scholarly journals A genetically validated approach to detect inorganic polyphosphates in plants

2019 ◽  
Author(s):  
Jinsheng Zhu ◽  
Sylvain Loubéry ◽  
Larissa Broger ◽  
Laura Lorenzo-Orts ◽  
Anne Utz-Pugin ◽  
...  
Keyword(s):  
Higher Plants ◽  
Extraction Methods ◽  
Binding Domain ◽  
Linear Polymers ◽  
Inorganic Polyphosphates ◽  
Inorganic Polyphosphate ◽  
E Coli ◽  
Staining Protocol ◽  
Dicotyledonous Plants ◽  
Eukaryotic Organisms

AbstractInorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. PolyPs represent important stores of phosphate and energy, and are abundantly found in many pro- and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP-specific dye and a polyP binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules inNicotiana benthamianaand Arabiopsis cells by heterologous expression ofE. colipolyphosphate kinase 1 (PPK1). Over-expression of PPK1 but not of a catalytically impaired version of the enzyme lead to severe growth phenotypes, suggesting that ATP-dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1 expressing Arabidopsis lines with plants expressing the polyP-binding domain ofE. coliexopolyphosphatase (PPX1c), which co-localized with PPK1-generated polyP granules. These granules were stained by the polyP-specific dye JC-D7 and appeared as electron dense structures in transmission electron microscopy (TEM) sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono- and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP-rich acidocalicisomes in the green algaeChlamydomonas reinhardtii.Together, our experiments suggest that higher plants may not contain large polyPs stores.Significance StatementA chemical dye and an inorganic polyphosphate binding domain are shown to specifically label inorganic polyphosphate granules in transgenic Arabidopsis lines and Chlamydomonas acidocalcisomes. Using these tools, we show that in contrast to many prokaryotic and eukaryotic organisms, higher plants do not seem to contain large inorganic polyphosphate stores.

scholarly journals Co-Translational Insertion of Aquaporins into Liposome for Functional Analysis via an E. coli Based Cell-Free Protein Synthesis System

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1325 ◽  
Author(s):  
Ke Yue ◽  
Tran Nam Trung ◽  
Yiyong Zhu ◽  
Ralf Kaldenhoff ◽  
Lei Kai
Keyword(s):  
Functional Analysis ◽  
Membrane Proteins ◽  
Fluorescent Protein ◽  
Water Channel ◽  
New Approach ◽  
E Coli ◽  
Straightforward Method ◽  
Lipid Environment ◽  
Green Fluorescent ◽  
Eukaryotic Organisms

Aquaporins are important and well-studied water channel membrane proteins. However, being membrane proteins, sample preparation for functional analysis is tedious and time-consuming. In this paper, we report a new approach for the co-translational insertion of two aquaporins from Escherichia coli and Nicotiana tabacum using the CFPS system. This was done in the presence of liposomes with a modified procedure to form homogenous proteo-liposomes suitable for functional analysis of water permeability using stopped-flow spectrophotometry. Two model aquaporins, AqpZ and NtPIP2;1, were successfully incorporated into the liposome in their active forms. Shifted green fluorescent protein was fused to the C-terminal part of AqpZ to monitor its insertion and status in the lipid environment. This new fast approach offers a fast and straightforward method for the functional analysis of aquaporins in both prokaryotic and eukaryotic organisms.

Enhancement of protein synthesis by an inorganic polyphosphate in an E. coli cell-free system

2006 ◽  
Vol 64 (2) ◽  
pp. 241-249 ◽  
Author(s):  
Hiromichi Itoh ◽  
Yumi Kawazoe ◽  
Toshikazu Shiba
Keyword(s):  
Protein Synthesis ◽  
Coli Cell ◽  
Inorganic Polyphosphate ◽  
Free System ◽  
Cell Free System ◽  
E Coli

scholarly journals Binding From both sides: TolR and full-length OmpA bind and maintain the local structure of the E. coli cell wall

2018 ◽  
Author(s):  
Alister T. Boags ◽  
Firdaus Samsudin ◽  
Syma Khalid
Keyword(s):  
Cell Wall ◽  
Electrostatic Interactions ◽  
Cell Envelope ◽  
Binding Domain ◽  
Gram Negative Bacteria ◽  
Inner Membrane ◽  
E Coli ◽  
Non Covalent Interactions ◽  
Covalent Interactions

