Diversity analysis of endophytes with antimicrobial and antioxidant potential from Viola odorata: an endemic plant species of the Himalayas.

Viola odorata, also known as "Banafshah" in high altitudes of Himalayas, is well known for its pharmaceutical importance in Ayurvedic and Unani medicinal system. The plant is a source of various drugs for its anti-inflammatory, diaphoretic, diuretic, emollient, expectorant, antipyretic, and laxative properties. The endophytes of plants have been reported for their role in modulating various physiological and biological processes of the host plants. In the present study, a total of 244 endophytes were isolated in pure cultures from the roots of Viola odorata, and genetic diversity was evaluated using amplified ribosomal DNA restriction analysis (ARDRA) and enterobacterial repetitive intergenic consensus (ERIC). The molecular fingerprinting revealed variation among various rRNA types among morphologically different endophytes based on ARDRA and ERIC-PCR. The screening of endophytes showed antimicrobial activity of 11 bacterial isolates and one actinomycete SGA9 against various pathogens Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. The antioxidant activity revealed the majority of the bacterial isolates able to scavenge the free radical in the range of 10-50% and 8 bacterial isolates in the range of 50-85%. Principal component analysis separated eight isolates away from the central eclipse and form a separate group based on antimicrobial and antioxidant potential. The identification of these eight isolates showed affiliation with different species of the genus Enterobacter, Microbacterium, Pseudomonas, Rhizobium, and Streptomyces. This is the first report on the characterization of endophytic bacteria and actinomycetes from endemic Viola odorata. Results suggested that these endophytes could be explored for the production of antimicrobial and antioxidant products.

Phylogenetic and Structural Analysis of Bacterial Nitrilases for the Biodegradation of Nitrile Compounds.

BACKGROUND: Microbial nitrilases play a vital role in the biodegradation of nitrilecontaining pollutants, effluent treatments in chemical and textile industries, and the biosynthesis of Indole-3-acetic acid (IAA) from tryptophan in plants. However, the lack of structural information limits the correlation between its activity and substrate specificity. METHODS: The present study involves the genome mining of bacteria for the distribution and diversity of nitrilases, their phylogenetic analysis and structural characterization for motifs/ domains, followed by interaction with substrates. RESULTS: Here, we mined the bacterial genomes for nitrilases and correlated their functions to hypothetical, uncharacterized, or putative ones. The comparative genomics revealed four AcNit, As7Nit, Cn5Nit and Cn9Nit predicted nitrilases encoding genes as uncharacterized subgroups of the nitrilase superfamily. The annotation of these nitrilases encoding genes revealed relatedness with nitrilase hydratases and cyanoalanine hydratases. At the proteomics level, the motif analysis of these protein sequences predicted a single motif of 20-28 aa, with glutamate (E), lysine (K) and cysteine (C) residues as a part of catalytic triad along with several other residues at the active site. The structural analysis of the nitrilases revealed geometrical and close conformation in the form of α-helices and ß-sheets arranged in a sandwich structure. The catalytic residues constituted the substrate binding pocket and exhibited the broad nitrile substrate spectra for aromatic and aliphatic nitriles-containing compounds. The aromatic amino acid residues Y159 in the active site were predicted to be responsible for substrate specificity. The substitution of non-aromatic alanine residue in place of Y159 completely disrupted the catalytic activity for indole-3-acetonitrile (IAN). CONCLUSION: The present study reports genome mining and simulation of structure-function relationship for uncharacterized bacterial nitrilases and their role in the biodegradation of pollutants and xenobiotics, which could be of applications in different industrial sectors.

Genomics of prokaryotic extremophiles to unfold the mystery of survival in extreme environments.

