Chitin Degradation Research Articles

Lytic Polysaccharide Monooxygenases as Chitin-Specific Virulence Factors in Crayfish Plague.

The oomycete pathogen Aphanomyces astaci, also known as “crayfish plague”, is an obligate fungal-like parasite of freshwater crustaceans and is considered responsible for the ongoing decline of native European crayfish populations. A. astaci is thought to secrete a wide array of effectors and enzymes that facilitate infection, however their molecular mechanisms have been poorly characterized. Here, we report the identification of AA15 lytic polysaccharide monooxygenases (LPMOs) as a new group of secreted virulence factors in A. astaci. We show that this enzyme family has greatly expanded in A. astaci compared to all other oomycetes, and that it may facilitate infection through oxidative degradation of crystalline chitin, the most abundant polysaccharide found in the crustacean exoskeleton. These findings reveal new roles for LPMOs in animal–pathogen interactions, and could help inform future strategies for the protection of farmed and endangered species.

Seafood Processing Chitin Waste for Electricity Generation in a Microbial Fuel Cell Using Halotolerant Catalyst Oceanisphaera arctica YHY1

In this study, a newly isolated halotolerant strain Oceanisphaera arctica YHY1, capable of hydrolyzing seafood processing waste chitin biomass, is reported. Microbial fuel cells fed with 1% chitin and 40 g L−1 as the optimum salt concentration demonstrated stable electricity generation until 216 h (0.228 mA/cm2). N-acetyl-D-glucosamine (GlcNAc) was the main by-product in the chitin degradation, reaching a maximum concentration of 192.01 mg g−1 chitin at 120 h, whereas lactate, acetate, propionate, and butyrate were the major metabolites detected in the chitin degradation. O. arctica YHY1 utilized the produced GlcNAc, lactate, acetate, and propionate as the electron donors to generate the electric current. Cyclic voltammetry (CV) investigation revealed the participation of outer membrane-bound cytochromes, with extracellular redox mediators partly involved in the electron transfer mechanism. Furthermore, the changes in structural and functional groups in chitin after degradation were analyzed using FTIR and XRD. Therefore, the ability of O. arctica YHY1 to utilize waste chitin biomass under high salinities can be explored to treat seafood processing brine or high salt wastewater containing chitin with concurrent electricity generation.

Draft Genome Sequence of the Chitin-Degrading Psychrotolerant Bacterium Pedobacter jejuensis TN23, Isolated from Antarctic Soil.

ABSTRACTPedobacter jejuensis TN23 was isolated from soil from Terra Nova Bay, Victoria Land, Antarctica. The assembled draft genome size is 4,795,808 bp, and it contains a total of 4,095 genes with 3,970 coding sequences, including genes putatively involved in the degradation of chitin.

Comparative Analyses of the Gut Microbiome of Two Fox Species, the Red Fox (Vulpes Vulpes) and Corsac Fox (Vulpes Corsac), that Occupy Different Ecological Niches.

The gut microbiome is integral for the host's living and environmental adaptation and crucially important for understanding host adaptive mechanisms. The red fox (Vulpes vulpes) dominates a wider ecological niche and more complicated habitat than that of the corsac fox (V. corsac). However, the adaptive mechanisms (in particular, the gut microbiome responsible for this kind of difference) are still unclear. Therefore, we investigated the gut microbiome of these two species in the Hulunbuir grassland, China, and evaluated their microbiome composition, function, and adaptive mechanisms. We profiled the gut microbiome and metabolism function of red and corsac foxes via 16S rRNA gene and metagenome sequencing. The foxes harbored species-specific microbiomes and functions that were related to ecological niche and habitat. The red fox had abundant Bacteroides, which leads to significant enrichment of metabolic pathways (K12373 and K21572) and enzymes related to chitin and carbohydrate degradation that may help the red fox adapt to a wider niche. The corsac fox harbored large proportions of Blautia, Terrisporobacter, and ATP-binding cassette (ABC) transporters (K01990, K02003, and K06147) that can help maintain corsac fox health, allowing it to live in harsh habitats. These results indicate that the gut microbiome of the red and corsac foxes may have different abilities which may provide these species with differing capabilities to adapt to different ecological niches and habitats, thus providing important microbiome data for understanding the mechanisms of host adaptation to different niches and habitats.

