Chitinase Synthesis Research Articles

Genomics, Proteomics, and Antifungal Activity of Chitinase from the Antarctic Marine Bacterium Curtobacterium sp. CBMAI 2942.

This study aimed to evaluate the genomic profile of the Antarctic marine Curtobacterium sp. CBMAI 2942, as well as to optimize the conditions for chitinase production and antifungal potential for biological control. Assembly and annotation of the genome confirmed the genomic potential for chitinase synthesis, revealing two ChBDs of chitin binding (Chi C). The optimization enzyme production using an experimental design resulted in a 3.7-fold increase in chitinase production. The chitinase enzyme was identified by SDS-PAGE and confirmed through mass spectrometry analysis. The enzymatic extract obtained using acetone showed antifungal activity against the phytopathogenic fungus Aspergillus sp. series Nigri CBMAI 1846. The genetic capability of Curtobacterium sp. CBMAI 2942 for chitin degradation was confirmed through genomic analysis. The basal culture medium was adjusted, and the chitinase produced by this isolate from Antarctica showed significant inhibition against Aspergillus sp. Nigri series CBMAI 1846, which is a tomato phytopathogenic fungus. This suggests that this marine bacterium could potentially be used as a biological control of agricultural pests.

In vitro Acaricidal Activity of Serratia Ureilytica Against the Dust Mite Tyrophagus Putrescentiae and Identification of Genes Related to Biocontrol.

The present study evaluated the acaricidal activity of three Serratia strains isolated from Mimosa pudica nodules in the Lancandon zone Chiapas, Mexico. The analysis of the genomes based on the Average Nucleotide Identity, the phylogenetic relationships allows the isolates to be placed in the Serria ureilytica clade. The size of the genomes of the three strains is 5.4Mb, with a GC content of 59%. The Serratia UTS2 strain presented the highest mortality with 61.41% against Tyrophagus putrescentiae followed by the Serratia UTS4 strain with 52.66% and Serratia UTS3 with 47.69% at 72h at a concentration of 1X109 cell/mL. In the bioinformatic analysis of the genomes, genes related to the synthesis of chitinases, proteases and cellulases were identified, which have been reported for the biocontrol of mites. It is the first report of S. ureilytica with acaricidal activity, which may be an alternative for the biocontrol of stored products with high fat and protein content.

Bioprospecting Study of Plant Growth Promoting Rhizospheric Bacteria from Oil Palm Plantation as Biological Control Agent of Ganoderma boninense.

<b>Background and Objective:</b> The prioritisation of oil palm studies involves the exploration of novel bacterial isolates as possible agents for suppressing <i>Ganoderma boninense</i>. The objective of this study was to evaluate and characterise the potential of rhizospheric bacteria, obtained from the rhizosphere of oil palm plants, in terms of their ability to demonstrate anti-<i>Ganoderma </i>activity. <b>Materials and Methods:</b> The study began by employing a dual culture technique to select hostile bacteria. Qualitative detection was performed to assess the antifungal activity, as well as the synthesis of chitinase and glucanase, from certain isolates. The candidate strains were molecularly identified using 16S-rRNA ribosomal primers, specifically the 27F and 1492R primers. <b>Results:</b> The findings of the study indicated that the governmental plantation exhibited the highest ratio between diazotroph and indigenous bacterial populations in comparison to the other sites. Out of a pool of ninety bacterial isolates, a subset of twenty-one isolates demonstrated the ability to impede the development of <i>G. boninense</i>, as determined using a dual culture experiment. Twenty-one bacterial strains were found to exhibit antifungal activity. Nine possible bacteria were found based on the sequence analysis. These bacteria include <i>Burkholderia territorii</i> (RK2, RP2, RP3, RP5), <i>Burkholderia stagnalis</i> (RK3), <i>Burkholderia cenocepacia</i> (RP1), <i>Serratia marcescens</i> (RP13) and <i>Rhizobium multihospitium</i> (RU4). <b>Conclusion:</b> The findings of the study revealed that a significant proportion of the bacterial population exhibited the ability to perform nitrogen fixation, indole-3-acetic acid (IAA) production and phosphate solubilization. However, it is worth noting that <i>Rhizobium multihospitium</i> RU4 did not demonstrate the capacity for phosphate solubilization, while <i>B. territory</i> RK2 did not exhibit IAA production.

