Temperature as an autoecological factor of chitinolytic microbial complex formation in soils

The dynamics of carbon dioxide emission from soil was studied during chitinolytic succession induced by humidification and chitin introduction at different temperatures (5, 27, and 50°C) using gas chromatography. The abundance and biomass of the chitinolytic bacterial and actinomycete complex in soil were evaluated by luminescent microscopy. Active development of the chitinolytic microbial complexes was observed at all studied temperatures. The most active growth of chitinolytic microorganisms was observed at high temperature during early succession and at low temperature during late succession. High and low temperatures provided for active development of the chitinolytic microbial complex in soils confined to warm climatic zones (brown desert-steppe soil) and soils of temporary zones (gray forest soil). Actinomycetes demonstrated the most active growth among chitinolytic microorganisms in the studied soil samples both at low and high temperatures.     

Specificity of the chitinolytic microbial complex of soils incubated at different temperatures

The structural and functional specificity of the chitinolytic microbial complex changes dramatically depending on the incubation temperature of soil microcosms. It was shown that the highest rates of chitin degradation occurred in desert soils at high temperatures (50°C); in the moderate and northern zones, these rates peaked at lower temperatures (5°C). The role of prokaryotes as the main chitin degraders in soils incubated at high temperatures, with fungi more actively participating in chitin decomposition at low temperatures, was shown for the first time. Fluorescent in situ hybridization (FISH) revealed the predominance of actinomycetes in the metabolically active chitinolytic prokaryotic complex of desert soils (high temperatures); in the soils of the northern latitudes (low temperatures), proteobacteria prevailed. The relationship between the taxonomic position of the dominant members of the chitinolytic complex of soil microorganisms, isolated in pure cultures with the dominant phylogenetic groups and the sequence types obtained by using molecular biological techniques (FISH) was revealed.     

Succession of chitinolytic microorganisms in chernozem soil

The chitinolytic prokaryotic and eukaryotic microbial complex of chernozem soil has been investigated in the course of a succession initiated by the introduction of chitin and humidification. The dynamics of the cell numbers of chitinolytic microorganisms and of their biomass was assessed by fluorescent microscopy and by inoculation of selective media. Emission of carbon dioxide and nitrous oxide, as well as dinitrogen fixation, was assessed by gas chromatography. It was found that, when the succession was initiated by the introduction of both chitin and humidification, it resulted in greater cell numbers and biomass of chitinolytic microorganisms and higher levels of CO2 and N2O emission and of nitrogen fixation than when the succession was initiated by humidification alone. As compared to the control samples, a significant (twofold) increase in the prokaryote cell number and biomass was found on the fourth day of the succession initiated by humidification and introduction of chitin. One week after the initiation of succession, the fungal biomass and length of mycelium were twice as high as those in the control samples. These results led to the conclusion that chitin utilization in chernozem soil starts during the initial stages of succession and is performed by both prokaryotic and eukaryotic microorganisms.     

Use of chitin for controlling plant plant-parasitic nematodes

Summary Bean and tomato seedlings, treated with different amounts of ammonia and chitin, and inoculated with the root-knot nematode,Meloidogyne javanica, were reared in two consecutive 35-day growth of shoot and root and the condition of the root systems of both plant species and degree of infection byM. javanica were decreased by increasing amounts of ammonia. Chitin caused a relatively small reduction in gall formation but almost no changes in fresh shoot weights. The effect of chitin on plant growth and nematode attack were also compared in irradiated and non-irradiated soil. In the first cycle galling index of the chitin treated plant was similar to that of untreated plants maintained in the irradiated soil conditions, while in the non-irradiated soil, chitin treatment reduced galling index. In the second cycle, chitin treatment reduced galling index in both irradiated and non-irradiated soils, especially in the latter, where galling index greatly decreased compared with the non-treated plants. Differences in fresh shoot weight between nematode-infected and nematode-free plants amended with chitin were greater under non-irradiated than irradiated conditions, especially in the second cycle. In non-irradiated soil, a higher level of chitinolytic microorganisms, particularly actinomycetes, was found in the second cycle. Contribution from the Agricultural Research Organization (ARO), Bet Dagan, Israel. No. 1520-E, 1985 series.     