SUMMARYWe present a molecular modeling and simulation study of the of the E. coli cell envelope, with a particular focus on the role of TolR, a native protein of the E. coli inner membrane in interactions with the cell wall. TolR has been proposed to bind to peptidoglycan, but the only structure of this protein thus far is in a conformation in which the putative peptidoglycan binding domain is not accessible. We show that a model of the extended conformation of the protein in which this domain is exposed, binds peptidoglycan largely through electrostatic interactions. We show that non-covalent interactions of TolR and OmpA with the cell wall, from the inner membrane and outer membrane sides respectively, maintain the position of the cell wall even in the absence of Braun’s lipoprotein. When OmpA is truncated to remove the peptidoglycan binding domain, TolR is able to pull the cell wall down towards the inner membrane. The charged residues that mediate the cell-wall interactions of TolR in our simulations, are conserved across a number of species of Gram-negative bacteria.

scholarly journals Inorganic Polyphosphate, Exopolyphosphatase, andPho84-Like Transporters May Be Involved in Copper Resistance inMetallosphaera sedulaDSM 5348T

Archaea ◽  
2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Matías Rivero ◽  
Constanza Torres-Paris ◽  
Rodrigo Muñoz ◽  
Ricardo Cabrera ◽  
Claudio A. Navarro ◽  
...  
Keyword(s):  
Present Report ◽  
Resistance Mechanism ◽  
Metal Resistance ◽  
Copper Resistance ◽  
Site Directed Mutagenesis ◽  
Pcr Analysis ◽  
Linear Polymers ◽  
Inorganic Polyphosphate ◽  
Metallosphaera Sedula

Polyphosphates (PolyP) are linear polymers of orthophosphate residues that have been proposed to participate in metal resistance in bacteria and archaea. In addition of having a CopA/CopB copper efflux system, the thermoacidophilic archaeonMetallosphaera sedulacontains electron-dense PolyP-like granules and a putative exopolyphosphatase (PPXMsed,Msed_0891) and four presumedpho84-like phosphate transporters (Msed_0846,Msed_0866,Msed_1094, andMsed_1512) encoded in its genome. In the present report, the existence of a possible PolyP-based copper-resistance mechanism inM. sedulaDSM 5348Twas evaluated.M. sedulaDSM 5348Taccumulated high levels of phosphorous in the form of granules, and its growth was affected in the presence of 16 mM copper. PolyP levels were highly reduced after the archaeon was subjected to an 8 mM CuSO4shift. PPXMsedwas purified, and the enzyme was found to hydrolyze PolyPin vitro. Essential residues for catalysis of PPXMsedwere E111 and E113 as shown by a site-directed mutagenesis of the implied residues. Furthermore,M. sedula ppx,pho84-like, andcopTMAgenes were upregulated upon copper exposure, as determined by qRT-PCR analysis. The results obtained support the existence of a PolyP-dependent copper-resistance system that may be of great importance in the adaptation of this thermoacidophilic archaeon to its harsh environment.

scholarly journals Biological Roles of Ornithine Aminotransferase (OAT) in Plant Stress Tolerance: Present Progress and Future Perspectives

2018 ◽  
Vol 19 (11) ◽  
pp. 3681 ◽  
Author(s):  
Alia Anwar ◽  
Maoyun She ◽  
Ke Wang ◽  
Bisma Riaz ◽  
Xingguo Ye
Keyword(s):  
Abiotic Stresses ◽  
Higher Plants ◽  
Plant Stress ◽  
Pathogen Resistance ◽  
Ornithine Aminotransferase ◽  
Plant Tolerance ◽  
Crop Varieties ◽  
Drought And Salt Stresses ◽  
Eukaryotic Organisms ◽  
Daunting Challenge

