Mutant Gossypium universal stress protein-2 (GUSP-2) gene confers resistance to various abiotic stresses in E. coli BL-21 and CIM-496-Gossypium hirsutum

AbstractGossypium arboreum is considered a rich source of stress-responsive genes and the EST database revealed that most of its genes are uncharacterized. The full-length Gossypium universal stress protein-2 (GUSP-2) gene (510 bp) was cloned in E. coli and Gossypium hirsutum, characterized and point mutated at three positions, 352–354, Lysine to proline (M1-usp-2) & 214–216, aspartic acid to serine (M2-usp-2) & 145–147, Lysine to Threonine (M3-usp-2) to study its role in abiotic stress tolerance. It was found that heterologous expression of one mutant (M1-usp-2) provided enhanced tolerance against salt and osmotic stresses, recombinant cells have higher growth up to 10-5dilution in spot assay as compared to cells expressing W-usp-2 (wild type GUSP-2), M2-usp-2 and M3-usp-2 genes. M1-usp-2 gene transcript profiling exhibited significant expression (8.7 fold) in CIM-496-Gossypium hirsutum transgenic plants and enhance drought tolerance. However, little tolerance against heat and cold stresses in bacterial cells was observed. The results from our study concluded that the activity of GUSP-2 was enhanced in M1-usp-2 but wipe out in M2-usp-2 and M3-usp-2 response remained almost parallel to W-usp-2. Further, it was predicted through in silico analysis that M1-usp-2, W-usp-2 and M3-usp-2 may be directly involved in stress tolerance or function as a signaling molecule to activate the stress adaptive mechanism. However, further investigation will be required to ascertain its role in the adaptive mechanism of stress tolerance.

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CLONING AND EXPRESSION OF UNIVERSAL STRESS PROTEIN 2 (USP2) GENE IN ESCHERICHIA COLI

Journal of Humanities, Social and Management Sciences (JHSMS) ◽

10.54112/bcsrj.v2021i1.48 ◽

2021 ◽

Vol 2021 (1) ◽

Author(s):

A Akram ◽

K Arshad ◽

MN Hafeez

Keyword(s):

Abiotic Stress ◽

Stress Tolerance ◽

Protein Gene ◽

Abiotic Stress Tolerance ◽

Stress Protein ◽

Crop Plants ◽

Cloning And Expression ◽

Major Type ◽

Transgenic Crop ◽

Universal Stress Protein

Different types of abiotic stresses inhibit the normal growth of plants by changing their physical biochemical, morphological, and molecular traits. It links to the polygenic traits, which is controlled with the help of different genes, due to this polygenetic the manipulation of foreign genetic makeup is very difficult. Drought stress is the very major type of threat to reduce the yield of cash crops in Pakistan and as well as in all over the world. Gene manipulation is the solution to face this problem by producing genetically modified crop plants that have the ability to survive in drought conditions. Universal stress protein gene has been already identified in bacteria which showed its response under stressed conditions, by manipulation of universal stress protein gene. It was found from our study that the bacterial cells transformed with the USP2 gene isolated from cotton induced abiotic stress tolerance under heat, osmotic, and salt stress. It was suggested from our findings that the USP2 gene could be used to produce abiotic stress tolerance transgenic crop plants to enhance crop plant yield and quality.

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Universal stress protein‐like gene from mulberry enhances abiotic stress tolerance in Escherichia coli and transgenic tobacco cells

Plant Biology ◽

10.1111/plb.13311 ◽

2021 ◽

Author(s):

K. H. Dhanyalakshmi ◽

K. N. Nataraja

Keyword(s):

Escherichia Coli ◽

Abiotic Stress ◽

Stress Tolerance ◽

Transgenic Tobacco ◽

Abiotic Stress Tolerance ◽

Stress Protein ◽

Tobacco Cells ◽

Universal Stress Protein

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Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches

Current Genomics ◽

10.2174/1389202921999200424181239 ◽

2020 ◽

Vol 21 (4) ◽

pp. 295-310 ◽

Cited By ~ 1

Author(s):

Antra Chatterjee ◽

Shilpi Singh ◽

Ruchi Rai ◽

Shweta Rai ◽

L.C. Rai

Keyword(s):

Abiotic Stress ◽

Stress Tolerance ◽

Iron Acquisition ◽

Abiotic Stress Tolerance ◽

In Silico Analysis ◽

Hypothetical Protein ◽

E Coli ◽

Anabaena Pcc7120

Background: Cyanobacteria are excellent model to understand the basic metabolic processes taking place in response to abiotic stress. The present study involves characterization of a hypothetical protein Alr0765 of Anabaena PCC7120 comprising CBS-CP12 domain and deciphering its role in abiotic stress tolerance. Methods: Molecular cloning, heterologous expression and protein purification using affinity chromatography was performed to obtain native purified protein Alr0765. 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 redox sensitivity of the target protein. Transcript level under different abiotic stresses as well as spot assay, CFU count, ROS level and cellular H2O2 level were used to show 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 the purified protein was a dimer. Binding possibility of Alr0765 with ATP was observed with almost 6-fold increment in relative fluorescence during TNP-ATP binding assay with a ƛ 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 affirms the iron acquisition property of protein. Moreover, generation of redox potential of 146 mV by Alr0765 suggested its probable role in maintaining 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, 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 cellular ROS level and total cellular H2O2 concentration validated 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 provide tolerance against multiple abiotic stresses.

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Inactivation of PadR, the Repressor of the Phenolic Acid Stress Response, by Molecular Interaction with Usp1, a Universal Stress Protein from Lactobacillus plantarum, in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.00774-09 ◽

2009 ◽

Vol 75 (16) ◽

pp. 5273-5283 ◽

Cited By ~ 28

Author(s):

Jérôme Gury ◽

Hélène Seraut ◽

Ngoc Phuong Tran ◽

Lise Barthelmebs ◽

Stéphanie Weidmann ◽

...

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Lactobacillus Plantarum ◽

Phenolic Acid ◽

Stress Protein ◽

Acid Stress ◽

Universal Stress Protein ◽

E Coli ◽

Gram Positive Bacteria ◽

Acid Stress Response

ABSTRACT The phenolic acid decarboxylase gene padA is involved in the phenolic acid stress response (PASR) in gram-positive bacteria. In Lactobacillus plantarum, the padR gene encodes the negative transcriptional regulator of padA and is cotranscribed with a downstream gene, usp1, which encodes a putative universal stress protein (USP), Usp1, of unknown function. The usp1 gene is overexpressed during the PASR. However, the role and the mechanism of action of the USPs are unknown in gram-positive bacteria. Therefore, to gain insights into the role of USPs in the PASR; (i) a usp1 deletion mutant was constructed; (ii) the two genes padR and usp1 were coexpressed with padA under its own promoter as a reporter gene in Escherichia coli; and (iii) molecular in vitro interactions between the PadR, Usp1, and the padA promoter were studied. Although the usp1 mutant strain retained phenolic acid-dependent PAD activity, it displayed a greater sensitivity to strong acidic conditions compared to that of the wild-type strain. PadR cannot be inactivated directly by phenolic acid in E. coli recombinant cultures but is inactivated by Usp1 when the two proteins are coexpressed in E. coli. The PadR inactivation observed in recombinant E. coli cells was supported by electrophoretic mobility shift assays. Although Usp1 seems not to be absolutely required for the PASR, its capacity to inactivate PadR indicates that it could serve as an important mediator in acid stress response mechanisms through its capacity to interact with transcriptional regulators.

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