RNA sequencing analysis reveals the role of choline acetyltransferase in regulating development, reproduction, and insecticide susceptibility in Tribolium castaneum (Coleoptera: Tenebrionidae)

AbstractCholine acetyltransferase (Tcchat) is essential for metamorphosis, reproduction, and susceptibility to insecticide in Tribolium castaneum. However, the mechanisms through which Tcchat contributes to these biological processes remain poorly understood. Here we report the findings of RNA sequencing‐based transcriptome profiling of T. castaneum following RNA interference against Tcchat. In all, 173 differentially expressed genes were identified: 120 upregulated genes and 53 downregulated genes. Gene Ontology analysis classified the genes into 30 functional groups. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the genes were distributed over 22 different pathways. Silencing Tcchat reduced the activity of endopeptidase and inhibited regulation of protein decomposition in the beetles. Hence, knockdown of Tcchat inhibited the synthesis of chitinase and serine protease, which disturbed embryonic development in T. castaneum, thus affecting its development and reproduction. RNA interference of Tcchat also suppressed the synthesis of cytochrome P450 in the extracellular region and adversely affected normal functioning of the lysosomal pathway, which might have hindered the insects' ability to recognize, phagocytose, and degrade exogenous toxic substances, resulting in increased mortality. Furthermore, knockdown of Tcchat increased the susceptibility of larvae to carbamate and organophosphate insecticides, which supports the notion that Tcchat is indeed involved in susceptibility to insecticide in T. castaneum. Finally, Tcchat knockdown adults showed progressively reduced locomotor activity. These results provide novel insights into biological processes affected by Tcchat and provide a potential theoretical basis for the biological control of T. castaneum.

Alteration of gut microbiota composition and function of Indian major carp, rohu (Labeo rohita) infected with Argulus siamensis

Gut microbiome homeostasis is critical in preventing diseases. However, the effect of disease on gut microbiota assembly remains unclear. At present, there are no reports on the composition and functional analysis of intestinal microbiota of Indian major carp, rohu (L. rohita) infected with ectoparasite, Argulus. In this study, we analysed and compared the intestinal microbiota of healthy and Argulus-infected rohu by 16S rRNA amplicon sequencing. Argulus infection could significantly influence the diversity and richness of the gut microbiota. However, abundance of Actinobacteria and Patescibacteria were enriched significantly in Argulus-infected fish. Venn diagram revealed that there were many more unique genera in the infected group as compared to control fish. The genera, Stenotrophomonas and Pirellula were significantly increased in infected fish while the abundance of Reyranella was decreased. LEfSe analysis showed a significant enrichment in abundances of 11 taxa in healthy group and 17 taxa in infected group. Furthermore, genera Rubellimicrobium, Dielma, Hyphomicrobium, Reyranella, Streptomyces and Cloacibacterium performed the best in differentiating between both the groups. Predicted microbiota function by PICRUSt revealed that the gut microbiota of infected fish was mainly associated with enriched synthesis of chitinases, chitin binding proteins, osmoprotectant proteins and sulfatases enzymes. There was a positive association between the structural and functional composition of the gut microbiota. The results indicated that the Argulus infection could affect the intestinal microbiota composition and function of rohu.

Nematicidal Activity of the Endophyte Serratia ureilytica against Nacobbus aberrans in Chili Plants (Capsicum annuum L.) and Identification of Genes Related to Biological Control.

The genus Serratia is widely distributed in soil, water, plants, animals, invertebrates, and humans. Some species of this genus have antifungal, antibacterial, and nematicidal activity. In this work, the nematicidal activity of the endophytic strain of Serratia sp. in chili, Capsicum annuum L., is reported, where at a bacterial concentration of 4 × 109 cel/mL, the penetration of nematodes into the roots significantly decreased by 91 and 55% at 7 and 21 days after inoculation. This bacterial concentration also significantly decreased the number of galls, eggs, egg masses and reproduction factor produced by Nacobbus aberrans in Chili plants, with respect to the control where this bacterial strain was not applied. In the analysis of the genome of the strain, based on average nucleotide identity (ANI), the isolate could be affiliated to the species Serratia ureilytica. The size of the genome is 5.4 Mb, with a 59.3% content of GC. Genes related to the synthesis of chitinases, siderophores, proteases C, serralisins, hemolysin, and serrawettin W2 that have been reported for biocontrol of nematodes were identified in the genome. It is the first report of Serratia ureilytica with nematicidal activity. Based on these results of nematicidal activity, this strain can be evaluated in the field as an alternative in the biocontrol of Nacobbus aberrans in chili cultivation.