Mycophagous mites and their internal associated bacteria cooperate to digest chitin in soil

The greater bulk of soil nitrogen is immobilized in chitinous cell walls of fungi. Mycophagous soil mites participate in chitin decomposition and, hence, in the subsequent mobilization of nitrogen. The source of the chitinolytic enzymes was searched in this study. A multimethodical approach was designed for these studies. Histology, plating and identification of bacteria from mite homogenate and, finally, homogenate and bacterial treatment of the soil fungi were applied. Here the presence and activity of chitinolytic bacteria inside mycophagous mites are reported. These bacteria form an extraintestinal group within the mite’s body and pass their enzymes into the mite’s gut. Our results demonstrate that true mycophagous mites, defined by their ability to digest chitin (i.e. the fungal cell wall), achieve this through internal “cooperation” with chitinolytic bacteria that provide the necessary chitinolytic enzymes. The nitrogen from chitin is thus made available to other soil organisms and plants.     

Description of the phylogenetic structure of hydrolytic prokaryotic complex in the soils

With the help of the molecular-biological method of cell hybridization in situ (FISH), the abundance of a physiologically active hydrolytic prokaryotic complex in chernozem and gley-podzolic soils is determined. The total proportion of metabolically active cells, which were detected by hybridization with universal probes as representatives of the domains Bacteria and Archaea, in samples of the studied soil, was from 38% for chernozem up to 78% for gley-podzolic soil of the total number of cells. The differences in the structure of chitinolytic and pectinolytic prokaryotic soil complexes are detected. Along with the high abundance of Actinobacteria and Firmicutes in the soils with chitin, an increase in phylogenetic groups such as Alphaproteobacteria and Bacteroidetes is observed.     

Assessment of chitin decomposer diversity within an upland grassland

The breakdown of chitin within an acidic upland grassland was studied. The aim was to provide a molecular characterisation of microorganisms involved in chitin degradation in the soil using soil microcosms and buried litter bags containing chitin. The investigation involved an examination of the effects of liming on the microbial communities within the soil and their chitinolytic activity. Microcosm experiments were designed to study the influence of lime and chitin enrichment on the grassland soil bacterial community ex situ under controlled environmental conditions. Bacterial and actinomycete counts were determined and total community DNA was extracted from the microcosms and from chitin bags buried at the experimental site. PCR based on specific 16S rRNA target sequences provided products for DGGE analysis to determine the structure of bacterial and actinomycete communities. Chitinase activity was assessed spectrophotometrically using chitin labelled with remazol brilliant violet. Both liming and chitin amendment increased bacterial and actinomycete viable counts and the chitinase activity. DGGE band patterns confirmed changes in bacterial populations under the influence of both treatments. PCR products amplified from DNA isolated from chitin bags were cloned and sequenced. Only a few matched known species but a prominent coloniser of chitin proved to be Stenotrophomonas maltophilia.     

Microbial transformation of chitin in soil under anaerobic conditions

Anaerobic chitinolytic complex was studied in three soil types: chernozem, gray forest soil, and chestnut soil. The abundance and biomass of anaerobic chitinolytic microbial complex of fungi, bacteria, and actinomycetes were evaluated by luminescent microscopy. The dynamics of methane emission from soil during chitinolytic succession was studied by gas chromatography. All three studied microbial groups proved to participate in chitin transformation in soil under anaerobic conditions. The highest biomass growth was observed among prokaryotes, particularly actinomycetes, whose biomass doubled. The increase in methane emission during chitinolytic succession was most pronounced in soils with low organic matter content.     

