scholarly journals AzuR From the SmtB/ArsR Family of Transcriptional Repressors Regulates Metallothionein in Anabaena sp. Strain PCC 7120

2022 ◽  
Vol 12 ◽  
Author(s):  
T. V. Divya ◽  
Celin Acharya
Keyword(s):  
Metal Binding ◽  
Cadmium Toxicity ◽  
Cadmium Stress ◽  
Negative Regulation ◽  
Size Exclusion ◽  
Transcriptional Repressors ◽  
Mobility Shift ◽  
Putative Promoter Region ◽  
Native Strain ◽  
Pcc 7120

Metallothioneins (MTs) are cysteine-rich, metal-sequestering cytosolic proteins that play a key role in maintaining metal homeostasis and detoxification. We had previously characterized NmtA, a MT from the heterocystous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of Anabaena NmtA by AzuR (Alr0831) belonging to the SmtB/ArsR family of transcriptional repressors. There is currently no experimental evidence for any functional role of AzuR. It is observed that azuR is located within the znuABC operon but in the opposite orientation and remotely away from the nmtA locus. Sequence analysis of AzuR revealed a high degree of sequence identity with Synechococcus SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in Escherichia coli and purified it by chitin affinity chromatography. Far-UV circular dichroism spectroscopy indicated that the recombinant AzuR protein possessed a properly folded structure. Glutaraldehyde cross-linking and size-exclusion chromatography revealed that AzuR exists as a dimer of ∼28 kDa in solution. Analysis of its putative promoter region [100 bp upstream of nmtA open reading frame (ORF)] identified the presence of a 12–2–12 imperfect inverted repeat as the cis-acting element important for repressor binding. Electrophoretic mobility shift assays (EMSAs) showed concentration-dependent binding of recombinant dimeric AzuR with the promoter indicating that NmtA is indeed a regulatory target of AzuR. Binding of AzuR to DNA was disrupted in the presence of metal ions like Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Pb2+, and Mn2+. The metal-dependent dissociation of protein–DNA complexes suggested the negative regulation of metal-inducible nmtA expression by AzuR. Overexpression of azuR in its native strain Anabaena 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of Anabaena MT by an α5 SmtB/ArsR metalloregulator AzuR.

scholarly journals Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1230
Author(s):  
Sawsan Napthine ◽  
Chris H. Hill ◽  
Holly C. M. Nugent ◽  
Ian Brierley
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Stop Codon ◽  
Leukemia Virus ◽  
Size Exclusion ◽  
Human Immunodeficiency Virus Replication ◽  
Mobility Shift ◽  
Ribosomal Frameshifting ◽  
Stop Codon Readthrough

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

scholarly journals FarRRegulates the farAB-Encoded Efflux Pump of Neisseriagonorrhoeae via an MtrR RegulatoryMechanism

2003 ◽  
Vol 185 (24) ◽  
pp. 7145-7152 ◽  
Author(s):  
E.-H. Lee ◽  
C. Rouquette-Loughlin ◽  
J. P. Folster ◽  
W. M. Shafer
Keyword(s):  
Regulatory Mechanism ◽  
Efflux Pump ◽  
Regulatory Protein ◽  
Transcriptional Repressors ◽  
Sequence Database ◽  
Mobility Shift ◽  
Pump System ◽  
Regulatory Cascade ◽  
Genome Sequence Database ◽  
Gel Mobility Shift

ABSTRACT The farAB operon of Neisseria gonorrhoeae encodes an efflux pump which mediates gonococcal resistance to antibacterial fatty acids. It was previously observed that expression of the farAB operon was positively regulated by MtrR, which is a repressor of the mtrCDE-encoded efflux pump system (E.-H. Lee and W. M. Shafer, Mol. Microbiol. 33:839-845, 1999). This regulation was believed to be indirect since MtrR did not bind to the farAB promoter. In this study, computer analysis of the gonococcal genome sequence database, lacZ reporter fusions, and gel mobility shift assays were used to elucidate the regulatory mechanism by which expression of the farAB operon is modulated by MtrR in gonococci. We identified a regulatory protein belonging to the MarR family of transcriptional repressors and found that it negatively controls expression of farAB by directly binding to the farAB promoter. We designated this regulator FarR to signify its role in regulating the farAB operon. We found that MtrR binds to the farR promoter, thereby repressing farR expression. Hence, MtrR regulates farAB in a positive fashion by modulating farR expression. This MtrR regulatory cascade seems to play an important role in adjusting levels of the FarAB and MtrCDE efflux pumps to prevent their excess expression in gonococci.

