CRISPR-Cas system: from diagnostic tool to potential antiviral treatment.

This mini review focuses on the diagnosis and treatment of virus diseases using Crisper-Cas technology. The present paper describes various strategies involved in diagnosing diseases using Crispr-Cas-based assays. Additionally, CRISPR-Cas systems offer great potential as new therapeutic tools for treating viral infections including HIV, Influenza, and SARS-CoV-2. There are several major challenges to be overcome before this technology can be applied routinely in clinical settings, such as finding a suitable delivery tool, toxicity, and immunogenicity, as well as off-target effects. This review also discusses ways to deal with the challenges associated with Crisper-Cas technology. KEY POINTS: • Crisper technology is being applied to diagnose infectious and non-infectious diseases. • A new generation of CRISPR-Cas-based assays has been developed which detect pathogens within minutes, providing rapid diagnosis of diseases. • Crispr-Cas tools can be used to combat viral infections, specifically HIV, influenza, and SARS-CoV-2.

Microbial lipases and their industrial applications: a comprehensive review.

Lipases are very versatile enzymes, and produced the attention of the several industrial processes. Lipase can be achieved from several sources, animal, vegetable, and microbiological. The uses of microbial lipase market is estimated to be USD 425.0 Million in 2018 and it is projected to reach USD 590.2 Million by 2023, growing at a CAGR of 6.8% from 2018. Microbial lipases (EC 3.1.1.3) catalyze the hydrolysis of long chain triglycerides. The microbial origins of lipase enzymes are logically dynamic and proficient also have an extensive range of industrial uses with the manufacturing of altered molecules. The unique lipase (triacylglycerol acyl hydrolase) enzymes catalyzed the hydrolysis, esterification and alcoholysis reactions. Immobilization has made the use of microbial lipases accomplish its best performance and hence suitable for several reactions and need to enhance aroma to the immobilization processes. Immobilized enzymes depend on the immobilization technique and the carrier type. The choice of the carrier concerns usually the biocompatibility, chemical and thermal stability, and insolubility under reaction conditions, capability of easy rejuvenation and reusability, as well as cost proficiency. Bacillus spp., Achromobacter spp., Alcaligenes spp., Arthrobacter spp., Pseudomonos spp., of bacteria and Penicillium spp., Fusarium spp., Aspergillus spp., of fungi are screened large scale for lipase production. Lipases as multipurpose biological catalyst has given a favorable vision in meeting the needs for several industries such as biodiesel, foods and drinks, leather, textile, detergents, pharmaceuticals and medicals. This review represents a discussion on microbial sources of lipases, immobilization methods increased productivity at market profitability and reduce logistical liability on the environment and user.

Lipase catalysis in organic solvents: advantages and applications.

Lipases are industrial biocatalysts, which are involved in several novel reactions, occurring in aqueous medium as well as non-aqueous medium. Furthermore, they are well-known for their remarkable ability to carry out a wide variety of chemo-, regio- and enantio-selective transformations. Lipases have been gained attention worldwide by organic chemists due to their general ease of handling, broad substrate tolerance, high stability towards temperatures and solvents and convenient commercial availability. Most of the synthetic reactions on industrial scale are carried out in organic solvents because of the easy solubility of non-polar compounds. The effect of organic system on their stability and activity may determine the biocatalysis pace. Because of worldwide use of lipases, there is a need to understand the mechanisms behind the lipase-catalyzed reactions in organic solvents. The unique interfacial activation of lipases has always fascinated enzymologists and recently, biophysicists and crystallographers have made progress in understanding the structure-function relationships of these enzymes. The present review describes the advantages of lipase-catalyzed reactions in organic solvents and various effects of organic solvents on their activity.

Identification of new members of the MAPK gene family in plants shows diverse conserved domains and novel activation loop variants.

