The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.     

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Chitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Enzymatic deacetylation of chitin by extracellular chitin deacetylase from a newly screened Mortierella sp. DY-52

Among more than a hundred colonies of fungi isolated from soil samples, DY-52 has been screened as an extracellular chitin deacetylase (CDA) producer. The isolate was further identified as Mortierella sp., based on the morphological properties and the nucleotide sequence of its 18S rRNA gene. The fungus exhibited maximal growth in yeast peptone glucose (YPD) liquid medium containing 2% of glucose at pH 5.0 and 28 degrees C with 150 rpm. The CDA activity of DY-52 was maximal (20 U/mg) on the 3rd day of culture in the same medium. The CDA was inducible by addition of glucose and chitin. The enzyme contained two isoforms of molecular mass 50 kDa and 59 kDa. This enzyme showed a maximal activity at pH 5.5 and 60 degrees C. In addition, it had a pH stability range of 4.5-8.0 and a temperature stability range of 4-40 degrees C. The enzyme was enhanced in the presence of Co2+ and Ca2+. Among various substrates tested, WSCT-50 (water-soluble chitin, degree of deacetylation 50%), glycol chitin, and crab chitosan (DD 71-88%) were deacetylated. Moreover, the CDA can handle N-acetylglucosamine oligomers (GlcNAc)2-7.     

虾青素高产菌株的选育

采用激光（Laser）、紫外线（UV）、亚硝基胍（NTG）及其复合诱变法夫酵母菌,再用高浓度葡萄糖培养基选育经复合诱变的法夫酵母菌。结果表明：与原始出发菌相比,菌株经诱变后其虾青素产量有显著提高,其中复合诱变的是青素产量可达7.26μm/ml。诱变高产菌株遗传稳定性好。     

甘蓝夜蛾两种不同类型几丁质脱乙酰基酶基因的克隆与组织特异性表达

几丁质脱乙酰基酶(chitin deacetylase,CDA)是昆虫几丁质降解酶中的一种酶,可以将几丁质转化为壳聚糖,在昆虫几丁质代谢中具有重要作用.本研究以甘蓝夜蛾Mamestra brassicae5龄幼虫虫体为材料提取总RNA,利用RT-PCR和RACE技术,分别扩增得到甘蓝夜蛾的2类不同几丁质脱乙酰基酶基因的...     

Insertional inactivation of virR in Streptococcus pyogenes M49 demonstrates that VirR functions as a positive regulator of ScpA, FcRA, OF, and M protein

Abstract Several reports have shown that Streptococcus pyogenes strains which produce opacity factor (OF+) have diverged significantly from OF⁻ serotypes. This study questions whether several surface proteins of an OF+ culture are regulated by the positive regulatory protein VirR, in a manner similar to OF~ strains. Interruption of the virR region of an OF⁺ S. pyogenes (strain CS101, M type 49) was performed using a temperature-sensitive plasmid containing a fragment of virR . Interruption of the virR region produced cultures with (indétectable amounts of M49 and ScpA proteins, and reduced the yield of FcRA protein. In addition, mutants had a significant reduction in detectable opacity factor. These results suggest that virR functions as a positive regulator of a variety of surface proteins in OF⁺ strains.     

Biological control of rice root-knot nematode,Meloidogyne graminicola through mixture of Pseudomonas fluorescens strains