The effect of chitin on the biological activity of Bacillus subtilis strains

The aim of the work was to assess the effect of various forms of chitin and chitosan during submerged cultivation of Bacillus subtilis strains, which form the basis of the laboratory sample Vitaplan, CL, on the synthesis of chitinase, as well as on the antagonistic activity and inducing effect of B. subtilis strains in the pathosystems of wheat - Cochliobolus sativus and Puccinia recondita f. sp. tritici. The inclusion of chitin in the form of dry powder or chitin and chitosan in the form of a colloidal suspension into the medium for deep cultivation of bacteria showed that only colloidal chitin increased the antagonistic activity of B. subtilis strains against test cultures of Alternaria solani and Clavibacter michiganensis. The ability of B. subtilis strains to synthesize extracellular chitinase when cultivated in a medium containing colloidal chitin was established. A higher fungistatic effect of the laboratory sample Vitaplan CL + colloidal chitin against Cohliobolus sativus was revealed as compared to the original sample. It was shown that the laboratory sample Vitaplan, CL + colloidal chitin increases the resistance of wheat to dark brown spot and brown rust 1.5–2.0 times more effectively as compared to Vitaplan, CL. As a result of the research, a laboratory sample of Vitaplan, CL + colloidal chitin was obtained with increased antagonistic and inducing activity as compared to Vitaplan, CL.

Biosynthesis, statistical optimization and molecular modeling of chitinase from crab shell wastes by a mangrove associated actinobacterium Streptomyces olivaceus (MSU3) using Box-Behnken design and its antifungal effects

The present study was undertaken to investigate biosynthesis, production through statistical optimization, purification and molecular modeling of chitinase by a mangrove associated actinobacterium Streptomyces olivaceus (MSU3) under submerged fermentation process. Initially, the substrate chitin was extracted from crab shell wastes through standard procedure and the nature of extracted chitin was characterized by FT-IR, XRD and SEM analysis. Simultaneously, the chitinase by the chosen actinobacterium was biosynthesized and statistically optimized by Box-Behnken design. The chitinase was effectively produced under optimized conditions like NaCl (3.05% w/v), pH 6.5, temperature 47 °C and 2.75% inoculum size for 90 h with maximum level of coefficient (R2 = 0.9680) and more significance (P < 0.0001). In purification process, the enzyme expressed 680.0 IU total activity, 5.09 fold purification and 32.06% yield with the molecular mass of 52 kDa on SDS-PAGE. The three dimensional structure of the predicted chitinase protein was encoded with 536 amino acids. The maximum activity of purified chitinase was documented using the fermentation medium constituted with 1 mM Fe3+, 2.0% chitin and 2.5% NaCl at pH 8 and the temperature at 40 °C. The Km and Vmax values of the purified chitinase exhibited 2.24 mg/ml and 284.80 μg/min/mg, respectively. The purified chitinase expressed antifungal activity with the maximum (20.0 mm) zone of inhibition against Aspergillus brasiliensis. Based on the study, it could be authenticated that the chosen actinobacterium is a novel isolate for chitinase synthesis and also it acts as an antifungal agent.

Chitosan Resistance of Bacteria and Micromycetes Differing in Ability to Produce Extracellular Chitinases and Chitosanases

Effect of chitosan on growth and development of bacterial and fungal strains differing in their ability to synthesize chitinases and chitosanases was explored. Bacterial cultures simultaneously producing chitinases and chitosanases were found to be more resistant to soluble chitosan than both the strains synthesizing only chitinases and inactive bacteria. Paenibacillus ehimensis strain IB-739, which was characterized by inducible synthesis of chitinases and chitosanases, exhibited the highest resistance (minimal bactericidal chitosan concentration 14−15 mg/mL). No direct correlation was revealed between the level of chitosanase production by bacteria and their resistance to chitosan. Sublethal concentrations of chitosan promoted the growth of several bacterial strains; the most intensive growth of chitosan-degrading bacteria correlated with the maximal values of their chitosanase production. Within the concentration range studied (≤16 mg/mL), no inhibitory action of chitosan against exo-1,4-β-glucosaminindase-producing strain Penicillium sp. IB-37-2A was observed. Compared to inactive fungal cultures, the growth of this fungus was significantly activated in presence of chitosan; the maximal concentration of this polysaccharide resulted in a sixfold increase of Penicillium sp. IB-37-2A biomass yield.