BACKGROUND: Mitogen Activated Protein Kinase (MAPK) signaling is of critical importance in plants and other eukaryotic organisms. The MAPK cascade plays an indispensible role in the growth and development of plants, as well as in biotic and abiotic stress responses. The MAPKs are constitute the most downstream module of the three tier MAPK cascade and are phosphorylated by upstream MAP kinase kinases (MAPKK), which are in turn are phosphorylated by MAP kinase kinase kinase (MAPKKK). The MAPKs play pivotal roles in regulation of many cytoplasmic and nuclear substrates, thus regulating several biological processes. RESULTS: A total of 589 MAPKs genes were identified from the genome wide analysis of 40 species. The sequence analysis has revealed the presence of several N- and C-terminal conserved domains. The MAPKs were previously believed to be characterized by the presence of TEY/TDY activation loop motifs. The present study showed that, in addition to presence of activation loop TEY/TDY motifs, MAPKs are also contain MEY, TEM, TQM, TRM, TVY, TSY, TEC and TQY activation loop motifs. Phylogenetic analysis of all predicted MAPKs were clustered into six different groups (group A, B, C, D, E and F), and all predicted MAPKs were assigned with specific names based on their orthology based evolutionary relationships with Arabidopsis or Oryza MAPKs. CONCLUSION: We conducted global analysis of the MAPK gene family of plants from lower eukaryotes to higher eukaryotes and analyzed their genomic and evolutionary aspects. Our study showed the presence of several new activation loop motifs and diverse conserved domains in MAPKs. Advance study of newly identified activation loop motifs can provide further information regarding the downstream signaling cascade activated in response to a wide array of stress conditions, as well as plant growth and development.

Integration of bioinformatics to biodegradation.

Bioinformatics and biodegradation are two primary scientific fields in applied microbiology and biotechnology. The present review describes development of various bioinformatics tools that may be applied in the field of biodegradation. Several databases, including the University of Minnesota Biocatalysis/Biodegradation database (UM-BBD), a database of biodegradative oxygenases (OxDBase), Biodegradation Network-Molecular Biology Database (Bionemo) MetaCyc, and BioCyc have been developed to enable access to information related to biochemistry and genetics of microbial degradation. In addition, several bioinformatics tools for predicting toxicity and biodegradation of chemicals have been developed. Furthermore, the whole genomes of several potential degrading bacteria have been sequenced and annotated using bioinformatics tools.

Bacterial degradation of chlorophenols and their derivatives.

Chlorophenols (CPs) and their derivatives are persistent environmental pollutants which are used in the manufacture of dyes, drugs, pesticides and other industrial products. CPs, which include monochlorophenols, polychlorophenols, chloronitrophenols, chloroaminophenols and chloromethylphenols, are highly toxic to living beings due to their carcinogenic, mutagenic and cytotoxic properties. Several physico-chemical and biological methods have been used for removal of CPs from the environment. Bacterial degradation has been considered a cost-effective and eco-friendly method of removing CPs from the environment. Several bacteria that use CPs as their sole carbon and energy sources have been isolated and characterized. Additionally, the metabolic pathways for degradation of CPs have been studied in bacteria and the genes and enzymes involved in the degradation of various CPs have been identified and characterized. This review describes the biochemical and genetic basis of the degradation of CPs and their derivatives.

Biotransformation and chemotaxis of 4-chloro-2-nitrophenol by Pseudomonas sp. JHN.

Pseudomonas sp. JHN decolourized and biotransformed 4-chloro-2-nitrophenol (4C2NP) in the presence of additional carbon source. The effect of the various concentrations of the 4C2NP was studied on the decolourization of 4C2NP by Pseudomonas sp. JHN. It was observed that strain JHN decolourized and biotransformed 4C2NP up to concentration of 0.6 mM. Gas chromatography and gas chromatography-mass spectrometry detected 5-chloro-2-methylbenzoxazole as a major metabolite of the co-metabolism of 4C2NP. Furthermore, strain JHN exhibits positive chemotaxis toward 4C2NP based on the drop plate and capillary assays. This is the first report of the chemotaxis toward 4C2NP by any bacterium.