The organisms surviving in extreme environments deploy system support including self-protection, and energy distribution to counter extreme environmental stresses. The biological adaptations provide clues about the metabolic networks and regulatory circuits involved in their success in survival to extreme environments of these organisms. Besides this, genes and proteins of these extremophiles have gained worldwide attention of researchers, due to their immense biotechnological importance including source of novel enzymes and biomolecules for applications in industrial processes. Therefore, obtaining an insight into genomic aspects is of vital importance for basic and applied research. Genome wide studies showed that the microbes living in extreme habitats reorganize their genome using insertion, expansion or reduction of genome size, gene reshuffling through displacements and genes reorganization, G+C skewness in the genome, horizontal transfer of genes, change in polyploidy level, and preference for codon in genes that assists during adaptations to environmental extremes. For example, the comparative genomics studies revealed a significant loss of genes in acidophiles than in alkaliphiles and smaller genome size of thermophiles in comparison to psychrophiles. The genomic adaptations in halotolerance include polyploidy, battery of genes for the biosynthesis of organic osmolytes, mechanism of inorganic osmolytes acquisition and role of inorganic osmolytes and transporter system. Furthermore, it is evident that local niche specific adaptations also play a key role during adaptations to extreme environments. All these adaptations maintain extremophiles as operational units and provide them a competitive advantage over their counterparts. The review article describes the genomic multifaceted adaptation at genomic and physiological levels of extremophiles that assists in reshaping the prokaryotic extremophiles during adaptations to extreme environments to obtain a competitive edge.

Genomic Organization of Streptomyces flavotricini NGL1 and Streptomyces erythrochromogenes HMS4 Reveals Differential Plant Beneficial Attributes and Laccase Production Capabilities.

The genus Streptomyces has been explored in industrial sectors due to its endurance to environmental stresses, the production of a plethora of biomolecules, the biological remediation of soils, and alleviating plant stresses. The whole genome of NGL1 and HMS4 was sequenced due to the specific laccase activity against 2,6-dimethoxyphenol (2,6-DMP) and differential plant beneficial attributes. The deduced genome of 8.85 Mbp and 7.73 Mbp in size with a G+C content of 72.03% and 72.3% was obtained for NGL1 and HMS4, respectively. A total of 8438 and 7322 protein coding genes, 155 (130 tRNA, 25 rRNA) and 145 tRNA (121 tRNA, 24 rRNA) coding genes were predicted in NGL1 and HMS4, respectively. The comparative genomics of NGL1 and HMS4 showed 185 and 162 genes encoding for carbohydrate-active enzymes, respectively. The genomic ability of these strains to encode carbohydrate-active enzymes, laccase, and diversity of BGCs, along with plant beneficial attributes to suppress the plant pathogens can be used for several industrial and agricultural applications.

Biology and molecular interactions of Parastagonospora nodorum blotch of wheat.

MAIN CONCLUSION: Parastagonospora nodorum is one of the important necrotrophic pathogens of wheat which causes severe economical loss to crop yield. So far, a number of effectors of Parastagonospora nodorum origin and their target interacting genes on the host plant have been characterized. Since targeting effector-sensitive gene carefully can be helpful in breeding for resistance. Therefore, constant efforts are required to further characterize the effectors, their interacting genes, and underlying biochemical pathways. Furthermore, to develop effective counter-strategies against emerging diseases, continuous efforts are required to determine the qualitative resistance that demands to screen of diverse genotypes for host resistance. Stagonospora nodorum blotch also refers to as Stagonospora glume blotch and leaf is caused by Parastagonospora nodorum. The pathogen deploys necrotrophic effectors for the establishment and development on wheat plants. The necrotrophic effectors and their interaction with host receptors lead to the establishment of infection on leaves and extensive lesions formation which either results in host cell death or suppression/activation of host defence mechanisms. The wheat Stagonospora nodorum interaction involves a set of nine host gene-necrotrophic effector interactions. Out of these, Snn1-SnTox1, Tsn1-SnToxA and Snn-SnTox3 are one of the most studied interaction, due to its role in the suppression of reactive oxygen species production, regulating the cytokinin content through ethylene-dependent wayduring initial infection stage. Further, although the molecular basis is not fully unveiled, these effectors regulate the redox state and influence the ethylene biosynthesis in infected wheat plants. Here, we have discussed the biology of the wheat pathogen Parastagonospora nodorum, role of its necrotrophic effectors and their interacting sensitivity genes on the redox state, how they hijack the resistance mechanisms, hormonal regulated immunity and other signalling pathways in susceptible wheat plants. The information generated from effectors and their corresponding sensitivity genes and other biological processes could be utilized effectively for disease management strategies.

