scholarly journals Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides

2017 ◽  
Vol 199 (19) ◽  
Author(s):  
Chi Ho Chan ◽  
Caleb E. Levar ◽  
Fernanda Jiménez-Otero ◽  
Daniel R. Bond
Keyword(s):  
Metal Oxides ◽  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Electrical Current ◽  
Electron Acceptors ◽  
Geobacter Sulfurreducens ◽  
Hydrogen Electrode ◽  
Single Experiment ◽  
Content Type ◽  
Genes Encoding

ABSTRACT Geobacter sulfurreducens generates electrical current by coupling intracellular oxidation of organic acids to the reduction of proteins on the cell surface that are able to interface with electrodes. This ability is attributed to the bacterium's capacity to respire other extracellular electron acceptors that require contact, such as insoluble metal oxides. To directly investigate the genetic basis of electrode-based respiration, we constructed Geobacter sulfurreducens transposon-insertion sequencing (Tn-Seq) libraries for growth, with soluble fumarate or an electrode as the electron acceptor. Libraries with >33,000 unique insertions and an average of 9 insertions/kb allowed an assessment of each gene's fitness in a single experiment. Mutations in 1,214 different genomic features impaired growth with fumarate, and the significance of 270 genes unresolved by annotation due to the presence of one or more functional homologs was determined. Tn-Seq analysis of −0.1 V versus standard hydrogen electrode (SHE) electrode-grown cells identified mutations in a subset of genes encoding cytochromes, processing systems for proline-rich proteins, sensory networks, extracellular structures, polysaccharides, and metabolic enzymes that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of select genes identified via Tn-Seq revealed a new putative porin-cytochrome conduit complex (extABCD) crucial for growth with electrodes, which was not required for Fe(III) oxide reduction. In addition, four mutants lacking components of a putative methyl-accepting chemotaxis–cyclic dinucleotide sensing network (esnABCD) were defective in electrode colonization but grew normally with Fe(III) oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to Fe(III) oxides. IMPORTANCE Since metal oxide electron acceptors are insoluble, one hypothesis is that cells sense and reduce metals using the same molecular mechanisms used to form biofilms on electrodes and produce electricity. However, by simultaneously comparing thousands of Geobacter sulfurreducens transposon mutants undergoing electrode-dependent respiration, we discovered new cytochromes and chemosensory proteins supporting growth with electrodes that are not required for metal respiration. This supports an emerging model where G. sulfurreducens recognizes surfaces and forms conductive biofilms using mechanisms distinct from those used for growth with metal oxides. These findings provide a possible explanation for studies that correlate electricity generation with syntrophic interspecies electron transfer by Geobacter and reveal many previously unrecognized targets for engineering this useful capability in other organisms.

scholarly journals Genome scale mutational analysis of Geobacter sulfurreducens reveals distinct molecular mechanisms for respiration of poised electrodes vs. Fe(III) oxides

2016 ◽  
Author(s):  
Chi Ho Chan ◽  
Caleb E. Levara ◽  
Fernanda Jiménez-Oteroa ◽  
Daniel R. Bond
Keyword(s):  
Molecular Mechanisms ◽  
Mutational Analysis ◽  
Electron Acceptors ◽  
Geobacter Sulfurreducens ◽  
Electrode Surfaces ◽  
Genes Encoding ◽  
Generation Mechanisms ◽  
Extracellular Structures ◽  
Transposon Insertions ◽  
Genome Scale

AbstractGeobacter sulfurreducens generates electricity by coupling intracellular oxidation of organic acids with electron transfer to the cell exterior, while maintaining a conductive connection to electrode surfaces. This unique ability has been attributed to the bacterium’s capacity to also respire extracellular terminal electron acceptors that require contact, such as insoluble metal oxides. To expand the molecular understanding of electricity generation mechanisms, we constructed Geobacter sulfurreducens transposon mutant (Tn-Seq) libraries for growth with soluble fumarate or an electrode surface as the electron acceptor. Mutant libraries with over 33,000 unique transposon insertions and an average of 9 transposon insertions per kb allowed identification of 1,214 genomic features essential for growth with fumarate, including over 270 genes with one or more functional homologs that could not be resolved by previous annotation or in silico modeling. Tn-Seq analysis of electrode-grown cells identified mutations in over 50 genes encoding cytochromes, processing systems for proline-rich proteins, sensory systems, extracellular structures, polysaccharides, metabolic enzymes and hypothetical proteins that caused at least a 50% reduction in apparent growth rate. Scarless deletion mutants of genes identified via Tn-Seq revealed a new putative c-type cytochrome conduit complex (extABCD) essential for growth with electrodes, which was not required for Fe(III)-oxide reduction. In addition, mutants lacking components of a putative methyl-accepting chemotaxis/cyclic dinucleotide sensing network (esnABCD) were defective in electrode growth, but grew normally with Fe(III)-oxides. These results suggest that G. sulfurreducens possesses distinct mechanisms for recognition, colonization, and reduction of electrodes compared to other environmental electron acceptors.

