemical process using chitin, a polysaccharide present in abundance in the exoskeleton of crustaceans, as a substrate. In the present study, we report the potential of an eco-friendly biological process for the production of NAG using recombinant bacterial enzymes, chitinase (CHI) and chitobiase (CHB). The treatment of chitin with recombinant CHI alone produced 8% NAG and 72% chitobiose, a homodimer of NAG. However, supplementation of the reaction mixture with another recombinant enzyme, CHB, resulted in approximately six fold increase in NAG production. The product, NAG, was confirmed by HPLC, TLC and ESI-MS studies. Conditions are being optimized for increased production of NAG from chitin.

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.

Abundance of bacterial genes encoding for proteases and chitinases in the rhizosphere of three different potato cultivars

In terrestrial ecosystems, the majority of soil N is present in organic macromolecules like proteins, nucleic acids and chitin. Because these compounds cannot be assimilated by plants, plant growth is often N-limited. Therefore, the hydrolysis of proteins and chitin is particularly important in making N available for plants. In this study, we investigated the influence of different potato cultivars and different plant developmental stages on the abundance of genes encoding for alkaline and neutral metalloproteases (apr, npr) as well as bacterial chitinases (chiA) in the rhizosphere using quantitative real-time PCR in a greenhouse trial and a field study. It could be clearly demonstrated that abundance pattern was mainly affected by the plant vegetation stage, whilst the used plant genotype had only a minor influence on the development of the two functional populations.

An antifungal chitinase produced by Bacillus subtilis using chitin waste as a carbon source

The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish-processing waste. In this study, shrimp and crab shell powder, prepared by treating shrimp- and crab-processing waste by boiling and crushing, was used as a substrate for the isolation of an antifungal chitinase-producing microorganism. Bacillus subtilis NPU 001, a strain isolated from soil samples, excreted a chitinase when cultured in a medium containing 2% (w/v) shrimp and crab shell powder as the major carbon source. The chitinase, which was purified by sequential chromatography, had a Mw of 31 kDa and a pI of 5.4. The purified chitinase (2 mg ml−1) inhibited hyphal extension of the fungus Fusarium oxysporum. Compared with other known bacterial chitinases, the unique characteristics of NPU 001 chitinase include antifungal activity against plant-pathogenic fungi and the production of chitotriose as the major enzymatic hydrolysate from colloidal chitin.

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.