Streptomyces: host for refactoring of diverse bioactive secondary metabolites.

Microbial secondary metabolites are intensively explored due to their demands in pharmaceutical, agricultural and food industries. Streptomyces are one of the largest sources of secondary metabolites having diverse applications. In particular, the abundance of secondary metabolites encoding biosynthetic gene clusters and presence of wobble position in Streptomyces strains make it potential candidate as a native or heterologous host for secondary metabolite production including several cryptic gene clusters expression. Here, we have discussed the developments in Streptomyces strains genome mining, its exploration as a suitable host and application of synthetic biology for refactoring genetic systems for developing chassis for enhanced as well as novel secondary metabolites with reduced genome and cleaned background.

Genome wide underpinning of antagonistic and plant beneficial attributes of Bacillus sp. SBA12.

The plant beneficial microorganisms in rhizosphere are promising candidates to counter plant biotic and abiotic stresses. In the present study, Bacillus sp. SBA12 antagonistic to plant pathogenic fungi such as Phytophthora infestans and Sclerotinia sclerotiorum was explored. The complete genome of SBA12 comprises 5,297,566 bp size with GC content 49% and a total of 5698 genes. The genomic organization revealed presence of several biosynthetic clusters for antibiotics, siderophore and various other bioactive compounds such as lanthipeptide, LAP, bacteriocin, siderophore, NRP and terpenes. The gene cluster for fengycin known for its role as elicitors in inducing plant immunity is also reported. Besides this, lytic enzymes chitinases, ß-glucanases and proteases involved in pathogen suppression, acid and alkaline phosphatases, deaminases responsible for the phosphate solubilization and release of ammonia are also reported. The genomic organization of SBA12 revealed several hallmarks for plant growth promotion and pathogen suppression activities.

Molecular imprints of plant beneficial Streptomyces sp. AC30 and AC40 reveal differential capabilities and strategies to counter environmental stresses.

Streptomyces and their biomolecules are well explored for antibiotics production, bioremediation and alleviating the plant stresses due to their plant beneficial attributes. Therefore, due to plethora of biological attributes, the accurate portraying of molecular capabilities of these microorganisms at genomic level is of paramount importance. Here, we have evaluated biochemical attributes of two Streptomyces sp. AC30and AC40 for different plant beneficial activities which are antagonistic to Fusarium oxysporum, Alternaria solani, Sclerotinia sclerotium and Phytopthora infestans. In parallel, the draft genomes of these strains were deduced to understand their genomic capabilities using Illumina platform. The complete genome of AC30and AC40 were 11,284,599 bp and 12,636,188 bp in size with total G + C content of 62.36 and 54.75 %, respectively. Overall, higher number of genes (14,024) was reported for AC40 as compared to AC30 (12,476). The comparative genome organization revealed sharing of a few biosynthetic clusters as well as some exclusive biosynthetic clusters among both the strains. Further, expansion in the chitinases and glucanases was found in the genome of AC40. In addition, genes for 3-phytase and glycosyl hydrolase family 19 were restricted to AC40 only. The comparative genome study revealed presence of plant induced nitrilase in AC40 which is predicted for its role in IAA biosynthesis, release of ammonia, biotransformation of nitrile compounds to corresponding acids and bioremediation of soil containing nitrile compounds. For IAA and secondary metabolites biosynthesis, flavin-dependent monooxygenase, a rate limiting factor in Trp-dependent auxin biosynthesis pathway was found exclusive to AC30 genome. The comparative study revealed the diversification of few pathways/strategies to suppress plant pathogens and promote plant growth by Streptomyces strains.