The plant growth promoting rhizobacterium, Pseudomonas fluorescens strains PF1, TDK1, and PY15 were evaluated individually and in combinations for their efficacy against root-knot nematode, Meloidogyne graminicola, in rice plants under in vitro, glass house and field conditions. Culture filtrates of these strains either individually or as mixture inhibited egg hatching and caused mortality of juveniles of M. graminicola in vitro. The efficacy was more pronounced when filtrates of the strain were used as mixtures than as individual strains. Mixtures of P. fluorescens strains signficantly reduced M. graminicola infestation when applied as bacterial suspensions through seed treatment. The higher activity of peroxidase and chitinase enzymes was observed in plants treated with P. fluorescens mixtures than the plants treated with individual strains, two strain mixtures and untreated control. In field trials on rice, talc formulations of the P. fluorescens strains individually as well as mixtures were evaluated as seed treatment, soil treatment and combination of both. A mixture of the three strains was the most effective when applied either as seed + soil treatment or as seed treatment alone. The introduced P. fluorescens strains survived endophytically on rice roots. The application of the P. fluorescens mixture PF1 + TDK1 + PY15 in seed + soil treatment resulted in higher grain yield which provided a 27.3% increase over the control followed by P. fluorescens mixture PF1 + TDK1 + PY15 in seed treatment alone, which increased the grain yield of rice by 24.7% compared to the control.     

Adaptive Evolution of Mus Apobec3 Includes Retroviral Insertion and Positive Selection at Two Clusters of Residues Flanking the Substrate Groove

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity.     

Domain organization and phylogenetic analysis of proteins from the chitin deacetylase gene family of Tribolium castaneum and three other species of insects

A bioinformatics investigation of four insect species with annotated genome sequences identified a family of genes encoding chitin deacetylase (CDA)-like proteins, with five to nine members depending on the species. CDAs (EC 3.5.1.41) are chitin-modifying enzymes that deacetylate the beta-1,4-linked N-acetylglucosamine homopolymer. Partial deacetylation forms a heteropolysaccharide that also contains some glucosamine residues, while complete deacetylation produces the homopolymer chitosan, consisting exclusively of glucosamine. The genomes of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, the malaria mosquito, Anopheles gambiae, and the honey bee, Apis mellifera contain 9, 6, 5 and 5 genes, respectively, that encode proteins with a chitin deacetylase motif. The presence of alternative exons in two of the genes, TcCDA2 and TcCDA5, increases the protein diversity further. Insect CDA-like proteins were classified into five orthologous groups based on phylogenetic analysis and the presence of additional motifs. Group I enzymes include CDA1 and isoforms of CDA2, each containing in addition to a polysaccharide deacetylase-like catalytic domain, a chitin-binding peritrophin-A domain (ChBD) and a low-density lipoprotein receptor class A domain (LDLa). Group II is composed of CDA3 orthologs from each insect species with the same domain organization as group I CDAs, but differing substantially in sequence. Group III includes CDA4s, which have the ChBD domain but do not have the LDLa domain. Group IV comprises CDA5s, which are the largest CDAs because of a very long intervening region separating the ChBD and catalytic domains. Among the four insect species, Tribolium is unique in having four CDA genes in group V, whereas the other insect genomes have either one or none. Most of the CDA-like proteins have a putative signal peptide consistent with their role in modifying extracellular chitin in both cuticle and peritrophic membrane during morphogenesis and molting.     

The chitosan yield of zygomycetes at their optimum harvesting time

Fungi are a promising alternative source of chitosan. Fungi can be manipulated to give chitosan of more consistent and desired physico-chemical properties compared to chitosan obtained from crustacean sources. Chitosan was extracted from the mycelia of Rhizopus oryzae USDB 0602 at various phases of growth. The growth phase which produced the most extractable chitosan was determined to be the late exponential phase. In contrast to previous work on the screening of chitosan from fungal sources, mycelia of the fungi used in this study were harvested at their late exponential growth phase instead of at a fixed incubation time. The amount of extractable chitosan varied widely among the fungal strains. Gongronella butleri USDB 0201 was found to produce the highest amount of extractable chitosan per ml of substrate, followed by Cunninghamella echinulata and Gongronella butleri USDB 0428. However, in terms of yield of chitosan per unit mycelia mass, C. echinulata was the best strain among all fungi in the experiment. Therefore, besides G. butleri USDB 0201, C. echinulata can also be considered to be used in the commercial production of chitosan.     