Metabolic pathway for degradation of 2-chloro-4-aminophenol by Arthrobacter sp. SPG.

A degradation pathway of 2-chloro-4-aminophenol (2C4AP) was studied in an Arthrobacter sp. SPG that utilized 2C4AP as its sole source of carbon and energy. The 2C4AP degradation was initiated by a 2C4AP-deaminase that catalyzed the conversion of 2C4AP into chlorohydroquinone (CHQ) with removal of ammonium ion. In the next step, a CHQ-dehalogenase dehalogenated CHQ to hydroquinone (HQ) that cleaved into γ-hydroxymuconic semialdehyde by a HQ-dioxygenase. The 2C4AP degradation was also investigated in sterile and non-sterile soil microcosms using strain SPG. The results show that the SPG cells degraded 2C4AP more rapidly in sterile soil than non-sterile soil. Our studies showed that strain SPG may be used for bioremediation of 2C4AP-contaminated sites. This is the first report of the 2C4AP degradation by any bacteria.

Industrial applications of fungal lipases: a review.

Fungal lipases (triacylglycerol acyl hydrolases EC 3.1.1.3) are significant industrial enzymes and have several applications in a number of industries and fields. Fungal lipases are found in several species of fungi and yeast. These enzymes are carboxylic acid esterases, categorized under the serine hydrolase family, and do not require any cofactor during the catalyzing of the reactions. It was also noticed that processes including the extraction and purification of lipases from fungi are comparatively easier and cheaper than other sources of lipases. In addition, fungal lipases have been classified into three chief classes, namely, GX, GGGX, and Y. Fungal lipases have applications not only in the hydrolysis of fats and oils (triglycerides) but are also involved in synthetic reactions such as esterification, acidolysis, alcoholysis, interesterification, and aminolysis. The production and activity of fungal lipases are highly affected by the carbon source, nitrogen source, temperature, pH, metal ions, surfactants, and moisture content. Therefore, fungal lipases have several industrial and biotechnological applications in many fields such as biodiesel production, ester synthesis, production of biodegradable biopolymers, formulations of cosmetics and personal care products, detergent manufacturing, degreasing of leather, pulp and paper production, textile industry, biosensor development, and drug formulations and as a diagnostic tool in the medical sector, biodegradation of esters, and bioremediation of wastewater. The immobilization of fungal lipases onto different carriers also helps in improving the catalytic activities and efficiencies of lipases by increasing thermal and ionic stability (in organic solvents, high pH, and temperature), being easy to recycle, and inducing the volume-specific loading of the enzyme onto the support, and thus, these features have proved to be appropriate for use as biocatalysts in different sectors.

Bioengineering of fungal endophytes through the CRISPR/Cas9 system.

The CRISPR/Cas9 system is a genome-editing tool that allows for precise and efficient modifications to the DNA of a cell. This technology can be used in endophytic fungi, which live within plants and can have beneficial effects on their host, making them important for agriculture. Using CRISPR/Cas9, researchers can introduce specific genetic changes into endophytic fungal genomes, allowing them to study the function of genes, improve their plant-growth-promoting properties, and create new, more beneficial endophytes. This system works by using the Cas9 protein, which acts as a pair of molecular scissors, to cut DNA at specific locations determined by a guide RNA. Once the DNA is cut, the cell's natural repair mechanisms can be used to insert or delete specific genes, allowing for precise editing of the fungal genome. This article discusses the mechanism and applications of CRISPR/Cas9 to fungal endophytes.

Metabolism of 4-chloro-2-nitrophenol in a gram-positive bacterium, Exiguobacterium sp. PMA.

