Current Genomics - Volume 21, Issue 4, 2020

Genomic and proteomic advances in extremophile microorganism studies are increasingly demonstrating their ability to produce a variety of enzymes capable of converting biomass into bioenergy. Such microorganisms are found in environments with nutritional restrictions, anaerobic environments, high salinity, varying pH conditions and extreme natural environments such as hydrothermal vents, soda lakes, and Antarctic sediments. As extremophile microorganisms and their enzymes are found in widely disparate locations, they generate new possibilities and opportunities to explore biotechnological prospecting, including biofuels (biogas, hydrogen and ethanol) with an aim toward using multi-omics tools that shed light on biotechnological breakthroughs.

Since the last few decades, the promiscuous and uncontrolled use of plastics led to the accumulation of millions of tons of plastic waste in the terrestrial and marine environment. It elevated the risk of environmental pollution and climate change. The concern arises more due to the reckless and unscientific disposal of plastics containing high molecular weight polymers, viz., polystyrene, polyamide, polyvinylchloride, polypropylene, polyurethane, and polyethylene, etc. which are very difficult to degrade. Thus, the focus is now paid to search for efficient, eco-friendly, low-cost waste management technology. Of them, degradation of non-degradable synthetic polymer using diverse microbial agents, viz., bacteria, fungi, and other extremophiles become an emerging option. So far, very few microbial agents and their secreted enzymes have been identified and characterized for plastic degradation, but with low efficiency. It might be due to the predominance of uncultured microbial species, which consequently remain unexplored from the respective plastic degrading milieu. To overcome this problem, metagenomic analysis of microbial population engaged in the plastic biodegradation is advisable to decipher the microbial community structure and to predict their biodegradation potential in situ. Advancements in sequencing technologies and bioinformatics analysis allow the rapid metagenome screening that helps in the identification of total microbial community and also opens up the scope for mining genes or enzymes (hydrolases, laccase, etc.) engaged in polymer degradation. Further, the extraction of the core microbial population and their adaptation, fitness, and survivability can also be deciphered through comparative metagenomic study. It will help to engineer the microbial community and their metabolic activity to speed up the degradation process.

Background: This study was carried out to classify the diversity of the deep marine psychrotolerant actinomycetes sp. nov., in the Bay of Bengal and exploit the production of coldactive industrial and pharmaceutical biomolecules. Objective: 1) Characterization, optimum the growth conditions and classify the diversity of the novel isolated deep marine psychrotolerant actinomycetes sp from the Bay-of-Bengal. 2) Screening for industrially important biocatalysts and determine the antimicrobial activities against the five dreadful pathogens. 3) The differential expression profiling of the candidate genes to regulate the biosynthesis of selected enzymes. Methods: The cold-adapted actinomycetes were isolated from the deep marine water collections at 1200 mts below the surface in Bay-of-Bengal. The phenotypic and genotypic characterizations have been carried out to understand the persistent diversity of this novel marine psychrotolerant actinomycetes species. The production of cold-active enzymes, such as amylase, cellulase, lipase, pectinase, and L-asparaginase, were screened and the expression profiling genes were determined by using qRT PCR. The antibacterial and antifungal activities have also been investigated. Results: A total number of 37 novel actinomycetes were isolated and the phenotypic and genotypic characterizations identified the genus, dominated by Streptomyces (17 distinct sub-groups) as the major group, followed by Micromonospora, Actinopolyspora, Actinosynnema, Streptoverticillium, Saccharopolyspora, Nocardiopsis, and Nocardia. The optimum growth and abundant mycelium formation are observed at 15°C to 20°C and also capability for thriving at 4°C. All the isolates exhibited a significant role in the production of biocatalysts, and the antagonistic activities were also noted against five major selected pathogens. Conclusion: The Streptomyces from the Bay-of-Bengal have high biosynthetic potential and can serve as a good resource for the exploration of bioactive natural products.

Background: The presence of anthraquinone (Disperse blue 64) and azodyes (Acid yellow 17) in a waterbody are considered among the most dangerous pollutants. Methods: In this study, two different isolated microbes, bacterium and fungus, were individually and as a co-culture applied for the degradation of Disperse Blue 64 (DB 64) and Acid Yellow 17 (AY 17) dyes. The isolates were genetically identified based upon 16S (for bacteria) and ITS/5.8S (for fungus) rRNA genes sequences as Pseudomoans aeruginosa and Aspergillus flavus, respectively. Results: The fungal/bacterial consortium exhibited a higher percentage of dyes degradation than the individual strains, even at a high concentration of 300 mg/L. Azoreductase could be identified as the main catabolic enzyme and the consortium could induce azoreductase enzyme in the presence of both dyes. However, the specific substrate which achieved the highest azoreductase specific activity was Methyl red (MR) (3.5 U/mg protein). The tentatively proposed metabolites that were detected by HPLC/MS suggested that the reduction process catalyzed the degradation of dyes. The metabolites produced by the action consortium on two dyes were safe on Vicia faba and Triticum vulgaris germination and health of seedlings. Toxicity of the dyes and their degradation products on the plant was different according to the type and chemistry of these compounds as well as the type of irrigated seeds. Conclusion: We submit that the effective microbial degradation of DB64 and AY17 dyes will lead to safer metabolic products.

