scholarly journals Functional and Expression Analyses of the cop Operon, Required for Copper Resistance in Agrobacterium tumefaciens

2009 ◽  
Vol 191 (16) ◽  
pp. 5159-5168 ◽  
Author(s):  
Sirikan Nawapan ◽  
Nisanart Charoenlap ◽  
Anchalee Charoenwuttitam ◽  
Panatda Saenkham ◽  
Skorn Mongkolsuk ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Copper Ions ◽  
Protein A ◽  
Copper Resistance ◽  
Copper Chaperone ◽  
Phytopathogenic Bacterium ◽  
Ion Concentrations ◽  
Increased Sensitivity ◽  
Cop Operon

ABSTRACT The copper resistance determinant copARZ, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium Agrobacterium tumefaciens. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a copR-dependent fashion. Analysis of the copARZ promoter revealed a putative CopR binding box located within the spacer of the −35 and −10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the copARZ operon or copZ reduces the level of resistance to copper but not to other metal ions. Also, the copARZ operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional copZ does not affect the ability of copper ions to induce the copARZ promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the copARZ operon as a component of the copper resistance machinery in A. tumefaciens.

The RSP_2889 gene product of Rhodobacter sphaeroides is a CueR homologue controlling copper-responsive genes

Microbiology ◽  
2011 ◽  
Vol 157 (12) ◽  
pp. 3306-3313 ◽  
Author(s):  
Verena Peuser ◽  
Jens Glaeser ◽  
Gabriele Klug
Keyword(s):  
Gene Product ◽  
Rhodobacter Sphaeroides ◽  
Copper Ions ◽  
Copper Metabolism ◽  
Photosynthetic Bacterium ◽  
Copper Resistance ◽  
Copper Chaperone ◽  
Promoter Regions ◽  
Low Copy Number ◽  
Increased Sensitivity

Metal homeostasis is important in all living cells in order to provide sufficient amounts of metal ions for biological processes but to prevent toxic effects by excess amounts. Here we show that the gene product of RSP_2889 of the facultatively photosynthetic bacterium Rhodobacter sphaeroides is homologous to CueR, a regulator of copper metabolism in Escherichia coli and other bacteria. CueR binds to the promoter regions of genes for a copper-translocating ATPase and for a copper chaperone and is responsible for their high expression when cells are exposed to elevated levels of copper ions. While deletion of RSP_2889 has no significant effect on copper resistance, expression from a low-copy-number plasmid mediates increased sensitivity to copper.

scholarly journals Copper chaperone blocks amyloid formation via ternary complex

2018 ◽  
Vol 51 ◽  
Author(s):  
Istvan Horvath ◽  
Tony Werner ◽  
Ranjeet Kumar ◽  
Pernilla Wittung-Stafshede
Keyword(s):  
Control Systems ◽  
Protein Misfolding ◽  
Ternary Complex ◽  
Copper Ions ◽  
Copper Chaperone ◽  
Amyloid Formation ◽  
Cellular Environment ◽  
Copper Site ◽  
Amyloidogenic Protein

AbstractProtein misfolding in cells is avoided by a network of protein chaperones that detect misfolded or partially folded species. When proteins escape these control systems, misfolding may result in protein aggregation and amyloid formation. We here show that aggregation of the amyloidogenic protein α-synuclein (αS), the key player in Parkinson's disease, is controlled by the copper transport protein Atox1 in vitro. Copper ions are not freely available in the cellular environment, but when provided by Atox1, the resulting copper-dependent ternary complex blocks αS aggregation. Because the same inhibition was found for a truncated version of αS, lacking the C-terminal part, it appears that Atox1 interacts with the N-terminal copper site in αS. Metal-dependent chaperoning may be yet another manner in which cells control its proteome.

scholarly journals Molecular insights into the copper-sensitive operon repressor in Acidithiobacillus caldus

2021 ◽  
Author(s):  
Shaoxiang Hou ◽  
Yanjun Tong ◽  
Hailin Yang ◽  
Shoushuai Feng
Keyword(s):  
Binding Affinity ◽  
Dissociation Constants ◽  
Copper Ions ◽  
Copper Resistance ◽  
Size Exclusion ◽  
Transcriptional Analysis ◽  
Copper Stress ◽  
Acidithiobacillus Caldus ◽  
Competition Assays

The copper-sensitive operon repressor (CsoR) family is the main Cu(I)-sensing family, which is widely distributed, and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥ 3 g/L copper ions). Here, we identified CsoR Ac in hyper-copper-resistant Acidithiobacillus caldus , a type strain used in the bioleaching process of copper ores. CsoR Ac possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli . Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoR Ac monomer in vitro combined with atomic absorption spectroscopy analysis. Cu I -CsoR Ac and apo-CsoR Ac share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants ( K D = 2.26 × 10 −18 M and 0.53 × 10 −15 M) and Cu(I) binding affinity of apo-CsoR Ac were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoR Ac did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants shared the same order of magnitude of Cu(I) binding affinity with apo-CsoR Ac . Moreover, apo-CsoR Ac could bind to the DNA fragment P08430 in vitro , although with low affinity. Finally, a working model was proposed to illustrate putative copper resistance mechanisms in A. caldus . Importance Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus . Here, we characterized a major Cu(I)-sensing family protein, CsoR Ac , which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator, bathocuproine disulfonate. In particular, CsoR family proteins were only identified in A. caldus , rather than A. ferrooxidans and A. thiooxidans , which are both type strains used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

scholarly journals The Helicobacter pylori CrdRS Two-Component Regulation System (HP1364/HP1365) Is Required for Copper-Mediated Induction of the Copper Resistance Determinant CrdA