BACKGROUND: Chloronitrophenols (CNPs) are widely used in the synthesis of dyes, drugs and pesticides, and constitute a major group of environmental pollutants. 4-Chloro-2-nitrophenol (4C2NP) is an isomer of CNPs that has been detected in various industrial effluents. A number of physicochemical methods have been used for treatment of wastewater containing 4C2NP. These methods are not as effective as microbial degradation, however. RESULTS: A 4C2NP-degrading bacterium, Exiguobacterium sp. PMA, which uses 4C2NP as the sole carbon and energy source was isolated from a chemically-contaminated site in India. Exiguobacterium sp. PMA degraded 4C2NP with the release of stoichiometeric amounts of chloride and ammonium ions. The effects of different substrate concentrations and various inoculum sizes on degradation of 4C2NP were investigated. Exiguobacterium sp. PMA degraded 4C2NP up to a concentration of 0.6 mM. High performance liquid chromatography and gas chromatography-mass spectrometry identified 4-chloro-2-aminophenol (4C2AP) and 2-aminophenol (2AP) as possible metabolites of the 4C2NP degradation pathway. The crude extract of 4C2NP-induced PMA cells contained enzymatic activity for 4C2NP reductase and 4C2AP dehalogenase, suggesting the involvement of these enzymes in the degradation of 4C2NP. Microcosm studies using sterile and non-sterile soils spiked with 4C2NP were carried out to monitor the bioremediation potential of Exiguobacterium sp. PMA. The bioremediation of 4C2NP by Exiguobacterium sp. PMA was faster in non-sterilized soil than sterilized soil. CONCLUSIONS: Our studies indicate that Exiguobacterium sp. PMA may be useful for the bioremediation of 4C2NP-contaminated sites. This is the first report of (i) the formation of 2AP in the 4C2NP degradation pathway by any bacterium and (iii) the bioremediation of 4C2NP by any bacterium.

Degradation of chlorinated nitroaromatic compounds.

Chlorinated nitroaromatic compounds (CNAs) are persistent environmental pollutants that have been introduced into the environment due to the anthropogenic activities. Bacteria that utilize CNAs as the sole sources of carbon and energy have been isolated from different contaminated and non-contaminated sites. Microbial metabolism of CNAs has been studied, and several metabolic pathways for degradation of CNAs have been proposed. Detoxification and biotransformation of CNAs have also been studied in various fungi, actinomycetes and bacteria. Several physicochemical methods have been used for treatment of wastewater containing CNAs; however, these methods are not suitable for in situ bioremediation. This review describes the current scenario of the degradation of CNAs.

Biotransformation of 4-chloro-2-nitrophenol into 5-chloro-2-methylbenzoxazole by a marine Bacillus sp. strain MW-1.

Decolourization, detoxification and biotransformation of 4-chloro-2-nitrophenol (4C2NP) by Bacillus sp. strain MW-1 were studied. This strain decolorized 4C2NP only in the presence of an additional carbon source. On the basis of thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS), 4-chloro-2-aminophenol, 4-chloro-2-acetaminophenol and 5-chloro-2-methylbenzoxazole were identified as metabolites. Resting cells depleted 4C2NP with stoichiometric formation of 5-chloro-2-methyl benzoxazole. This is the first report of the formation of 5-chloro-2-methylbenzoxazole from 4C2NP by any bacterial strain.

Chryseomicrobium imtechense gen. nov., sp. nov., a new member of the family Planococcaceae.