Bioprospecting Psychrotrophic Bacteria for Serine-Type Proteases from the Cold Areas of Western Himalayas.

The aim of present study was to analyze the prevalence of protease diversity among psychrotrophic bacteria in Lahaul and Spiti of the Western Himalayas. A total of 459 bacteria were screened and protease activity was observed in 150 isolates at 5 °C. Furthermore, 55 isolates showed protease activity up to pH 10 at 5 °C. Based on the hydrolytic zone, 22 isolates were selected for protease quantification. The protease activity varied from 58-377 U mL-1 at 10 °C, 49-396 U mL-1 at 28 °C and 31-407 U mL-1 at 37 °C. Similarly, protease activity ranged from 36-353 U mL-1 at pH 7, 40-306 U mL-1 at pH 9 and 33-304 U mL-1 at pH 10. The isolates were identified based on 16S rRNA gene sequencing and showed phylogenetic relationship to Arthrobacter belonging to the class Actinobacteria, Bacillus, Exiguobacterium, Paenibacillus, and Planomicrobium to Bacilli, and Pseudomonas, Serratia, and Stenotrophomonas to Gammaproteobacteria. Zymogram analysis revealed variations in protease isoforms ranging from 20 to 250 kDa which were strongly inhibited in the presence of phenylmethylsulfonyl fluoride, thus indicated serine-type nature. The extensive number of serine proteases among these bacteria was confirmed by annotating genomes of the reported genera for prevalence of protease isoforms. The properties of proteases including low-temperature activity with alkaline stability and detergent compatibility suggested their suitability as bio-additives in laundry.

Molecular and biotechnological aspects of secondary metabolites in actinobacteria.

The ability to produce plethora of secondary metabolites and enzymes for pharmaceutical, agricultural and biotechnological applications make actinobacteria one of the most explored microbes among prokaryotes. The secondary metabolites and lytic enzymes of actinobacteria are known for their role in various physiological, cellular and biological processes including environmental sensing, mineral acquisition and recycling, and establishing social communication. In addition, the basic scaffold of secondary metabolties derived from actinobacteria is a source of inspiration for chemists. Recent development in "gene to metabolites" and "metabolites to gene" based omics technologies have played major role in revealing the prevalence of silent gene clusters in the genome of actinobacteria. Moreover, the development in precision-based genome editing tools and use of artificial gene operon for pathway engineering have emerged as a key player in activation of these silent/cryptic gene clusters for novel metabolites at large scale which were previously found to be poorly expressed and difficult to characterize in lab conditions. The access to diverse uncharacterized biosynthetic gene clusters of different types and the leverage of modern gene editing tools for modulated expression of the operons would contribute to novel product discovery and product diversification compared to traditional way of mining metabolites. Here, in review article, we have discussed the taxonomic status, genomic potential of actinobacteria for various secondary metabolites and role of genetic engineering to explore these microbes for human welfare.

Trends in extracellular serine proteases of bacteria as detergent bioadditive: alternate and environmental friendly tool for detergent industry.

Proteases, one of the largest groups of industrial enzymes occupy a major share in detergent industry. To meet the existing demands, proteases with efficient catalytic properties are being explored from bacteria residing in extreme habitats. Alkaline proteases are also considered as promising candidates for industrial sectors due to the activity and stability under alkaline and harsh environment. Therefore, a systematic review on experimental studies of bacterial proteases was conducted with emphasis on purification, characterization, cloning and expression and their suitability as detergent additive. Relevant searches using a combination of filters/keywords were performed in the online databases; PubMed, Science Direct, Scopus and Web of Science. Over thousands of research papers, 71 articles in Scopus, 48 articles in Science Direct, 18 articles in PubMed and 8 articles in Web of Science were selected with regard to bacterial extracellular proteases till date. Selected articles revealed majority of the studies conducted between the years 2015 and 17 and were focused on purification of proteases from bacteria. Among microbes, a total of 41 bacterial genera have been explored with limited studies from extreme habitats. Majority of the studies have reported the involvement of subtilisin-like serine proteases with effective properties for detergent industries. The studies revealed shifting of trend from purification to cloning to genetic engineering to meet the industrial demands. The present systematic review describes the proteases from extremophilic bacteria and use of biotechnological techniques such as site-directed mutagenesis and codon optimization to engineer enzymes with better hot spots in the active sites to meet industrial challenges.