scholarly journals NanoSIMS imaging reveals metabolic stratification within current-producing biofilms

2019 ◽  
Vol 116 (41) ◽  
pp. 20716-20724 ◽  
Author(s):  
Grayson L. Chadwick ◽  
Fernanda Jiménez Otero ◽  
Jeffrey A. Gralnick ◽  
Daniel R. Bond ◽  
Victoria J. Orphan
Keyword(s):  
Secondary Ion Mass Spectrometry ◽  
Anaerobic Respiration ◽  
Electrical Current ◽  
Total Activity ◽  
Stable Isotope Probing ◽  
Electron Acceptors ◽  
Geobacter Sulfurreducens ◽  
Cellular Growth ◽  
Anode Surface ◽  
Redox Potentials

Metal-reducing bacteria direct electrons to their outer surfaces, where insoluble metal oxides or electrodes act as terminal electron acceptors, generating electrical current from anaerobic respiration. Geobacter sulfurreducens is a commonly enriched electricity-producing organism, forming thick conductive biofilms that magnify total activity by supporting respiration of cells not in direct contact with electrodes. Hypotheses explaining why these biofilms fail to produce higher current densities suggest inhibition by formation of pH, nutrient, or redox potential gradients; but these explanations are often contradictory, and a lack of direct measurements of cellular growth within biofilms prevents discrimination between these models. To address this fundamental question, we measured the anabolic activity of G. sulfurreducens biofilms using stable isotope probing coupled to nanoscale secondary ion mass spectrometry (nanoSIMS). Our results demonstrate that the most active cells are at the anode surface, and that this activity decreases with distance, reaching a minimum 10 µm from the electrode. Cells nearest the electrode continue to grow at their maximum rate in weeks-old biofilms 80-µm-thick, indicating nutrient or buffer diffusion into the biofilm is not rate-limiting. This pattern, where highest activity occurs at the electrode and declines with each cell layer, is present in thin biofilms (<5 µm) and fully grown biofilms (>20 µm), and at different anode redox potentials. These results suggest a growth penalty is associated with respiring insoluble electron acceptors at micron distances, which has important implications for improving microbial electrochemical devices as well as our understanding of syntrophic associations harnessing the phenomenon of microbial conductivity.

scholarly journals Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

2014 ◽  
Vol 82 (4) ◽  
pp. 1600-1605 ◽  
Author(s):  
Melissa A. Dean ◽  
Randall J. Olsen ◽  
S. Wesley Long ◽  
Adriana E. Rosato ◽  
James M. Musser
Keyword(s):  
Staphylococcus Aureus ◽  
Pathway Analysis ◽  
Molecular Mechanisms ◽  
Point Mutations ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Small Colony Variants ◽  
Content Type ◽  
Genes Encoding ◽  
Small Colony

ABSTRACTStaphylococcus aureussmall-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of fiveS. aureusSCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

scholarly journals Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli: TABLE 1

2015 ◽  
Vol 198 (1) ◽  
pp. 7-11 ◽  
Author(s):  
Regine Hengge ◽  
Michael Y. Galperin ◽  
Jean-Marc Ghigo ◽  
Mark Gomelsky ◽  
Jeffrey Green ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Content Type ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Systematic Nomenclature ◽  
K 12 ◽  
Encoding Genes

In recent years,Escherichia colihas served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely usedE. coliK-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenicE. colistrains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling inE. coli, we now propose a general and systematicdgcandpdenomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains ofE. coliin future studies.

scholarly journals Activity of Aztreonam in Combination with Avibactam, Clavulanate, Relebactam, and Vaborbactam against Multidrug-Resistant Stenotrophomonas maltophilia

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
M. Biagi ◽  
D. Lamm ◽  
K. Meyer ◽  
A. Vialichka ◽  
M. Jurkovic ◽  
...  
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Treatment Strategies ◽  
Multidrug Resistant ◽  
Content Type ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Novel Treatment Strategies