A Gram-stain-positive, rod-shaped, yellow, non-motile, non-spore-forming, strictly aerobic bacterial strain, designated MW 10(T), was isolated from seawater of the Bay of Bengal, India, and was subjected to a polyphasic taxonomic study. Analysis of the 16S rRNA gene sequence revealed that strain MW 10(T) showed highest similarity to the type strains of Psychrobacillus psychrodurans (96.15 %) and Psychrobacillus psychrotolerans (96.01 %) and showed less than 96 % similarity to members of the genera Paenisporosarcina, Planococcus, Sporosarcina and Planomicrobium. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain MW 10(T) formed a clade separate from members of closely related genera. The morphological, physiological and chemotaxonomic characteristics of strain MW 10(T) differed from those of members of closely related genera. The major fatty acid in strain MW 10(T) was iso-C(15 : 0) and the menaquinones were MK-7 (48.4 %), MK-8 (32.3 %), MK-7(H(2)) (13.7 %) and MK-6 (5.6 %). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unknown phospholipid, an unknown lipid and an unknown glycolipid. The cell-wall peptidoglycan type was l-Lys-d-Asp. The genomic DNA G+C content (53.4 mol%) of strain MW 10(T) was significantly different from those of members of closely related genera. On the basis of its morphological, physiological and chemotaxonomic characteristics as well as our phylogenetic analysis, we conclude that strain MW 10(T) is a member of a novel genus and species, for which the name Chryseomicrobium imtechense gen. nov., sp. nov., is proposed. The type strain of Chryseomicrobium imtechense is MW 10(T) ( = MTCC 10098(T)  = JCM 16573(T)).

Reductive dehalogenation mediated initiation of aerobic degradation of 2-chloro-4-nitrophenol (2C4NP) by Burkholderia sp. strain SJ98.

Burkholderia sp. strain SJ98 (DSM 23195) was previously isolated and characterized for degradation and co-metabolic transformation of a number nitroaromatic compounds. In the present study, we evaluated its metabolic activity on chlorinated nitroaromatic compounds (CNACs). Results obtained during this study revealed that strain SJ98 can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize it as sole source of carbon, nitrogen, and energy under aerobic conditions. The cells of strain SJ98 removed 2C4NP from the growth medium with sequential release of nearly stoichiometric amounts of chloride and nitrite in culture supernatant. Under aerobic degradation conditions, 2C4NP was transformed into the first intermediate that was identified as p-nitrophenol by high-performance liquid chromatography, LCMS-TOF, and GC-MS analyses. This transformation clearly establishes that the degradation of 2C4NP by strain SJ98 is initiated by "reductive dehalogenation"; an initiation mechanism that has not been previously reported for microbial degradation of CNAC under aerobic conditions.

Pathway for degradation of 2-chloro-4-nitrophenol in Arthrobacter sp. SJCon.

Degradation of 2-Chloro-4-nitrophenol (2C4NP) was studied by Arthrobacter sp. SJCon, isolated from the soil of a pesticide contaminated site. This strain utilized 2C4NP as sole source of carbon and energy and degraded 2C4NP with stoichiometric release of nitrite and chloride ions. A metabolite was detected during the study of 2C4NP degradation and identified as chlorohydroquinone (CHQ) by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS). Inhibition study using 2,2'-dipyridyl showed that CHQ is a terminal aromatic compound in degradation pathway of 2C4NP. CHQ dioxygenase activity was observed in the crude extract of 2C4NP induced cells of the strain SJCon that suggested the cleavage of the CHQ to maleylacetate (MA). Our study clearly showed that Arthrobacter sp. SJCon degraded 2C4NP via formation of CHQ that further cleaved to MA by CHQ dioxygenase. This mechanism of degradation of 2C4NP differs from previously reported degradation pathways of 2C4NP.

Development of ESAT-6 Based Immunosensor for the Detection of Mycobacterium tuberculosis.

Early secreted antigenic target of 6 kDa (ESAT-6) has recently been identified as a biomarker for the rapid diagnosis of tuberculosis. We propose a stable and reusable immunosensor for the early diagnosis of tuberculosis based on the detection and quantification of ESAT-6 via cyclic voltammetry (CV). The immunosensor was synthesized by polymerizing aniline dispersed with the reduced graphene oxide (rGO) and Ni nanoparticles, followed by surface modification of the electroconductive polyaniline (PANI) film with anti-ESAT-6 antibody. Physicochemical characterization of the prepared materials was performed by several analytical techniques, including FE-SEM, EDX, XRD, FT-IR, Raman, TGA, TPR, and BET surface area analysis. The antibody-modified Ni-rGO-PANI electrode exhibited an approximately linear response (R2 = 0.988) towards ESAT-6 during CV measurements over the potential range of -1 to +1 V. The lower detection limit for ESAT-6 was approximately 1.0 ng mL-1. The novelty of this study includes the development of the reusable Ni-rGO-PANI-based electrochemical immunosensor for the early diagnosis of tuberculosis. Furthermore, this study successfully demonstrates that electro-conductive PANI may be used as a polymeric substrate for Ni nanoparticles and rGO.