Molecular characterization of ß-endoglucanase from antagonistic Trichoderma saturnisporum isolate GITX-Panog (C) induced under mycoparasitic conditions.

The endoglucanase belonging to glycoside hydrolase family 61 are little studied. In present study, a ß-endoglucanase of ~37 kDa induced on autoclaved mycelium of Fusarium oxysporum was cloned and characterized. The molecular characterization of ß-endoglucanase encoding gene revealed presence of a single intron and an open reading frame of 1044-bp which encoded a protein of 347 amino acid residues. The phylogenetic analysis of Eglu revealed its similarity to endo-ß-glucanases of other Trichoderma spp. The catalytic site of ß-endoglucanase contained Asp, Asn, His and Tyr residues. The cDNA encoding ß-glucanase was cloned into E. coli and Pichia pastoris using pQUA-30 and pPIC9K vector system, respectively. The comparison of structure revealed that most similar structure to Eglu is Hypocrea jecorina template 5o2w.1.A of glycoside hydrolase family 61.The biochemical characterization of ß-endoglucanase purified from T. saturnisporum isolate and the recombinant protein expressed in E. coli and P. pastoris was active under acidic conditions with a pH optima of 5 and temperature optima of 60 °C. The purified and expressed enzyme preparation was able to inhibit growth of F.oxysporum at 1 × 105 spores/mL which clearly revealed its significance in plant pathogen suppression.

Chryseobacterium glaciei sp. nov., isolated from the surface of a glacier in the Indian trans-Himalayas.

A novel bacterial strain, IHBB 10212T, of the genus Chryseobacterium was isolated from a glacier near the Kunzum Pass located in the Lahaul-Spiti in the North-Western Himalayas of India. The cells were Gram-negative, aerobic, non-sporulating, single rods, lacked flagella, and formed yellow to orange pigmented colonies. The strain utilized maltose, trehalose, sucrose, gentibiose, glucose, mannose, fructose, mannitol, arabitol and salicin for growth. Flexirubin-type pigments were produced by strain IHBB 10212T. The 16S rRNA gene sequence analysis showed relatedness of strain IHBB 10212T to Chryseobacterium polytrichastri DSM 26899T (97.43 %), Chryseobacterium greenlandense CIP 110007T (97.29 %) and Chryseobacterium aquaticum KCTC 12483T (96.80 %). Iso-C15 : 0 and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) constituted the major cellular fatty acids. The polar lipids present were six unidentified aminolipids, one unidentified phospholipid and three unidentified lipids. MK-6 was identified as the major quinone. The DNA G+C content was 34.08  mol%. Digital DNA-DNA hybridization of strain IHBB 10212T with C. polytrichastri, C. greenlandense and C. aquaticum showed values far below the prescribed thresholds of 95 % for average nucleotide identity and 70 % for the Genome-to-Genome Distance Calculator for species delineation. Based on its differences from validly published Chryseobacterium species, strain IHBB 10212T is identified as a new species, for which the proposed name is Chryseobacterium glaciei sp. nov., with IHBB 10212T as the type strain (=MTCC 12457T=JCM 31156T=KACC 19170T).

Heterologous expression and structure-function relationship of low-temperature and alkaline active protease from Acinetobacter sp. IHB B 5011(MN12).