A Review on Chitinase Synthesis from varied sources and its Applications towards Environment

Chitin is the second most abundant biopolymer widely distributed in the marine and terrestrial environments. It is an insoluble linear polymer of beta-1,4-linked N-acetylglucosamine (GlcNAc). Chitin is abundantly present in the cell wall of fungi, exoskeleton of insects and crustacean cells. In nature, chitin exists as alpha, beta, and gamma crystalline forms. Degradation of chitin can be done using four enzyme systems: endochitinase; N-acetylglucosaminase; polysaccharide monooxygenase, and exochitinase. Endophytes isolated from marine algae have very good potent of producing chitinase and also have a wide application in the medical field like anticancer activity antimicrobial activity and many other industries like food, agriculture etc. Chitinases are applicable in agriculture fields to control pathogens. This review focuses on the major application of chitinase enzymes from different plant and microbial sources.

Biodegradation of chitinous substances and chitinase production by the soil actinomycete Streptomyces rimosus

A chitinolytic actinomycete was isolated from agricultural soil in the centre of Poland. It was identified as Streptomyces rimosus. Identification was based on standard biochemical tests and on 16S rRNA gene sequence analysis. The degradation of chitinous substances (shrimp shell waste, crab chitin powder and chitosan) was examined on the basis of oxygen consumption by applying OxiTop (WTW). The enzyme catalyzed the hydrolysis of the disaccharide 4-MU-(GlcNAc)3 most efficiently and was therefore classified as an endochitinase. The chitinase was purified from culture medium by fractionation with ammonium sulfate and affinity chromatography. The purified proteins were subjected to identification by mass spectrometry. Based on analysis of the resultant fragments of the amino acid sequence of chitinase produced by the examined bacteria, degenerate primers for PCR were designed. The results of our research prove that the substances which are metabolised by S. rimosus the most efficiently are chitosan and the shrimp shell waste. Chitinolytic activity examination showed that shrimp shell waste was the finest inducer of chitinase synthesis. The molecular mass of the purified enzyme was 63 kDa. The highest activity of chitinase was obtained at the temperature of 40°–45 °C. Optimal pH for chitinase activity was 7.0. The activity of the purified chitinase was stabilized by Mg2+ ions. The activity of the enzyme was inhibited by Hg2+ and Pb2+ ions. Chitinase inhibited the growth of the fungal phytopathogens Fusarium solani and Alternaria alternata. The nucleotide sequence of the amplified gene fragment proved to be similar to the sequence of chitinase-encoding genes of the glycoside hydrolases family 18.

Influences of Medium Composition on Mycelial Growth, Sporulation and Chitinase Secretion of Metarhizium anisopliae HY‐2

Metarhizium anisopliae HY‐2, an entomopathogenic fungus isolated from pest cadaver in South Korea, is applicable as a potent bio‐pesticide because of its insecticidal activity to Adoretus tenuimaculatus. Here we report this strain varied in its ability to grow under different nutritional and environmental factors. Temperature at 28°C and pH from 6 to 7 were obtained the best‐fit environmental requirements. Insect pupa and tryptone were found the optimal nitrogen sources for both mycelial growth and sporulation. The strain reached the most suitable effects at the carbon‐to‐nitrogen ratio from 10:1 to 20:1 when the carbon concentration was 4 g/ml provided by glucose and nitrogen supplied by tryptone. Additionally, strain HY‐2 produced an extracellular chitinase at a relatively high level (21.1 mU/ml) when cultivated for 20 days on a medium supplemented with colloidal chitin (1%) and insect pupa (0.5%), indicating that synthesis of chitinase is subject to induction by certain substrate.