A Piperine-Based Scaffold as a Novel Starting Point to Develop Inhibitors against the Potent Molecular Target OfChtI.

The insect chitinase OfChtI from the agricultural pest Ostrinia furnacalis (Asian corn borer) is a promising target for green insecticide design. OfChtI is a critical chitinolytic enzyme for the cuticular chitin degradation at the stage of molting. In this study, piperine, a natural amide compound isolated from black pepper, Piper nigrum L., was discovered for the first time to have inhibitory activity toward OfChtI. The compound-enzyme interaction was presumed to take place between the piperine benzo[d][1,3] dioxole skeleton and subsite -1 of the substrate-binding pocket of OfChtI. Hence, on the basis of the deduced inhibitory mechanism and crystal structure of the substrate-binding cavity of OfChtI, compounds 5a-f were designed and synthesized by introducing a butenolide scaffold into the lead compound piperine. The enzymatic activity assay indicated that compounds 5a-f (Ki = 1.03-2.04 μM) exhibited approximately 40-80-fold higher inhibitory activity than the lead compound piperine (I) (Ki = 81.45 μM) toward OfChtI. The inhibitory mechanism of the piperonyl butenolide compounds was elucidated by molecular dynamics, which demonstrated that the introduced butenolide skeleton improved the binding affinity to OfChtI. Moreover, the in vivo activity assay indicated that these compounds also displayed moderate insecticidal activity toward O. furnacalis. This work introduces the natural product piperine as a starting point for the development of novel insecticides targeting OfChtI.

Chitinases and Chitinase-Like Proteins as Therapeutic Targets in Inflammatory Diseases, with a Special Focus on Inflammatory Bowel Diseases.

Chitinases belong to the evolutionarily conserved glycosyl hydrolase family 18 (GH18). They catalyze degradation of chitin to N-acetylglucosamine by hydrolysis of the β-(1-4)-glycosidic bonds. Although mammals do not synthesize chitin, they possess two enzymatically active chitinases, i.e., chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase), as well as several chitinase-like proteins (YKL-40, YKL-39, oviductin, and stabilin-interacting protein). The latter lack enzymatic activity but still display oligosaccharides-binding ability. The physiologic functions of chitinases are still unclear, but they have been shown to be involved in the pathogenesis of various human fibrotic and inflammatory disorders, particularly those of the lung (idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, sarcoidosis, and asthma) and the gastrointestinal tract (inflammatory bowel diseases (IBDs) and colon cancer). In this review, we summarize the current knowledge about chitinases, particularly in IBDs, and demonstrate that chitinases can serve as prognostic biomarkers of disease progression. Moreover, we suggest that the inhibition of chitinase activity may be considered as a novel therapeutic strategy for the treatment of IBDs.

A Novel Nitrogen and Carbon Metabolism Regulatory Cascade Is Implicated in Entomopathogenicity of the Fungus Metarhizium robertsii.