Localization of metallothionein, heat shock protein (Hsp70), and superoxide dismutase expression in Hemidiaptomus roubaui (Copepoda, Crustacea) exposed to cadmium and heat stress

2007 ◽  
Vol 85 (3) ◽  
pp. 362-371 ◽  
Author(s):  
Martine Liberge ◽  
Roxane-M. Barthélémy
Keyword(s):  
Superoxide Dismutase ◽  
Heat Stress ◽  
Heat Shock ◽  
Metal Binding ◽  
Stress Proteins ◽  
Reproductive Function ◽  
Cadmium Stress ◽  
Specific Distribution ◽  
Freshwater Crustacean ◽  
Shock Proteins

Immunohistochemical methods were applied in the present study to investigate the expression of stress proteins such as metallothioneins (MT), which are metal-binding proteins, and heat shock proteins (Hsp70), as well as an antioxidant enzyme (superoxide dismutase, SOD), in the freshwater crustacean copepod Hemidiaptomus roubaui (Richard, 1888) exposed to cadmium or heat stress. The results show a tissue-specific distribution of MT-like protein after cadmium exposure in the brain and in the nerve cord. Cadmium stress did not provoke inducible Hsp70 or SOD expression. Unlike cadmium, heat stress induced the expression of Hsp70 and SOD in the shell glands, a structure involved in the reproductive function, and more particularly in the formation of the diapause egg envelope. MT expression is not induced in animals exposed to heat stress.

scholarly journals A Functional SNP in the Promoter of LBX1 Is Associated With the Development of Adolescent Idiopathic Scoliosis Through Involvement in the Myogenesis of Paraspinal Muscles

2021 ◽  
Vol 9 ◽  
Author(s):  
Leilei Xu ◽  
Zhenhua Feng ◽  
Zhicheng Dai ◽  
Wayne Y. W. Lee ◽  
Zhichong Wu ◽  
...  
Keyword(s):  
Adolescent Idiopathic Scoliosis ◽  
Idiopathic Scoliosis ◽  
Muscle Development ◽  
Luciferase Assay ◽  
Paraspinal Muscles ◽  
Mobility Shift ◽  
Putative Promoter Region ◽  
Mobility Shift Assay ◽  
First Time ◽  
Functional Snp

Previous studies have shown that LBX1 is associated with adolescent idiopathic scoliosis (AIS) in multiple populations. For the first time, rs1322330 located in the putative promoter region of LBX1 was found significantly associated with AIS in the Chinese population [p = 6.08 × 10–14, odds ratio (OR) = 1.42, 95% confidence interval of 1.03–1.55]. Moreover, the luciferase assay and electrophoretic mobility shift assay supported that the allele A of rs1322330 could down-regulate the expression of LBX1 in the paraspinal muscles of AIS. In addition, silencing LBX1 in the myosatellite cells resulted in significantly inhibited cell viability and myotube formation, which supported an essential role of LBX1 in muscle development of AIS. To summarize, rs1322330 may be a novel functional SNP regulating the expression of LBX1, which was involved in the etiology of AIS possibly via regulation of myogenesis in the paraspinal muscles.

scholarly journals Molecular Characterization of MexL, the Transcriptional Repressor of the mexJK Multidrug Efflux Operon in Pseudomonas aeruginosa

2005 ◽  
Vol 49 (5) ◽  
pp. 1844-1851 ◽  
Author(s):  
Rungtip Chuanchuen ◽  
Jared B. Gaynor ◽  
RoxAnn Karkhoff-Schweizer ◽  
Herbert P. Schweizer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Polyacrylamide Gel Electrophoresis ◽  
Size Exclusion ◽  
Mutant Protein ◽  
Mobility Shift ◽  
Promoter Sequences ◽  
Exclusion Chromatography ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Gel Mobility Shift ◽  
Carboxy Terminal

ABSTRACT The Pseudomonas aeruginosa mexJK efflux operon is constitutively expressed in mutants with defects in the upstream mexL gene, which encodes a repressor of the TetR family. MexL and a MexLA47D mutant protein were purified from Escherichia coli as fusion proteins with carboxy-terminal hexahistidine tags. Native polyacrylamide gel electrophoresis and size exclusion chromatography revealed that MexL is a tetramer in solution. MexL and MexLA47D oligomerization was confirmed using a genetic approach, and the MexLA47D mutant protein was not impaired in multimerization. Gel mobility shift and footprinting assays demonstrated that MexL, but not MexLA47D, binds specifically to the 94-bp mexL-mexJ intergenic region to sequences located between positions −84 and −20 from the mexJ initiation codon. MexL protected about 60 nucleotides on each strand, and the protected regions overlapped almost perfectly, a finding consistent with MexL regulating the expression of both mexL and mexJK, which was ascertained by gene fusion analyses. The protected region contains predicted −10 and −35 promoter sequences for both mexL and mexJ, with partially overlapping −10 regions. The mexL promoter assignment was verified by mapping the mexL transcription start site, and the mexJ promoter was localized to the predicted regions using lacZ fusions. The MexL-protected region contains two inverted GTATTT repeats, and their location in the protected region and overlap with the mexL and mexJ promoter sequences strongly support a role in MexL binding.