Use of chitin for controlling plant-parasitic nematodes

Cabbage plants were grown in soil amended with Clandosan (CLA) prepared from crustacean chitin (0.3% w/w). The plants were maintained in constant temperature tanks set to 15° or 30°C, in soils naturally infested with cyst nematodeHeterodera schachtii, or inoculated with the root-knot nematode,Meloidogyne javanica, respectively. At 30°C, after the first month following inoculation, CLA caused an increase in top fresh weight of plants but no reduction in nematode—induced root galling was recorded. However, when fresh plants were planted, CLA induced a large reduction in gall formation and caused an increase in top fresh weight of nematode-inoculated plants. At 15°C, CLA significantly affected the plants only after 60 days: an increase in top fresh weight and a reduction in the number of eggs per cyst were recorded. Ammonium was not detected in soil after 30 days, at 30°C, whereas at 15°C, CLA-treated soil contained twice as much ammonium as non-treated soil. After 60 days, ammonium was not detected at all. After 30 days nitrate concentrations in soil attained higher values at 30°C than at 15°C, whereas after 60 days high levels were detected only at 15°C. At 30°C, CLA induced an increase in the number of fungi, chitinolytic bacteria, and total amount of bacteria; at 15°C, such an increase was detected only with the chitinolytic microorganisms.Contribution from the Agricultural Research Organization (ARO), Bet Dagan, Israel No. 2196-E, 1987 series.     

Chitinolytic enzymes from<Emphasis Type="Italic">Clostridium aminovalericum</Emphasis>: Activity screening and purification

A strain isolated from the feces of takin was identified as Clostridium aminovalericum. In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase. The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin. Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively). Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL). The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27. At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27. The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer. At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted. However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated. The protein and enzyme recovery reached 90%.     

Thermophilic chitinolytic microorganisms of brown semidesert soil

In brown semidesert soil, thermophilic prokaryotic organisms identified as Streptomyces roseolilacinus and Silanimonas lenta were shown to play the main role in chitin transformation at 50°C. The phylogenetic positions of the isolated dominant chitinolytic microorganisms were determined on the basis of 16S rRNA gene sequencing. The consumption of chitin as a source of carbon and nitrogen by both the bacterium and the actinomycete was evident from considerable biomass accumulation, high emission of carbon dioxide, and presence in the medium of the chitinase exoenzyme.     

Chitin hydrolysis by Listeria spp., including L. monocytogenes

Listeria spp., including the food-borne pathogen Listeria monocytogenes, are ubiquitous microorganisms in the environment and thus are difficult to exclude from food processing plants. The factors that contribute to their multiplication and survival in nature are not well understood, but the ability to catabolize various carbohydrates is likely to be very important. One major source of carbon and nitrogen in nature is chitin, an insoluble linear beta-1,4-linked polymer of N-acetylglucosamine (GlcNAc). Chitin is found in cell walls of fungi and certain algae, in the cuticles of arthropods, and in shells and radulae of molluscs. In the present study, we demonstrated that L. monocytogenes and other Listeria spp. are able to hydrolyze alpha-chitin. The chitinolytic activity is repressed by the presence of glucose in the medium, suggesting that chitinolytic activity is subjected to catabolite repression. Activity is also regulated by temperature and is higher at 30 degrees C than at 37 degrees C. In L. monocytogenes EGD, chitin hydrolysis depends on genes encoding two chitinases, lmo0105 (chiB) and lmo1883 (chiA), but not on a gene encoding a putative chitin binding protein (lmo2467). The chiB and chiA genes are phylogenetically related to various well-characterized chitinases. The potential biological implications of chitinolytic activity of Listeria are discussed.     

Fungal chitinases and their biological role in the antagonism onto nematode eggs. A review