ABSTRACTThe entomopathogenic fungus Metarhizium robertsii can switch among parasitic, saprophytic, and symbiotic lifestyles in response to changing nutritional conditions, which is attributed to its extremely versatile metabolism. Here, we found that the Fus3–mitogen-activated protein kinase (MAPK) and the transcription factor regulator of nutrient selection 1 (RNS1) constitute a novel fungal cascade that regulates the degradation of insect cuticular lipids, proteins, and chitin to obtain nutrients for hyphal growth and enter the insect hemocoel for subsequent colonization. On the insect cuticle, Fus3-MAPK physically contacts and phosphorylates RNS1, which facilitates the entry of RNS1 into nuclei. The phosphorylated RNS1 binds to the DNA motif BM2 (ACCAGAC) in its own promoter to self-induce expression, which then activates the expression of genes for degrading cuticular proteins, chitin, and lipids. We further found that the Fus3-MAPK/RNS1 cascade also activates genes for utilizing complex and less-favored nitrogen and carbon sources (casein, colloid chitin, and hydrocarbons) that were not derived from insects, which is repressed by favored organic carbon and nitrogen nutrients, including glucose and glutamine. In conclusion, we discovered a novel regulatory cascade that controls fungal nitrogen and carbon metabolism and is implicated in the entomopathogenicity of M. robertsii.IMPORTANCE Penetration of the cuticle, the first physical barrier to pathogenic fungi, is the prerequisite for fungal infection of insects. In the entomopathogenic fungus Metarhizium robertsii, we found that the Fus3–mitogen-activated protein kinase (MAPK) and the transcription factor regulator of nutrient selection 1 (RNS1) constitute a novel cascade that controls cuticle penetration by regulating degradation of cuticular lipids, proteins, and chitin to obtain nutrients for hyphal growth and entry into the insect hemocoel. In addition, during saprophytic growth, the Fus3-MAPK/RNS1 cascade also activates utilization of complex and less-favored carbon and nitrogen sources that are not derived from insects. The homologs of Fus3-MAPK and RNS1 are widely found in ascomycete filamentous fungi, including saprophytes and pathogens with diverse hosts, suggesting that the regulation of utilization of nitrogen and carbon sources by the Fus3-MAPK/RNS1 cascade could be widespread. Our work provides significant insights into regulation of carbon and nitrogen metabolism in fungi and fungal pathogenesis in insects.

Chaperone-assisted soluble expression and characterization of chitinase chiZJ408 in Escherichia coli BL21 and the chitin degradation by recombinant enzyme

The chaperone-assisted soluble expression and characterization of high molecular weight chitinase chiZJ408 in Escherichia coli BL21 were investigated. Chitin degradation products by chitinase chiZJ408 were analyzed. The results indicated that the introduction of the chaperone GroELS promoted the correct folding of chitinase chiZJ408 and increased its soluble expression by 14.9% (p < 0.05) in E. coli BL21. The optimal pH and temperature of chitinase chiZJ408 were respectively 6.0 and 50 °C. Chitinase chiZJ408 was stable at pHs of 4.0 ∼ 7.0 and at below 40 °C. Mg2+and Ca2+ had a significant impact on improving the activity of chitinase chiZJ408. Chitinase chiZJ408 was demonstrated to be exochitinase that cleaved the β-1,4-glycosidic bond of the chitin chain to generate only N,N’-diacetylchitobiose. This study broadens our understanding of chitinase and provides a basis for solving the problem of inclusion body formed by long fragment gene expression in E. coli.

The holobiont transcriptome of teneral tsetse fly species of varying vector competence

BackgroundTsetse flies are the obligate vectors of African trypanosomes, which cause Human and Animal African Trypanosomiasis. Teneral flies (newly eclosed adults) are especially susceptible to parasite establishment and development, yet our understanding of why remains fragmentary. The tsetse gut microbiome is dominated by two Gammaproteobacteria, an essential and ancient mutualist Wigglesworthia glossinidia and a commensal Sodalis glossinidius. Here, we characterize and compare the metatranscriptome of teneral Glossina morsitans to that of G. brevipalpis and describe unique immunological, physiological, and metabolic landscapes that may impact vector competence differences between these two species.ResultsAn active expression profile was observed for Wigglesworthia immediately following host adult metamorphosis. Specifically, ‘translation, ribosomal structure and biogenesis’ followed by ‘coenzyme transport and metabolism’ were the most enriched clusters of orthologous genes (COGs), highlighting the importance of nutrient transport and metabolism even following host species diversification. Despite the significantly smaller Wigglesworthia genome more differentially expressed genes (DEGs) were identified between interspecific isolates (n = 326, ~ 55% of protein coding genes) than between the corresponding Sodalis isolates (n = 235, ~ 5% of protein coding genes) likely reflecting distinctions in host co-evolution and adaptation. DEGs between Sodalis isolates included genes involved in chitin degradation that may contribute towards trypanosome susceptibility by compromising the immunological protection provided by the peritrophic matrix. Lastly, G. brevipalpis tenerals demonstrate a more immunologically robust background with significant upregulation of IMD and melanization pathways.ConclusionsThese transcriptomic differences may collectively contribute to vector competence differences between tsetse species and offers translational relevance towards the design of novel vector control strategies.