Plant tolerance to biotic and abiotic stresses is complicated by interactions between different stresses. Maintaining crop yield under abiotic stresses is the most daunting challenge for breeding resilient crop varieties. In response to environmental stresses, plants produce several metabolites, such as proline (Pro), polyamines (PAs), asparagine, serine, carbohydrates including glucose and fructose, and pools of antioxidant reactive oxygen species. Among these metabolites, Pro has long been known to accumulate in cells and to be closely related to drought, salt, and pathogen resistance. Pyrroline-5-carboxylate (P5C) is a common intermediate of Pro synthesis and metabolism that is produced by ornithine aminotransferase (OAT), an enzyme that functions in an alternative Pro metabolic pathway in the mitochondria under stress conditions. OAT is highly conserved and, to date, has been found in all prokaryotic and eukaryotic organisms. In addition, ornithine (Orn) and arginine (Arg) are both precursors of PAs, which confer plant resistance to drought and salt stresses. OAT is localized in the cytosol in prokaryotes and fungi, while OAT is localized in the mitochondria in higher plants. We have comprehensively reviewed the research on Orn, Arg, and Pro metabolism in plants, as all these compounds allow plants to tolerate different kinds of stresses.

The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain

2004 ◽  
Vol 37 (5) ◽  
pp. 741-749 ◽  
Author(s):  
Yoshimi Umemura ◽  
Tomoko Ishiduka ◽  
Rie Yamamoto ◽  
Muneharu Esaka
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Higher Plants ◽  
Binding Domain ◽  
Dna Binding Domain

The DNA binding domain and bending angle of E. coli CAP protein

Cell ◽  
1986 ◽  
Vol 47 (6) ◽  
pp. 995-1005 ◽  
Author(s):  
Huei-Nin Liu-Johnson ◽  
Marc R. Gartenberg ◽  
Donald M. Crothers
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Bending Angle ◽  
E Coli

scholarly journals Hg(II) sequestration and protection by the MerR metal-binding domain (MBD)

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 709-719 ◽  
Author(s):  
Jie Qin ◽  
Lingyun Song ◽  
Hassan Brim ◽  
Michael J. Daly ◽  
Anne O. Summers
Keyword(s):  
Cell Surface ◽  
Metal Binding ◽  
Deinococcus Radiodurans ◽  
Biochemical Properties ◽  
Binding Domain ◽  
Mercury Resistance ◽  
E Coli ◽  
Metal Binding Domain ◽  
Single Polypeptide ◽  
Physiological And Biochemical

MerR, the metalloregulator of the bacterial mercury resistance (mer) operon, binds Hg(II) with high affinity. To study the mechanism of metal-induced activation, a small protein was previously engineered embodying in a single polypeptide the metal-binding domain (MBD) ordinarily formed between two monomers of MerR. Here the physiological and biochemical properties of MBD expressed on the cell surface or in the cytosol were examined, to better understand the environments in which specific metal binding can occur with this small derivative. Over 20 000 surface copies of MBD were expressed per Escherichia coli cell, with metal stoichiometries of ∼1·0 Hg(II) per MBD monomer. Cells expressing MBD on their surface in rich medium bound 6·1-fold more Hg(II) than those not expressing MBD. Although in nature cells use the entire mer operon to detoxify mercury, it was interesting to note that cells expressing only MBD survived Hg(II) challenge and recovered more quickly than cells without MBD. Cell-surface-expressed MBD bound Hg(II) preferentially even in the presence of a 22-fold molar excess of Zn(II) and when exposed to equimolar Cd(II) in addition. MBD expressed in the cystosol also afforded improved survival from Hg(II) exposure for E. coli and for the completely unrelated bacterium Deinococcus radiodurans.

scholarly journals A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

2006 ◽  
Vol 72 (1) ◽  
pp. 245-251 ◽  
Author(s):  
Ju-Yuan Zhang ◽  
Jie Zou ◽  
Qiyu Bao ◽  
Wen-Li Chen ◽  
Li Wang ◽  
...  
Keyword(s):  
Arthrospira Platensis ◽  
Biochemical Properties ◽  
Salt Sensitivity ◽  
E Coli ◽  
Multiple Origins ◽  
Manic Depressive ◽  
Sodium Toxicity ◽  
Function Relationship ◽  
Eukaryotic Organisms

ABSTRACT 3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg2+ for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li+ (50% inhibitory concentration [IC50] = 3.6 mM) but only slightly sensitive to Na+ (IC50 = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li+ and Na+, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li+ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.

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