2005 ◽  
Vol 187 (13) ◽  
pp. 4683-4688 ◽  
Author(s):  
Barbara Waidner ◽  
Klaus Melchers ◽  
Frank Nils Stähler ◽  
Manfred Kist ◽  
Stefan Bereswill
Keyword(s):  
Helicobacter Pylori ◽  
Copper Ions ◽  
Response Regulator ◽  
Copper Resistance ◽  
Resistance Determinant ◽  
Upstream Region ◽  
Regulation System ◽  
Direct Role ◽  
H Pylori

ABSTRACT Here we describe that the Helicobacter pylori sensor kinase produced by HP1364 and the response regulator produced by HP1365 and designated CrdS and CrdR, respectively, are both required for transcriptional induction of the H. pylori copper resistance determinant CrdA by copper ions. CrdRS-deficient mutants lacked copper induction of crdA expression and were copper sensitive. A direct role of CrdR in transcriptional regulation of crdA was confirmed by in vitro binding of CrdR to the crdA upstream region. A 21-nucleotide sequence located near the crdA promoter was shown to be required for CrdR binding.

scholarly journals Mammalian Copper Chaperone Cox17p Has an Essential Role in Activation of Cytochrome c Oxidase and Embryonic Development

2002 ◽  
Vol 22 (21) ◽  
pp. 7614-7621 ◽  
Author(s):  
Yoshinori Takahashi ◽  
Koichiro Kako ◽  
Shin-ichi Kashiwabara ◽  
Akio Takehara ◽  
Yoshiko Inada ◽  
...  
Keyword(s):  
Essential Role ◽  
Copper Ions ◽  
Mouse Embryos ◽  
Copper Chaperone ◽  
Succinate Dehydrogenase Activity ◽  
Copper Transporter ◽  
A Cell ◽  
Deficient Mice ◽  
Mammalian System

ABSTRACT Cox17p is essential for the assembly of functional cytochrome c oxidase (CCO) and for delivery of copper ions to the mitochondrion for insertion into the enzyme in yeast. Although this small protein has already been cloned or purified from humans, mice, and pigs, the function of Cox17p in the mammalian system has not yet been elucidated. In vitro biochemical data for mammalian Cox17p indicate that the copper binds to the sequence -KPCCAC-. Although mouse embryos homozygous for COX17 disruption die between embryonic days E8.5 and E10, they develop normally until E6.5. This phenotype is strikingly similar to embryos of Ctr1(−/−), a cell surface copper transporter, in its lethality around the time of gastrulation. COX17-deficient embryos exhibit severe reductions in CCO activity at E6.5. Succinate dehydrogenase activity and immunoreactivities for anti-COX subunit antibodies were normal in the COX17(−/−) embryos, indicating that this defect was not caused by the deficiency of other complexes and/or subunits but was caused by impaired CCO activation by Cox17p. Since other copper chaperone (Atox1 and CCS)-deficient mice show a more moderate defect, the disruption of the COX17 locus causes the expression of only the phenotype of Ctr1(−/−). We found that the activity of lactate dehydrogenase was also normal in E6.5 embryos, implying that the activation of CCO by Cox17p may not be essential to the progress of embryogenesis before gastrulation.

Inhibition of Platelet Aggregation by Ethanol in Vitro Shows Specificity for Aggregating Agent Used and Is Influenced by Platelet Lipid Composition

1982 ◽  
Vol 48 (01) ◽  
pp. 049-053 ◽  
Author(s):  
C G Fenn ◽  
J M Littleton
Keyword(s):  
Platelet Aggregation ◽  
Lipid Composition ◽  
Saturated Fatty Acids ◽  
Calcium Ionophore ◽  
Cholesterol Content ◽  
Membrane Lipid ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Increased Sensitivity

SummaryEthanol at physiologically tolerable concentrations inhibited platelet aggregation in vitro in a relatively specific way, which may be influenced by platelet membrane lipid composition. Aggregation to collagen, calcium ionophore A23187 and thrombin (low doses) were often markedly inhibited by ethanol, adrenaline and ADP responses were little affected, and aggregation to exogenous arachidonic acid was actually potentiated by ethanol. Aggregation to collagen, thrombin and A23187 was inhibited more by ethanol in platelets enriched with saturated fatty acids than in those enriched with unsaturated fats. Platelets enriched with cholesterol showed increased sensitivity to ADP, arachidonate and adrenaline but this increase in cholesterol content did not appear to influence the inhibition by ethanol of platelet responses. The results suggest that ethanol may inhibit aggregation by an effect on membrane fluidity and/or calcium mobilization resulting in decreased activity of a membrane-bound phospholipase.

Human Copper Chaperone Atox1 Translocates to the Nucleus but does not Bind DNA In Vitro

2015 ◽  
Vol 22 (6) ◽  
pp. 532-538 ◽  
Author(s):  
Dana Kahra ◽  
Tanumoy Mondol ◽  
Moritz Niemiec ◽  
Pernilla Wittung-Stafshede
Keyword(s):  
Copper Chaperone

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

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