Transcriptional profile of oil palm pathogen, Ganoderma boninense, reveals activation of lignin degradation machinery and possible evasion of host immune response

BackgroundThe white-rot fungi in the genus Ganoderma interact with both living and dead angiosperm tree hosts. Two Ganoderma species, a North American taxon, G. zonatum and an Asian taxon, G. boninense, have primarily been found associated with live palm hosts. During the host plant colonization process, a massive transcriptional reorganization helps the fungus evade the host immune response and utilize plant cell wall polysaccharides.ResultsA publicly available transcriptome of G. boninense - oil palm interaction was surveyed to profile transcripts that were differentially expressed in planta. Ten percent of the G. boninense transcript loci had altered expression as it colonized oil palm plants one-month post inoculation. Carbohydrate active enzymes (CAZymes), particularly those with a role in lignin degradation, and auxiliary enzymes that facilitate lignin modification, like cytochrome P450s and haloacid dehalogenases, were up-regulated in planta. Several lineage specific proteins and secreted proteins that lack known functional domains were also up-regulated in planta, but their role in the interaction could not be established. A slowdown in G. boninense respiration during the interaction can be inferred from the down-regulation of proteins involved in electron transport chain and mitochondrial biogenesis. Additionally, pathogenicity related genes and chitin degradation machinery were down-regulated during the interaction indicating G. boninense may be evading detection by the host immune system.ConclusionsThis analysis offers an overview of the dynamic processes at play in G. boninense - oil palm interaction and provides a framework to investigate biology of Ganoderma fungi across plantations and landscape.

Physiological characterization of chitin synthase A responsible for the biosynthesis of cuticle chitin in Culex pipiens pallens (Diptera: Culicidae)

BackgroundThe pathogens transmitted by mosquitoes to humans and animals cause several emerging and resurgent infectious diseases. Increasing insecticide resistance requires rational action to control the target vector population. Chitin is indispensable for insect growth and development and absent from vertebrates and higher plants. Chitin synthase A (CHSA) is a crucial enzyme in chitin synthesis; therefore, identifying and characterizing how CHSA determines chitin content may contribute to the development of novel vector control strategies.ResultsThe injection of small interfering RNA targeting CHSA (siCHSA) to knockdown CHSA transcripts in larval, pupal and adult stages of Culex pipiens pallens resulted in the appearance of different lethal phenotypes. When larval and pupal stages were injected with siCHSA, CHSA knockdown prevented larval molting, pupation and adult eclosion, and affected the production of chitin and chitin degradation, which resulted in an ecdysis defect phenotype of mosquitoes. When siCHSA was injected into mosquitoes in the adult stage, CHSA knockdown also affected the laminar organization of the mesoderm and the formation of pseudo-orthogonal patterns of the large fibers of the endoderm.ConclusionWe provide a systematic and comprehensive description of the effects of CHSA on morphogenesis and metamorphosis. The results show that CHSA not only affects chitin synthesis during molting, but also might be involved in chitin degradation. Our results further show that CHSA is important for the structural integrity of the adult mosquito cuticle.Graphical

RNAi-Mediated Silencing of the Chitinase 5 Gene for Fall Webworm (Hyphantria cunea) Can Inhibit Larval Molting Depending on the Timing of dsRNA Injection.