Quantitative analysis of chitinase gene expression in chickpea

The synthesis of chitinases is one of the strategies evolved in higher plants to resist stresses. To investigate the role of chitinases in the interaction between chickpea (Cicer arietinum L.) and pathogens, the expression of two classes of chitinase, I and III, was monitored in infected and non-infected plants with different disease resistance (resistant MCC10 and sensitive MCC724 genotypes). The expression levels of these genes in chickpea were studied at different times after inoculation with Fusarium oxysporum f. sp. ciceri isolate 85. Semi-quantitative reverse transcription PCR was used to study the differential induction of the chitinase genes. Expression levels of chitinase I in non-infected and infected MCC724 plants were higher than those in MCC10. Since chitinase III may play an effective role in plant defense, its expression levels were quantified by reverse transcription PCR. Chitinase III was induced in MCC724 within 6 h and in MCC10 in 48 h after inoculation. However, the expression levels of chitinase III in MCC10 non-infected plants were significantly higher than in MCC724. The results showed that chitinase III can be effective to enhance resistance to F. oxysporum f. sp. ciceri isolate 85.

Effect of Medium Composition on the Synthesis of Chitinase and Chitin Deacetylase from Thermophilic Paenibacillus sp.Hul

To investigate the effects of nutritional components and initial pH on chitinase and chitin deacetylase production by thermophilic Paenibacillus sp. Hul, and provide basis for fermentation of enzymes and preparation of chito- oligosaccharides. The shake flask culture was employed to research the culture conditions of thermophilic Paenibacillus sp. Hu1 for producing chitinase and chitin deacetylase. The results show that the optimal carbon source was chitin powder with 35g/L; while yeast extract power was the optimal nitrogen source with 10g/L. It was beneficial to synthesize chitinase and chitin deacetylase when the initial pH was 6.0. In addition, the synthesis of chitinase and chitin deacetylase was inhibited by Cu2+, and activated by Ca2+. However, Mg2+ and Zn2+ did not show significant inhibitory or activation effects on the synthesis of the two enzymes. The hydrolysate components were analyzed using chitin as substrate with the crude enzyme, and the result suggested that enzymatic hydrolysates were mainly centralized in chito-oligosaccharides with polymerization number less than hexaose.

Microbial Synthesis of Chitinase in Solid Cultures and Its Potential as a Biocontrol Agent Against Phytopathogenic Fungus &lt;I&gt;Colletotrichum gloeosporioides&lt;/I&gt;

Antifungal activity of chitinase can be effectively utilized in biologic pest control strategies. Because solid-state cultivation has been termed a cost-effective means for fungal growth and metabolite production, chitinase production by Trichoderma harzianum was studied using wheat bran-based solid medium containing 1% colloidal chitin. Chitinase synthesis was found to be growth associated because maximum enzyme (5.4 U/g of dry substrate) and biomass production occurred at 72 h. Substrate moisture had a critical impact on chitinase production; five grams of medium having an initial moisture content of 68.4% when incubated for 72 h increased the enzyme yield to 9.3 U/g of dry substrate. Optimization of colloidal chitin concentration showed that improvements in chitinase yield and maximum activity were attained with a 2% (w/w) concentration. Supplementation of additional nitrogen sources also influenced enzyme production, and the best yield was obtained with yeast extract. The effect of crude chitinase on hyphal morphology of the phytopathogenic fungus Colletotrichum gloeosporioides was swelling as well as lysis of hyphal wall, depending on the age of the mycelium. Studies of pH and thermal stability showed that crude culture filtrate was active over pH 4.0-6.0 and retained about 48.2% activity after 40 min of incubation at 40 degrees C.

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon Thermococcus chitonophagus.

Thermococcus chitonophagus produces several, cellular and extracellular chitinolytic enzymes following induction with various types of chitin and chitin oligomers, as well as cellulose. Factors affecting the anaerobic culture of this archaeon, such as optimal temperature, agitation speed and type of chitin, were investigated. A series of chitinases, co-isolated with the major, cell membrane-associated endochitinase (Chi70), and a periplasmic chitobiase (Chi90) were subsequently isolated. In addition, a distinct chitinolytic activity was detected in the culture supernatant and partially purified. This enzyme exhibited an apparent molecular mass of 50 kDa (Chi50) and was optimally active at 80 degrees C and pH 6.0. Chi50 was classified as an exochitinase based on its ability to release chitobiose as the exclusive hydrolysis product of colloidal chitin. A multi-component enzymatic apparatus, consisting of an extracellular exochitinase (Chi50), a periplasmic chitobiase (Chi90) and at least one cell-membrane-anchored endochitinase (Chi70), seems to be sufficient for effective synergistic in vivo degradation of chitin. Induction with chitin stimulates the coordinated expression of a combination of chitinolytic enzymes exhibiting different specificities for polymeric chitin and its degradation products. Among all investigated potential inducers and nutrient substrates, colloidal chitin was the strongest inducer of chitinase synthesis, whereas the highest growth rate was obtained following the addition of yeast extract and/or peptone to the minimal, mineralic culture medium in the absence of chitin. In rich medium, chitin monomer acted as a repressor of total chitinolytic activity, indicating the presence of a negative feedback regulatory mechanism. Despite the undisputable fact that the multi-component chitinolytic system of this archaeon is strongly induced by chitin, it is clear that, even in the absence of any chitinous substrates, there is low-level, basal, constitutive production of chitinolytic enzymes, which can be attributed to the presence of traces of chito-oligosaccharides and other structurally related molecules (in the undefined, rich, non-inducing medium) that act as potential inducers of chitinolytic activity. The low, basal and constitutive levels of chitinase gene expression may be sufficient to initiate chitin degradation and to release soluble oligomers, which, in turn, induce chitinase synthesis.