The gene encoding protease from Acinetobacter sp. IHB B 5011(MN12) was cloned and expressed in Escherichia coli BL21(DE3). The nucleotide sequence revealed 1323bp ORF encoding 441 amino acids protein with molecular weight 47.2kDa. The phylogenetic analysis showed clustering of Alp protease with subtilisin-like serine proteases of S8 family. The amino acid sequence was comprised of N-terminal signal peptide 1-21 amino acids, pre-peptide 22-143 amino acids, peptidase S8 domain 144-434 amino acids, and pro-peptide 435-441 amino acids at C-terminus. Three constructs with signal peptide pET-Alp, without signal peptide pET-Alp1 and peptidase S8 domain pET-Alp2 were prepared for expression in E. coli BL21(DE3). The recombinant proteins Alp1 and Alp2 expressed as inclusion bodies showed ∼50kDa and ∼40kDa bands, respectively. The pre-propeptide ∼11kDa removed from Alp1 resulted in mature protein of ∼35kDa with 1738Umg-1 specific activity. The recombinant protease was optimally active at 40°C and pH 9, and stable over 10-70°C and 6-12pH. The activity at low-temperature and alkaline pH was supported by high R/(R+K) ratio, more glycine, less proline, negatively charged amino acids, less salt bridges and longer loops. These properties suggested the suitability of Alp as additive in the laundry.

Integrated Translatome and Proteome: Approach for Accurate Portraying of Widespread Multifunctional Aspects of Trichoderma.

Genome-wide studies of transcripts expression help in systematic monitoring of genes and allow targeting of candidate genes for future research. In contrast to relatively stable genomic data, the expression of genes is dynamic and regulated both at time and space level at different level in. The variation in the rate of translation is specific for each protein. Both the inherent nature of an mRNA molecule to be translated and the external environmental stimuli can affect the efficiency of the translation process. In biocontrol agents (BCAs), the molecular response at translational level may represents noise-like response of absolute transcript level and an adaptive response to physiological and pathological situations representing subset of mRNAs population actively translated in a cell. The molecular responses of biocontrol are complex and involve multistage regulation of number of genes. The use of high-throughput techniques has led to rapid increase in volume of transcriptomics data of Trichoderma. In general, almost half of the variations of transcriptome and protein level are due to translational control. Thus, studies are required to integrate raw information from different "omics" approaches for accurate depiction of translational response of BCAs in interaction with plants and plant pathogens. The studies on translational status of only active mRNAs bridging with proteome data will help in accurate characterization of only a subset of mRNAs actively engaged in translation. This review highlights the associated bottlenecks and use of state-of-the-art procedures in addressing the gap to accelerate future accomplishment of biocontrol mechanisms.

Elucidation of biocontrol mechanisms of Trichoderma harzianum against different plant fungal pathogens: Universal yet host specific response.

In the present study, different transcripts of Trichoderma harzianum ThHP-3 were evaluated for their response against four fungal pathogens Fusarium oxysporum, Colletotrichum capsici, Colletotrichum truncatum and Gloesercospora sorghi using RT-qPCR. The time course study of T. harzianum transcripts related to signal transduction, lytic enzymes, secondary metabolites and various transporters revealed variation in expression against four fungal pathogens. In a broader term, the transcripts were upregulated at various time intervals but the optimum expression of cyp3, abc, nrp, tga1, pmk, ech42 and glh20 varied with respect to host fungi. Additionally, the expression of transcripts related to transporters/cytochromes was also observed against Fusarium oxysporum after 96h whereas transcripts related to secondary metabolites and lytic enzymes showed significant difference in expression against Colletotrichum spp. from 72 to 96h. This is first study on transcriptomic response of T. harzianum against pathogenic fungi which shows their host specific response.

The relationship between capsular type and OmpA of Pasteurella multocida is associated with the outcome of disease.