Simple SummaryThe fall webworm, Hyphantria cunea, is a worldwide invasive pest causing serious ecological and economic damage. The use of RNAi is a feasible strategy for controlling this pest. In this study, we evaluated the importance of the chitinase 5 gene (HcCht5) in the development of H. cunea larvae. We found that the RNAi-mediated silencing of HcCht5 arrested molting and caused larval mortality depending on the dsRNA injection time. The silencing of HcCht5 down-regulated genes that were related to chitin metabolism, molting hormone signaling, and detoxification metabolism. Our findings indicate that HcCht5 is an important gene in regulating larval development and a promising target for RNAi-mediated pest management of the pest H. cunea.Chitinases, which are crucial enzymes required for chitin degradation and reconstruction, are often selectively considered to be effective molecular targets for pest control due to their critical roles in insect development. Although the Hyphantria cunea chitinase gene has been reported previously, its sequence characteristics, gene function, and feasibility as a potential target for pest management were absent. In the present study, we characterized the H. cunea chitinase gene and designated it HcCht5. Phylogenic and domain structure analysis suggested that HcCht5 contained the typical chitinase features and was clustered into chitinase group I. Tissue-specific and developmental expression pattern analysis with Real-Time Quantitative PCR (RT-qPCR) showed that HcCht5 was mainly expressed in the integument tissues and that the transcript levels peaked during molting. RNA interference (RNAi)-mediated silencing of HcCht5 caused 33.3% (2 ug) and 66.7% (4 ug) mortality rates after double-stranded RNA (dsRNA) injection. Importantly, the interference efficiency of HcCht5 depended on the injection time of double-stranded RNA (dsRNA), as the pre-molting treatment achieved molt arrest more effectively. In addition, transcriptome sequencing (RNA-seq) analysis of RNAi samples demonstrated silencing of the down-regulated HcCht5 genes related to chitin metabolism and molting hormone signaling, as well as genes related to detoxification metabolism. Our results indicate the essential role of HcCht5 in H. cunea development and detail the involvement of its gene function in the larval molting process.

Marinifaba aquimaris gen. nov., sp. nov., a novel chitin-degrading gammaproteobacterium in the family Alteromonadaceae isolated from seawater of the Mariana Trench.

A novel Gram-negative, rod-shaped, aerobic, oxidase-positive and catalase-negative bacterium, designated strain SM1970T, was isolated from a seawater sample collected from the Mariana Trench. Strain SM1970T grew at 15-37 oC and with 1-5% (w/v) NaCl. It hydrolyzed colloidal chitin, agar and casein but did not reduce nitrate to nitrite. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain SM1970T formed a distinct lineage close to the genusCatenovulum within the familyAlteromonadaceae, sharing the highest sequence similarity (93.6%) with type strain ofCatenovulum maritimum but < 93.0% sequence similarity with those of other known species in the classGammaproteobacteria. The major fatty acids of strain SM1970T were summed feature 3 (C16: 1ω7c and/or C16: 1ω6c), C16: 0 and summed feature 8 (C18: 1ω7c and/or C18: 1ω6c). The major polar lipids of the strain included phosphatidylethanolamine and phosphatidylglycerol and its main respiratory quinone was ubiquinone 8. The draft genome of strain SM1970T consisted of 77 scaffolds and was 4,172,146bp in length, containing a complete set of genes for chitin degradation. The average amino acid identity (AAI) values between SM1970T and type strains of knownCatenovulum species were 56.6-57.1% while the percentage of conserved proteins (POCP) values between them were 28.5-31.5%. The genomic DNA G + C content of strain SM1970T was 40.1mol%. On the basis of the polyphasic analysis, strain SM1970T is considered to represent a novel species in a novel genus of the familyAlteromonadaceae, for which the nameMarinifaba aquimaris is proposed with the type strain being SM1970T (= MCCC 1K04323T = KCTC 72844T).

Structures and functions of carbohydrate-active enzymes of chitinolytic bacteria Paenibacillus sp. str. FPU-7.

Chitin and its derivatives have valuable potential applications in various fields that include medicine, agriculture, and food industries. Paenibacillus sp. str. FPU-7 is one of the most potent chitin-degrading bacteria identified. This review introduces the chitin degradation system of P. str. FPU-7. In addition to extracellular chitinases, P. str. FPU-7 uses a unique multimodular chitinase (ChiW) to hydrolyze chitin to oligosaccharides on the cell surface. Chitin oligosaccharides are converted to N-acetyl-d-glucosamine by β-N-acetylhexosaminidase (PsNagA) in the cytosol. The functions and structures of ChiW and PsNagA are also summarized. The genome sequence of P. str. FPU-7 provides opportunities to acquire novel enzymes. Genome mining has identified a novel alginate lyase, PsAly. The functions and structure of PsAly are reviewed. These findings will inform further improvement of the sustainable conversion of polysaccharides to functional materials.