Chitin, the most abundant aminopolysaccharide in nature, is a rigid and resistant structural component that contributes to the mechanical strength of chitin-containing organisms. Chemically, it is a linear cationic heteropolysaccharide composed of N-acetyl-D-glucosamine and D-glucosamine units. The enzymatic degradation of chitin is performed by a chitinolytic system with synergistic and consecutive action. Diverse organisms (containing chitin or not) produce a great variety of chitinolytic enzymes with different specificities and catalytic properties. Their physiological roles involve nutrition, parasitism, chitin recycling, morphogenesis, and/or defense. Microorganisms, as the main environmental chitin degraders, constitute a very important natural source of chitinolytic enzymes. Nowadays, the most used method for pest and plant diseases control is the utilization of chemical agents, causative of significant environmental pollution. Social concern has generated the search for alternative control systems (i.e., biological control), which contribute to the generation of sustainable agricultural development. Interactions among the different organisms are the natural bases of biological control. Interest in chitinolytic enzymes in the field of biological control has arisen due to their possible involvement in antagonistic activity against pathogenic chitin-containing organisms. The absence of chitin in plants and vertebrate animals allows the consideration of safe and selective “target” molecules for control of chitin-containing pathogenic organisms. Fungi show appropriate characteristics as potential biological control agents of insects, fungi, and nematodes due to the production of fungal enzymes with antagonistic action. The antagonistic interactions between fungi and plant nematode parasites are among the most studied experimental models because of the high economic relevance. Fungi which target nematodes are known as nematophagous fungi. The nematode egg is the only structural element where the presence of chitin has been demonstrated. In spite of being one of the most resistant biological structures, eggs are susceptible to being attacked by egg-parasitic fungi. A combination of physical and chemical phenomena result in their complete destruction. The contribution of fungal chitinases to the in vitro rupture of the eggshell confirms their role as a pathogenic factor. Chitinases have been produced by traditional fermentation methods, which have been improved by optimizing the culture conditions for industrial processes. Although wild-type microorganisms constitute an alternative source of chitinolytic enzymes, the advances in molecular biology are allowing the genetic transformation of fungi to obtain strains with high capability as biocontrol agents. Simultaneously, a better understanding of rhizosphere interactions, additional to the discovery of new molecular biology tools, will allow the choosing of better alternatives for the biological control of nematodes in order to achieve an integrated management of the soil ecosystem.     

Multiple components and induction mechanism of the chitinolytic system of the hyperthermophilic archaeon <Emphasis Type="Italic">Thermococcus chitonophagus</Emphasis>

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°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.     

Mutual relationships between soils and biological carrier systems

Improved viability and antagonistic activity of biocontrol agents during soil inoculation is of crucial importance to their effective application. The chitinolytic bacterium Serratia marcescens was used as a model organism to study the efficacy of freeze-dried alginate beads (in comparison to their non-dried counterparts) as possible carriers for immobilized biocontrol agents. The release of bacteria and chitinolytic enzyme from alginate beads, before and during their application in soil, was examined, and the beads' physical properties characterized. Dispersal of the alginate bead-entrapped S. marcescens in the soil resulted in high soil cell densities throughout the 35 days of the experiment. Chitin inclusion in the beads resulted in significantly higher chitinolytic activity of S. marcescens, increased dry-bead porosity and decreased stiffness. Rehydration of the dried beads (after immersion in soil) resulted in a sixfold increase in weight due to water absorption. No significant differences were found in bacterial count inside the non-dried (gel) versus dried beads. However, higher cell densities and chitinase activity were detected in soil containing dried beads with chitin than in that containing their non-dried counterparts. The biological performance of S. marcescens was examined in the greenhouse: a free cell suspension reduced bean (Phaseolus vulgaris L.) disease by 10%, while immobilized bacteria found in the dried, chitin-containing beads reduced disease by 60%.     

Methodological aspects of assessing chitin utilization by soil microorganisms

A computational method for estimating specific activity of chitin decomposition by microorganisms is proposed. Spectrophotometric and gas chromatographic methods have been used to determine the rates of chitinase production, biomass accumulation, and carbon dioxide emission by pure cultures of microorganisms grown on a chitin-containing medium. Among dominants of the chitinolytic community of chernozem (Trichoderma viride, Stretomyces albolongus, Alcaligenes, and Arthrobacter), the highest chitinolytic activity is characteristic of prokaryotes. In brown desert-steppe soil, the main destructor prokaryotes are actinomycetes (S. roseolilacinus). The biomass of the fungus T. viride growth on the chitin-containing medium markedly exceeds that of prokaryotes, but the specific activity of respiration and chitinase production in actinomycetes S. roseolilacinus and S. albolongus is an order of magnitude higher than in T. viride.     

Production and purification of a chitinase from Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus

The chitinolytic properties of Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus, are reported. E. americana was shown to produce chitinolytic activity in the absence of chitin and in the presence of glucose and N-acetylglucosamine, indicating constitutive synthesis by these strains. A single 33-kDa protein with chitinolytic activity was purified to homogeneity from culture filtrates, by hydrophobic interaction chromatography using a phenyl-group substituted matrix. This enzyme, by virtue of differential activity against chromogenic chitooligosaccharides and against dye-labelled soluble carboxymethylated chitin (CM-chitin-RBV), was demonstrated to be an endochitinase. Our data suggest this 33-kDa chitinase appeared to be the only chitinolytic enzyme produced by E. americana, strains of which do not grow using chitin as a carbon source. The significance of these findings in the context of mushroom disease is discussed.     