Synthesis of Extracellular Chitinase by Wild-Type B-10 and Mutant M-1 Strains of Serratia marcescens

Molecular weights of extracellular chitinases from wild-type B-10 (62, 54, 43, 38, and 21 kDa) and mutant M-1 strains of Serratia marcescens (62, 52, 43, 38, and 21 kDa) were estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. In the absence of chitin inductors, chitinolytic enzymes were not found in the culture liquid of B-10, whereas M-1 cells produced the chitinase complex (to 470 pU/cell). Crystalline chitin insignificantly stimulated the synthesis of chitinases with molecular weights of 62, 54, and 21 kDa by B-10 (up to 20 pU/cell), but caused oversynthesis of all chitinases by the mutant strain (up to 2600 pU/cell). Colloidal chitin induced the production of chitinases by cells of both strains. Two peaks of chitinolytic activity were observed during cultivation of strains B-10 (350 and 450 pU/cell) and M-1 (2200 and 2400 pU/cell). The first peak of cell productivity was associated with biosynthesis of the chitinase complex. The second peak was related to the synthesis of enzymes with molecular weights of 54, 43, 38, and 21 kDa (B-10) or 43, 38, and 21 kDa (M-1).

The accumulation of proteins with chitinase activity in the culture media of the parent and mutant Serratia marcescens strain grown in the presence of mitomycin C

The study of the accumulation pattern of extracellular proteins with chitinase activity in the parent Serratia marcescens strain Bú 211 (ATCC 9986) grown in the presence of mitomycin C and its mutant strain with the constitutive synthesis of chitinases grown in the absence of the inducer showed that chitinase activity appeared in the culture liquids of both strains at the end of the exponential phase (4 h of growth) and reached a maximum in the stationary phase (18-20 h of growth). The analysis of the culture liquids (12 h of growth) by denaturing electrophoresis in PAAG followed by the protein renaturation step revealed the presence of four extracellular proteins with chitinase activity and molecular masses of 21, 38, 52, and 58 kDa.

Synthesis of chitinase in Streptomyces thermoviolaceus is regulated by a two-component sensor-regulator system

The chiS and chiR genes located upstream of the chitinase locus ( chi40) on the chromosome of Streptomyces thermoviolaceus OPC-520 were cloned and sequenced. The deduced amino acid sequences revealed that ChiS (390 amino acids, 40.9 kDa) and ChiR (213 amino acids, 22 kDa) show significant sequence similarities to histidine kinases and response regulators, respectively, of typical prokaryotic two-component regulatory systems. The extracellular chitinase activity of Streptomyces lividans 66 (pTSR2 (bearing chiS, chiR and chi40)) was significantly enhanced by a high dosage of the chiS and chiR genes.

Synthesis of chitinase in Streptomyces thermoviolaceus is regulated by a two-component sensor-regulator system.

The chiS and chiR genes located upstream of the chitinase locus (chi40) on the chromosome of Streptomyces thermoviolaceus OPC-520 were cloned and sequenced. The deduced amino acid sequences revealed that ChiS (390 amino acids, 40.9 kDa) and ChiR (213 amino acids, 22 kDa) show significant sequence similarities to histidine kinases and response regulators, respectively, of typical prokaryotic two-component regulatory systems. The extracellular chitinase activity of Streptomyces lividans 66 (pTSR2 (bearing chiS, chiR and chi40)) was significantly enhanced by a high dosage of the chiS and chiR genes.