Microbial responses to herbivory-induced vegetation changes in a high-Arctic peatland

Herbivory by barnacle geese (Branta leucopsis) alters the vegetation cover and reduces ecosystem productivity in high-Arctic peatlands, limiting the carbon sink strength of these ecosystems. Here we investigate how herbivory-induced vegetation changes affect the activities of peat soil microbiota using metagenomics, metatranscriptomics and targeted metabolomics in a comparison of fenced exclosures and nearby grazed sites. Our results show that a different vegetation with a high proportion of vascular plants developed due to reduced herbivory, resulting in a larger and more diverse input of polysaccharides to the soil at exclosed study sites. This coincided with higher sugar and amino acid concentrations in the soil at this site as well as the establishment of a more abundant and active microbiota, including saprotrophic fungi with broad substrate ranges, like Helotiales (Ascomycota) and Agaricales (Basidiomycota). A detailed description of fungal transcriptional profiles revealed higher gene expression for cellulose, hemicellulose, pectin, lignin and chitin degradation at herbivory-exclosed sites. Furthermore, we observed an increase in the number of genes and transcripts for predatory eukaryotes such as Entomobryomorpha (Arthropoda). We conclude that in the absence of herbivory, the development of a vascular vegetation alters the soil polysaccharide composition and supports larger and more active populations of fungi and predatory eukaryotes.

Enzymatic activity and virulence of Cordyceps locustiphila (Hypocreales: Cordycipitaceae) on the South American locust Schistocerca cancellata (Orthoptera: Acrididae)

The first barrier, when penetrating an insect host, encountered by microorganisms like fungi is insects cuticle; fungi produce a wide variety of extracellular enzymes involved in the degradation of protein, chitin, and lipids. The main objectives were to assay the enzymatic activity of Cordyceps locustiphila anamorph, in solid and liquid medium at different temperatures and also to evaluate the pathogenicity against the South American locust, Schistocerca cancellata. Conidia of C. locustiphila anamorph were adjusted to 1 × 104, 1 × 106 and 1 × 108 conidia/ml and mortality was recorded for S. cancellata nymphs. The enzymatic activities were determined through a plate test and liquid medium. When assessing the pathogenicity, the fungus caused the highest mortality (84.5 ± 3.5%) at a dose of 1 × 108 conidia/ml. The proteolytic activity showed the highest values (1.56 ± 0.21) (U) at 26 °C. The highest lipolytic activity (1.13 ± 0.36) (U) was observed at 26 °C, while the highest chitinolytic activity was of 0.85 ± 0.1 (U) at 4 °C. Significant differences were observed for enzymatic production on liquid medium, the highest values were recorded for chitinolytic activity (1.63 ± 0.04) (U) and the lowest for caseinolytic activity (0.04 ± 0.001) (U). The results obtained on mortality as well as the quality and variety of enzymes produced by C. locustiphila suggest that this fungus has features that make it a good candidate to be used as a biological agent for the control of the South American locust.

Genome-Wide Identification and Analysis of Chitinase-Like Gene Family in Bemisia tabaci (Hemiptera: Aleyrodidae).