Chitinolytic actinobacteria isolated from an Algerian semi-arid soil: development of an antifungal chitinase-dependent assay and GH18 chitinase gene identification

The purpose of this study was to explore the microbial potential of a semi-arid sandy soil from south-central Algeria in order to isolate new chitinolytic actinobacteria. This soil is subjected to high temperatures (up to 43 °C) and has low nutrient content. Strains were isolated by plating soil suspensions on Bennett and Colloidal Chitin (CCM) medium. An initial clustering of isolates was made through BOX-PCR genetic profiling. Next, a 16S rRNA gene sequencing of representative isolates was realized. We also identified optimum physicochemical conditions for chitinolytic activity. A rapid in vitro assay based on glucose catabolic repression was developed to select isolates having a chitinase-dependent antifungal activity against two phytopathogenic fungi. Gene identification of glycosyl hydrolase family 18 (GH18) permitted us to assess the divergence of chitinase genes. Forty isolates were obtained from the semi-arid sandy soil. The molecular identification permitted us to assign them to Streptomyces or Micromonospora genera with seven possibly new bacterial species. For chitinolytic activity, 100% of isolates were able to grow and degrade colloidal chitin at pH 7 and at a temperature ranging from 30 to 40 °C. We also observed that Micromonospora strains had atypical activity patterns, with a strong chitinase activity maintained at high temperature. Finally, three strains presented an interesting chitinolytic potential to reduce fungal growth with new GH18 sequences. This study presents a new rapid method to detect antifungal chitinase-dependent activity that allowed to identify potentially new species of actinobacteria and new GH18 gene sequences.     

The composition of the chitinolytic microbial complex and its effect on chitin decomposition at various humidity levels

The dynamics of assimilation of chitin by soil microorganisms (primarily prokaryotes) as a source of carbon and nitrogen has been determined by gas chromatography and fluorescence microscopy. The highest rates of chitin decomposition in chernozem were detected at humidity levels corresponding to the pressure of soil moisture (P) of -1.4 atm. The rate of microbial consumption of chitin is three times higher than that of the carbon of soil organic matter. Fluorescence microscopy revealed that an increase in the pressure of soil moisture from P = -10 atm to P = -0.7 atm resulted in a considerable increase in the proportion of the specific surface of mycelial bacteria (actinomycetes).     

Production of chitinolytic enzymes from a novel species ofAeromonas

A bacterial strain secreting potent chitinolytic activity was isolated from shrimp-pond water by enrichment culture using colloidal crab-shell chitin as the major carbon source. The isolated bacterium, designated asAeromonas sp No. 16 exhibited a rod-like morphology with a polar flagellum. Under optimal culture conditions in 500-ml shaker flasks, it produced a chitinolytic activity of 1.4 U ml^–1. A slightly higher enzymatic activity of 1.5 U ml^–1 was obtained when cultivation was carried out in a 5-liter jar fermentor using a medium containing crystalline chitin as the carbon source. The secretion of the enzyme(s) was stimulated by several organic nitrogenous supplements. Most carbon sources tested (glucose, maltose, N-acetylglucosamine, etc) enhanced cell growth, but they slightly inhibited enzyme secretion. Glucosamine (0.5% w/v) severely inhibited cell growth (16% of the control), but it did not significantly affect enzyme secretion. The production of chitinolytic enzymes was pH sensitive and was enhanced by increasing the concentration of colloidal chitin to 1.5%. The observed chitinolytic activity could be attributed to the presence of -N-acetylglucosaminidase and chitinase. Chitinase was purified by ammonium sulfate fractionation and preparative gel electrophoresis to three major bands on SDS-PAGE. An in-gel enzymatic activity assay indicated that all three bands possessed chitinase activity. Analysis of the enzymatic products indicated that the purified enzyme(s) hydrolyzed colloidal chitin predominantly to N,N-diacetyl-chitobiose and, to a much lesser extent, the mono-, tri, and tetramer of N-acetylglucosamine, suggesting that they are mainly endochitinases.