Peripheral enzymatic deacetylation of chitin and reprecipitated chitin particles

The enzymatic deacetylation of various chitin preparations was investigated using the fungal chitin deacetylase (CDA) isolated from Rhizopus oryzae growth medium. Specific extracellular enzyme activity after solid state fermentation was 10 times higher than that after submerged fermentation. Natural crystalline chitin is a very poor substrate for the enzyme, but showed a five-time better deacetylation after dissolution and reprecipitation. Chitin particles, enzymatically deacetylated for only 1% exhibited a strongly increased binding capacity towards ovalbumin, while maintaining the rigidity and insolubility of chitin in a moderate acidic environment. Because of the unique combination of properties, these CDA treated chitin materials were named "chit-in-osan". Chitinosan was shown to be an attractive matrix for column chromatography because no hydrogel formation was observed, that impaired the flow of eluent. Under the same conditions, partially deacetylated chitosan swelled and blocked the flow in the column.     

Selective antimicrobial action of chitosan against spoilage yeasts in mixed culture fermentations

The effect of chitosan on Saccharomyces cerevisiae (the yeast that carries out alcohol fermentation), Brettanomyces bruxellensis and Brettanomyces intermedius (contaminants of alcohol fermentations), was investigated. The effect of chitosan was tested on each yeast, as well as on mixed cultivations of S. cerevisiae + B. bruxellensis and S. cerevisiae + B. intermedius. Chitosan enhanced the lag period of both strains of Brettanomyces (80 h for B. bruxellensis and 170 h for B. intermedius with 6 and 2 g/l chitosan, respectively). The growth rate of S. cerevisiae was inversely proportional to the chitosan concentration; the former was 50% when 6 g/l polysaccharide was used. Moreover, in mixed cultivations of S. cerevisiae and Brettanomyces strains, it was found that both B. bruxellensis and B. intermedius failed to grow while growth of S. cerevisiae was not affected (using 3 and 6 g/l chitosan, respectively). An interesting collateral result was that the presence of chitosan accelerated the consumption of glucose in the mixed cultivations (60 h instead of 120 h).     

An Enzymatic Assay for High-Throughput Screening of Cytidine-Producing Microbial Strains

Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH₃, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058 - 10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more).     

Further studies on the subcellular distribution of Cd2+ in Cd-sensitive and Cd-resistant strains of Saccharomyces cerevisiae

When a Cd-resistant strain (301 N) and a Cd-sensitive strain (101 N) of Saccharomyces cerevisiae were incubated in medium containing Cd2+, a large proportion of the cellular Cd2+ was found in the cytosol of strain 301 N, but not in that of strain 101 N. Approximately 65% of the cellular Cd2+ was released from strain 301 N after treatment with chitosan, which affects cell membrane permeability. About 80% of the cellular Cd2+ released from strain 301 N by chitosan treatment was detected in a 30 000-10 000 molecular weight fraction prepared by ultrafiltration. The distribution of Cd2+ into the cytosol in strain 301 N was inhibited in the presence of cycloheximide. The proportion of cellular Cu2+ or Zn2+ present in the cytosol after incubation with these ions was similar for the two strains (about 40%).     

Production of fungal chitosan by solid substrate fermentation followed by enzymatic extraction

An improved method is described for the production of chitosan from mycelia of the fungus Gongronella butleri, grown by solid substrate fermentation on sweet potato. The chitosan was extracted subsequently by 11 M NaOH at 45 °C, and 0.35 M acetic acid at 95 °C. The resulting extract was clarified using a heat-stable, commercial -amylase. The yield (4–6 g/100 g mycelia) and relative number average molecular weight (44–54 kDa) of the chitosan increased with increasing duration of fungal growth up to the sixth day.     