Simple SummaryFor the sake of growth and development, old exuviums of Bemisia tabaci nymphs should be periodically substituted by new ones until a final emergence turns them into adults. During ecdysis, chitinases are of great importance in chitin degradation and turnover of cuticles, which provides us potential targets for RNA interference-based B. tabaci management. Therefore, we annotated 14 chitinase and chitinase-like genes in B. tabaci based on data of genome and transcriptomes. With the help of a nanomaterial-promoted RNAi method, we found that silencing of BtCht10, BtCht5, and BtCht7 resulted in significant increase of death rate on B. tabaci nymphs and the developmental duration was noticeably postponed for BtCht2-silenced nymphs.Chitinases are of great importance in chitin degradation and remodeling in insects. However, the genome-wide distribution of chitinase-like gene family in Bemsia tabaci, a destructive pest worldwide, is still elusive. With the help of bioinformatics, we annotated 14 genes that encode putative chitinase-like proteins, including ten chitinases (Cht), three imaginal disk growth factors (IDGF), and one endo-β-N-acetylglucosaminidase (ENGase) in the genome of the whitefly, B. tabaci. These genes were phylogenetically grouped into eight clades, among which 13 genes were classified in the glycoside hydrolase family 18 groups and one in the ENGase group. Afterwards, developmental expression analysis suggested that BtCht10, BtCht5, and BtCht7 were highly expressed in nymphal stages and exhibit similar expression patterns, implying their underlying role in nymph ecdysis. Notably, nymphs exhibited a lower rate of survival when challenged by dsRNA targeting these three genes via a nanomaterial-promoted RNAi method. In addition, silencing of BtCht10 significantly resulted in a longer duration of development compared to control nymphs. These results indicate a key role of BtCht10, BtCht5, and BtCht7 in B. tabaci nymph molting. Our research depicts the differences of chitinase-like family genes in structure and function and identified potential targets for RNAi-based whitefly management.

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A.

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

Chitin Synthesis and Degradation in Crustaceans: A Genomic View and Application.

Chitin is among the most important components of the crustacean cuticular exoskeleton and intestinal peritrophic matrix. With the progress of genomics and sequencing technology, a large number of gene sequences related to chitin metabolism have been deposited in the GenBank database in recent years. Here, we summarized the genes and pathways associated with the biosynthesis and degradation of chitins in crustaceans based on genomic analyses. We found that chitin biosynthesis genes typically occur in single or two copies, whereas chitin degradation genes are all multiple copies. Moreover, the chitinase genes are significantly expanded in most crustacean genomes. The gene structure and expression pattern of these genes are similar to those of insects, albeit with some specific characteristics. Additionally, the potential applications of the chitin metabolism genes in molting regulation and immune defense, as well as industrial chitin degradation and production, are also summarized in this review.

Plant growth-promoting potential of 'Myroides gitamensis' isolated from virgin soils of Punjab.

Phosphate-solubilizing (PS) and phosphate-mineralizing (PM) bacteria are considered vital for augmenting the plant growth through phosphorus mobilization and plant growth-promoting attributes. In the present study, a rhizospheric bacterium was isolated from the virgin land of Punjab, India and identified as 'Myroides gitamensis' BSH-3 through 16S rRNA sequencing. 'M. gitamensis' showed potential halo zone on Pikovskaya agar. The novelty of the study lies in the fact that plant growth-promoting potential of 'M. gitamensis' has not been studied earlier. It was able to solubilize 17.53-106.66µg/mL of tricalcium phosphate and demonstrated a promising potential of mineralizing sodium phytate corresponding to 44.6-94.70µg/mL at 28°C. Variable PS and PM activity was observed at temperature range of 15-42°C with the maximum activity observed at 28°C after 96h of incubation. The nitrogen fixation ability, hydrogen sulfide production, cellulose hydrolysis test and chitin degradation was found to be negative. High indole acetic acid (42.82µg/mL), gibberellic acid (72.93µg/mL), ammonia (22.58µg/mL) production, phytase activity (0.49 pi/mL/min) and comparable amount of siderophore (28.55%) and acid phosphate activity (0.606µM p-nitrophenol/ml/min) was shown by 'M. gitamensis'. Inoculation of wheat with 'M. gitamensis' in pot experiment showed increased shoot and root length by 30.58% and 38.32%. Fresh weight and dry weight was increased by 45.74% and 67.81%, respectively, compared to uninoculated control. These results demonstrate that 'M. gitamensis' has promising PS, PM and plant growth-promoting attributes to be used as a bio-inoculant to enhance plant growth and soil fertility.