The genes encoding OmpA of Pasteurella multocida recovered from diseased and apparently healthy animals have been characterized. The nucleotide sequence revealed ORFs of 1047-1077 bp encoding proteins of 349-360 amino acids. Domain analysis of OmpA showed signal peptide, N-terminal ompA domain and C-terminal ligand binding domain. The transmembrane topology of OmpA showed short turns at the periplasmic end and longer irregular loops at the extracellular end. The phylogenetic analysis based on OmpA showed affiliation of isolates to 7 groups representing different alleles. The identical segments in OmpA also suggested assortative recombination within classes IV, V and VI of distinct lineages. Principal component analysis separated isolates into groups based on capsular type and PmompA alleles. The alleles belonging to class VI exclusively associated with capsular type A, whereas class I-IV were associated with capsular type B. PmompA alleles in class V were recorded in both serogroups. PmompA6.1, 6.4 were distributed among strains with capsular type A, and PmompA6.2 and 6.3 among capsular type B. Despite internal OmpA variabilty, restrictive and well defined distribution was seen amongst P. multocida. A definitive association of "OmpA-capsular type" was observed with clinical status of animals. A cohort of pasteurellae comprising of OmpA(I-IV)-capB was recovered from diseased animals and OmpA(VI)-capA from healthy subjects. This study concludes that P. multocida with serogroup A and B from healthy and diseased animals represent distinct clusters also differentiated based on their OmpA-types and OmpA-capsular type relationship possibly determine the virulence and disease outcome.

Molecular cloning and characterization of ech46 endochitinase from Trichoderma harzianum.

In the present study, endochitinase of T. harzianum isolate-ThHP3 induced against mycelium of F. oxysporum was cloned, sequenced and characterized. The complete nucleotide sequence contained an ORF of 1293bp corresponding to 430 amino acids with 46kDa molecular weight and theoretical pI 5.59. The precursor protein contained 22 amino acids long signal peptide at N terminus. The domain architecture of endochitinase showed low complexity regions, presence of 1W9P domain specific to cyclopentapeptide and lack of carbohydrate binding modules. The ligand binding site of ech46 endochitinase was constituted by 10 amino acids. The cDNA encoding ech46 endochitinase was ligated into pET28a vector and transformed to E. coli BL21. The predicted molecular weight of recombinant endochitinase without signal peptide was 49.4kDa with a theoretical pI 6.67. SDS-PAGE analysis of purified 6xHis tagged protein showed a single band of 49kDa. The refolded enzyme was active under acidic conditions with a temperature and pH optima of 50°C and 4. Km and Vmax for recombinant endochitinase using 4-pNP-(GlcNAc)3 were 315.2±0.36µM and 0.140±0.08µMmin-1, respectively and the calculated kcat was 6.44min-1. The RT-qPCR revealed induction of ech46 by phytopathogenic fungi.

Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity.

Cloning, Expression, and Functional Characterization of Serine Protease Aprv2 from Virulent Isolate Dichelobacter nodosus of Indian Origin.

A gene encoding an extracellular protease from Dichelobacter nodosus was characterized and expressed in E. coli rosetta-gami (DE3). The nucleotide sequence analysis revealed an ORF of 1427 bp ecoding 475 amino acids long protein of calculated molecular weight 50.6 kDa and pI value 6.09. The phylogenetic analysis showed relatedness to subtilisin-like serine proteases of peptidase S8 family. The amino acid sequence analysis showed presence of N-terminal pre-peptide (1-23 aa), pro-peptide (24-160 aa), peptidase S8 domain (161-457 aa), and a C-terminal extension (458-475 aa). The gene harboring native signal peptide was expressed in pET-22b(+) for production of AprV2 recombinant protein. SDS-PAGE revealed the highest production of IPTG induced recombinant protein ∼37 kDa at 16 °C after 16 h. The purified protein after Ni-NTA affinity chromatography showed single protein band of ∼37 kDa which was also confirmed by the detection of blue coloured band of same size in Western blotting. The recombinant protein showed activity over broad temperature and pH range with optimum at 35 °C and pH 7.0. Similarly, the enzyme was stable over broad range 15-65 °C and 4-10 pH with maximum stability at 25 °C and pH 6. The activity of purified enzyme was also stimulated in the presence of Ca2+. The purified enzyme showed highest activity towards casein as compared to gelatin and BSA. These findings suggest AprV2 as an important candidate for industrial applications such as pharmaceuticals.