scholarly journals A cadmium stress-responsive gene AtFC1 confers plant tolerance to cadmium toxicity

2017 ◽  
Vol 17 (1) ◽  
Author(s):  
Jun Song ◽  
Sheng Jun Feng ◽  
Jian Chen ◽  
Wen Ting Zhao ◽  
Zhi Min Yang
Keyword(s):  
Cadmium Toxicity ◽  
Cadmium Stress ◽  
Plant Tolerance ◽  
Responsive Gene ◽  
Tolerance To Cadmium

scholarly journals Isolation and Characterization of the Vascular-specific 22-kDa Zn-binding Protein

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 475D-475
Author(s):  
Kathryn C. Taylor ◽  
Danielle R. Elli
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Binding Protein ◽  
Xylem Sap ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Ancestral Gene ◽  
Rangpur Lime ◽  
Rough Lemon ◽  
Isolation And Characterization

A 22-kDa Zn-binding protein (ZBP) was isolated from the phloem tissue and evacuated xylem sap of `Valencia' sweet orange [Citrus sinensis (L.) Osbeck] on rough lemon [C. jambhiri (L.)], as well as Valencia on Rangpur lime [Citrus limonia Osbeck]. Phloem and xylem Zn was associated with the 22 kDa ZBP. The Mr value of this ZBP was estimated to be 19,500 by size exclusion chromatography and 22,800 by SDS-PAGE. This protein was isolated with an isoelectric point of 7.5. Ion exchange chromatography demonstrated that 22-kDa ZBP was highly anionic, requiring 0.43 M NaCl for elution from QAE Sepharose. The 22-kDa ZBP appears unique to citrus, having no cross reaction with protein from several tissues from a range of plant species. Accumulation decreased under Zn-deficient conditions, was enhanced by osmotic stress, and the protein completely disappeared with wounding. Amino acid composition demonstrated that the protein was rich in aspartate, and glutamate; and contained 6 cysteine, and 4 histidine residues. These amino acids may be involved in metal binding. N-terminal amino acid sequencing demonstrated that the 22-kDa ZBP had identity with sporamin A&B precursors, Kunitz-type trypsin inhibitors, and miraculin. It is suggested that the genes that encode these proteins are derived from a common ancestral gene.

scholarly journals Impact of Exogenous Application of Salicylic Acid and Hydrogen Peroxide on Essential Nutrients and Silymarin in Silybum Marianum (L.) Gaertn Grown at Two Different Altitudes of Balochistan Under Cadmium Stress

2021 ◽  
Author(s):  
Mereen Nizar ◽  
Kanval Shaukat ◽  
Noreen Zahra ◽  
Abdul Samad ◽  
Mohammad Bilal Hafeez ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Salicylic Acid ◽  
Cadmium Toxicity ◽  
Foliar Spray ◽  
Cadmium Stress ◽  
Milk Thistle ◽  
Silybum Marianum ◽  
Essential Nutrients ◽  
Exogenous Application

Abstract Cadmium (Cd+2) is a potentially toxic element that inhibits growth and development of several species of plants along with Silybum marianum (L.) Gaertn which is an essential medicinal plant belonging to family Asteraceae. The exogenous application with 0.25µM Salicylic acid (SA) and 10µM hydrogen peroxide (H2O2) to ameliorate harmful effects of cadmium (500 µM) on milk thistle were studied that were grown at two different ecological zones of Balochistan province of Pakistan i.e. Quetta (Qta) and Turbat (Tbt). The design of experiment was Randomized Complete Block Design (RCBD) with three replicates. Application of SA and H2O2, priming (P), Foliar spray (FS) and combinational treatments (P+FS) were highly helpful in alleviating the negative role of cadmium toxicity. The essential nutrients i.e. nitrate (NO3-), calcium (Ca2+) and potassium (K) were affected by Cd+2 induced toxicity however, the substantial role of SA and H2O2 widely helped to reduce the cadmium stress and boosted up the plant nutrients content. In a nutshell, exogenous treatments of SA and H2O2 enhanced the yield potential along with highest silymarin contents in milk thistle seeds which is of prime significance for its medicinal importance in treatment of liver diseases. The data obtained in this study highly recommend the priming and foliar spray of SA and H2O2 on milk thistle plants, as the best solution to alleviative the cadmium toxicity which will ultimately leads to better growth and yield of the plants.

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