Chitin deacetylase: A potential target for Mythimna separata (Walker) control

Chitin deacetylase (CDA) is a hydrolytic enzyme that modifies chitin into chitosan in the body of insects. In this study, we obtained a full-length complementary DNA sequence (MsCDA1) from the oriental armyworm Mythimna separata by high-throughput sequencing. MsCDA1 is 1,952 bp long and includes 1,620 bp open reading frame encoding 539 amino acids. Analysis by quantitative real time polymerase chain reaction showed that MsCDA1 expression was higher at the adult stage than at earlier developmental stages. MsCDA1 was expressed in all larval tissues examined, in which the highest expression level was found in the midgut. The RNA interference (RNAi) suppressed MsCDA1 expression levels at 12, 24, and 48 hr after injection of double-stranded RNA (1–4 μg per larva) specific to MsCDA1. Under RNAi condition, CDA enzyme activity was significantly reduced and changes an ultramicroscopic structure of M. separata peritrophic matrix especially in its microfibrillar organization exhibiting loose network. In contrast, the surface of the peritrophic matrix was relatively smooth and well organized at control or low RNAi conditions. Moreover, RNAi of MsCDA1 expression impaired larval growth and development, occasionally leading to larval death. These results demonstrate that MsCDA1 plays a crucial role in maintaining peritrophic matrix integrity in M. separata.     

Chitosan from Syncephalastrum racemosum used as a film support for lipase immobilization

Chitosan from a native Mucoralean strain, Syncephalastrum racemosum, isolated from herbivorous dung (Northeast-Brazil), was used as a film support for lipase immobilization. S. racemosum showed highest chitosan yield (152 mg g dry mycelia weight(-1); 15.2% of dry mycelia weight) among the nine strains screened, which presented 89% D-glucosamine. A chitosan film was used for lipase (EC 3.1.1.3) immobilization using glutaraldehyde as a bifunctional agent. The immobilized lipase retained 47% (12.6 micromol s(-1) m(-2)) of its initial catalytic activity after four cycles of reaction. This result is comparable (same order of magnitude) to that of the enzyme immobilized on film made from commercially available crustacean chitosan.     

Functional properties of subunit interactions in human cytidine deaminase

An intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/W113F. F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions. In the presence of SDS, wild-type human CDA dissociates into enzymatically inactive monomers without intermediate forms via a non-cooperative transition. Extensive dialysis or dilution of the inactivated monomers restores completely the activity. Circular dichroism measurements show that the secondary/tertiary structure organization of each subunit is unaffected by the SDS concentration, while the mutation Phe/Trp causes weakening in quaternary structure. The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavours dissociation of the tetramer into subunits in the wild-type CDA, but not in mutant enzyme F137W/W113F. The absence of tyrosine fluorescence and the much higher quantum yield of the double mutant protein spectrum suggest the occurrence of an energy transfer effect between the protein subunits. This assumption is confirmed by the crystallographic studies on B.subtilis in which it is shown that three different subunits concur with the formation of each of the four active sites and that F125, homologous to the human CDA F137, is located at the interface between two different subunits contributing to the formation of active site.     

Recurrent mutation and selection for increased penicillin titre in Aspergillus nidulans.

Recurrent mutation and selection has been used to increase penicillin titre in two closely related strains of Aspergillus nidulans. A selection programme was initiated from each of the two strains (programmes A and B) and continued through six cycles of mutation and selection. Near-ultraviolet light in conjuction with 8-methoxypsoralen was employed as the sole mutagen throughout programme A and ethyl methanesulphonate as the sole mutagen throughout programme B. Excluding the first cycle of A, where only 50 strains were assayed, the selection programmes were identical. In each programme, 100 survivors were assayed for penicillin titre after each mutagenic treatment and, on the basis of a single yield test, the best five strains were picked and carried forward to the next cycle. In both selection programmes, a near 300% increase in penicillin titre was achieved. This yield advance illustrates the effectiveness for strain development of experimental designs involving successive cycles of mutagenesis with a single-stage screen and the selection of the top few percent survivors in each cycle.