Background: Cyanobacteria are excellent model to understand the basic metabolic processes taking place in response to abiotic stress. The present study involves the characterization of a hypothetical protein Alr0765 of Anabaena PCC7120 comprising the CBS-CP12 domain and deciphering its role in abiotic stress tolerance. Methods: Molecular cloning, heterologous expression and protein purification using affinity chromatography were performed to obtain native purified protein Alr0765. The energy sensing property of Alr0765 was inferred from its binding affinity with different ligand molecules as analyzed by FTIR and TNP-ATP binding assay. AAS and real time-PCR were applied to evaluate the iron acquisition property and cyclic voltammetry was employed to check the redox sensitivity of the target protein. Transcript levels under different abiotic stresses, as well as spot assay, CFU count, ROS level and cellular H2O2 level, were used to show the potential role of Alr0765 in abiotic stress tolerance. In-silico analysis of Alr0765 included molecular function probability analysis, multiple sequence analysis, protein domain and motif finding, secondary structure analysis, protein-ligand interaction, homologous modeling, model refinement and verification and molecular docking was performed with COFACTOR, PROMALS-3D, InterProScan, MEME, TheaDomEx, COACH, Swiss modeller, Modrefiner, PROCHECK, ERRAT, MolProbity, ProSA, TM-align, and Discovery studio, respectively. Results: Transcript levels of alr0765 significantly increased by 20, 13, 15, 14.8, 12, 7, 6 and 2.5 fold when Anabaena PCC7120 treated with LC50 dose of heat, arsenic, cadmium, butachlor, salt, mannitol (drought), UV-B, and methyl viologen respectively, with respect to control (untreated). Heterologous expression resulted in 23KDa protein observed on the SDS-PAGE. Immunoblotting and MALDI-TOF-MS/MS, followed by MASCOT search analysis, confirmed the identity of the protein and ESI/MS revealed that the purified protein was a dimer. Binding possibility of Alr0765 with ATP was observed with an almost 6-fold increment in relative fluorescence during TNP-ATP binding assay with a λ max of 538 nm. FTIR spectra revealed modification in protein confirmation upon binding of Alr0765 with ATP, ADP, AMP and NADH. A 10-fold higher accumulation of iron was observed in digests of E. coli with recombinant vector after induction as compared to control, which affirms the iron acquisition property of the protein. Moreover, the generation of the redox potential of 146 mV by Alr0765 suggested its probable role in maintaining the redox status of the cell under environmental constraints. As per CFU count recombinant, E. coli BL21 cells showed about 14.7, 7.3, 6.9, 1.9, 3 and 4.9 fold higher number of colonies under heat, cadmium (CdCl2), arsenic (Na3AsO4), salt (NaCl), UV-B and drought (mannitol) respectively compared to pET21a harboring E. coli BL21 cells. Deterioration in the cellular ROS level and total cellular H2O2 concentration validated the stress tolerance ability of Alr0765. In-silico analysis unraveled novel findings and attested experimental findings in determining the role of Alr0765. Conclusion: Alr0765 is a novel CBS-CP12 domain protein that maintains cellular energy level and iron homeostasis which provides tolerance against multiple abiotic stresses.

Background: Escherichia coli (E. coli) mazEF, a stress-induced toxin-antitoxin (TA) system, has been studied extensively. The MazF toxin is an endoribonuclease that cleaves RNAs at ACA sites. Thereby, under stress, the induced MazF generates a Stress-induced Translation Machinery (STM), composed of MazF processed mRNAs and selective ribosomes that specifically translate the processed mRNAs. Materials and Methods: Based on the data from the EcoCyc website of the National Center for Biotechnology Information (NCBI), the sequence of all E. coli MG1655 genes were scanned for ACA sites upstream from the initiation codons. Among these sequences, the fuzznuc program of the "European Molecular Biology Open Software Suite" (EMBOSS) was used to find the "ACA" pattern. The distribution of the ACA threonine codon, both in-frame and out-of-frame, was determined by using the HTML Script Program (Supplementary Material). Results: Here it is reported that for most of the E. coli proteins mediated by stress-induced MazF, the ACA threonine codon in their mRNAs is not in-frame but rather out-of-frame; in these same RNAs, the three synonymous threonine codons, ACG, ACU, and ACC, are in-frame. In contrast, for proteins translated by the canonical translation system, in the majority of mRNAs, the ACA codon is located in-frame. Conclusion: The described bias in the genetic code is a characteristic of E. coli genes specifying for stress-induced MazF-mediated proteins.