Draft genome sequence data of a 4-nitrophenol- degrading bacterium, Pseudomonas alloputida strain PNP.

A 4-nitrophenol-degrading bacterial strain PNP was isolated from pesticide-contaminated soil collected from Lucknow. Strain PNP utilized 0.5 mM 4-nitrophenol as its carbon source and degraded it completely within 24 h with stoichiometric release of nitrite ions. Strain PNP was associated with the genus Pseudomonas in a phylogentic tree and exhibited highest 16S rRNA gene sequence similarity to Pseudomonas juntendi BML3 (99.79%) and Pseudomonas inefficax JV551A3 (99.79%). Based on values of average nucleotide identity and digital DNA-DNA hybridization among strain PNP and its closely related type strains, it concluded that strain PNP belongs to Pseudomonas alloputida. The Illumina HiSeq platform was used to sequence the PNP genome. The draft genome sequence of Pseudomonas alloputida PNP was presented here. The total size of the draft assembly was 6,087,340 bp, distributed into 87 contigs with N50 value of 139502. The genome has an average GC content of 61.7% and contains 5461 coding sequences and 77 putative RNA genes. This Whole Genome Shotgun project has been submitted at DDBJ/ENA/GenBank under the accession JAGKJH000000000.

Draft genome sequence data of a chromium reducing bacterium, Bacillus licheniformis strain KNP.

A chromium-reducing bacterium designated as strain KNP was isolated from a sample collected from a tannery effluent of Kanpur, India. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain KNP belonged to the Bacillus genus and showed 100% similarity with Bacillus licheniformis. Furthermore, average nucleotide identity and digital DNA-DNA hybridization between strain KNP and its closely related strains confirmed its affiliation with Bacillus licheniformis species. Whole-genome sequencing of Bacillus licheniformis KNP was performed using the Illumina Hiseq platform. Here, we present the draft genome sequence of Bacillus licheniformis KNP. The total size of the draft assembly was 4,280,093 bp, distributed into 21 contigs with an N50 value of 4,186,229. The genome has 45.9% G + C content, 4255 coding sequences and 86 putative RNA genes. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JACDXS000000000. The version described in this paper is version JACDXS010000000.

Bacilli-Mediated Degradation of Xenobiotic Compounds and Heavy Metals.

Xenobiotic compounds are man-made compounds and widely used in dyes, drugs, pesticides, herbicides, insecticides, explosives, and other industrial chemicals. These compounds have been released into our soil and water due to anthropogenic activities and improper waste disposal practices and cause serious damage to aquatic and terrestrial ecosystems due to their toxic nature. The United States Environmental Protection Agency (USEPA) has listed several toxic substances as priority pollutants. Bacterial remediation is identified as an emerging technique to remove these substances from the environment. Many bacterial genera are actively involved in the degradation of toxic substances. Among the bacterial genera, the members of the genus Bacillus have a great potential to degrade or transform various toxic substances. Many Bacilli have been isolated and characterized by their ability to degrade or transform a wide range of compounds including both naturally occurring substances and xenobiotic compounds. This review describes the biodegradation potentials of Bacilli toward various toxic substances, including 4-chloro-2-nitrophenol, insecticides, pesticides, herbicides, explosives, drugs, polycyclic aromatic compounds, heavy metals, azo dyes, and aromatic acids. Besides, the advanced technologies used for bioremediation of environmental pollutants using Bacilli are also briefly described. This review will increase our understanding of Bacilli-mediated degradation of xenobiotic compounds and heavy metals.