ABSTRACT The intrinsic L1 metallo- and L2 serine-β-lactamases in Stenotrophomonas maltophilia make it naturally multidrug resistant and difficult to treat. There is a need to identify novel treatment strategies for this pathogen, especially against isolates resistant to first-line agents. Aztreonam in combination with avibactam has demonstrated potential, although data on other aztreonam–β-lactamase inhibitor (BLI) combinations are lacking. Additionally, molecular mechanisms for reduced susceptibility to these combinations have not been explored. The objectives of this study were to evaluate and compare the in vitro activities and to understand the mechanisms of resistance to aztreonam in combination with avibactam, clavulanate, relebactam, and vaborbactam against S. maltophilia. A panel of 47 clinical S. maltophilia strains nonsusceptible to levofloxacin and/or trimethoprim-sulfamethoxazole were tested against each aztreonam-BLI combination via broth microdilution, and 6 isolates were then evaluated in time-kill analyses. Three isolates with various aztreonam-BLI MICs were subjected to whole-genome sequencing and quantitative reverse transcriptase PCR. Avibactam restored aztreonam susceptibility in 98% of aztreonam-resistant isolates, compared to 61, 71, and 15% with clavulanate, relebactam, and vaborbactam, respectively. The addition of avibactam to aztreonam resulted in a ≥2-log10-CFU/ml decrease at 24 h versus aztreonam alone against 5/6 isolates compared to 1/6 with clavulanate, 4/6 with relebactam, and 2/6 with vaborbactam. Molecular analyses revealed that decreased susceptibility to aztreonam-avibactam was associated with increased expression of genes encoding L1 and L2, as well as the efflux pump (smeABC). Aztreonam-avibactam is the most promising BLI-combination against multidrug-resistant S. maltophilia. Decreased susceptibility may be due to the combination of overexpressed β-lactamases and efflux pumps. Further studies evaluating this combination against S. maltophilia are warranted.

scholarly journals Salt Stress-Induced Changes in the Transcriptome, Compatible Solutes, and Membrane Lipids in the Facultatively Phototrophic Bacterium Rhodobacter sphaeroides

2011 ◽  
Vol 77 (21) ◽  
pp. 7551-7559 ◽  
Author(s):  
Minoru Tsuzuki ◽  
Oleg V. Moskvin ◽  
Masayuki Kuribayashi ◽  
Kiichi Sato ◽  
Susana Retamal ◽  
...  
Keyword(s):  
Salt Stress ◽  
Rhodobacter Sphaeroides ◽  
Glycine Betaine ◽  
Mutational Analysis ◽  
Compatible Solutes ◽  
Phospholipid Composition ◽  
Compatible Solute ◽  
Content Type ◽  
Genes Encoding ◽  
Induced Changes

ABSTRACTResponses to NaCl stress were investigated in phototrophically grownAlphaproteobacterium Rhodobacter sphaeroidesby transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems,proVWXandbetS, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute inR. sphaeroides, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operonotsBA. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors,crpO(RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. WhencrpOwas provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network inR. sphaeroides.

scholarly journals An Inner Membrane Cytochrome Required Only for Reduction of High Redox Potential Extracellular Electron Acceptors

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Caleb E. Levar ◽  
Chi Ho Chan ◽  
Misha G. Mehta-Kolte ◽  
Daniel R. Bond
Keyword(s):  
Electron Transfer ◽  
Electron Acceptors ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Redox Potentials ◽  
Inner Membrane ◽  
Content Type ◽  
Transfer Strategies ◽  
Reducing Bacteria ◽  
Metal Reducing Bacteria

ABSTRACTDissimilatory metal-reducing bacteria, such asGeobacter sulfurreducens, transfer electrons beyond their outer membranes to Fe(III) and Mn(IV) oxides, heavy metals, and electrodes in electrochemical devices. In the environment, metal acceptors exist in multiple chelated and insoluble forms that span a range of redox potentials and offer different amounts of available energy. Despite this, metal-reducing bacteria have not been shown to alter their electron transfer strategies to take advantage of these energy differences. Disruption ofimcH, encoding an inner membranec-type cytochrome, eliminated the ability ofG. sulfurreducensto reduce Fe(III) citrate, Fe(III)-EDTA, and insoluble Mn(IV) oxides, electron acceptors with potentials greater than 0.1 V versus the standard hydrogen electrode (SHE), but theimcHmutant retained the ability to reduce Fe(III) oxides with potentials of ≤−0.1 V versus SHE. TheimcHmutant failed to grow on electrodes poised at +0.24 V versus SHE, but switching electrodes to −0.1 V versus SHE triggered exponential growth. At potentials of ≤−0.1 V versus SHE, both the wild type and theimcHmutant doubled 60% slower than at higher potentials. Electrodes poised even 100 mV higher (0.0 V versus SHE) could not triggerimcHmutant growth. These results demonstrate thatG. sulfurreducenspossesses multiple respiratory pathways, that some of these pathways are in operation only after exposure to low redox potentials, and that electron flow can be coupled to generation of different amounts of energy for growth. The redox potentials that trigger these behaviors mirror those of metal acceptors common in subsurface environments whereGeobacteris found.IMPORTANCEInsoluble metal oxides in the environment represent a common and vast reservoir of energy for respiratory microbes capable of transferring electrons across their insulating membranes to external acceptors, a process termed extracellular electron transfer. Despite the global biogeochemical importance of metal cycling and the ability of such organisms to produce electricity at electrodes, fundamental gaps in the understanding of extracellular electron transfer biochemistry exist. Here, we describe a conserved inner membrane redox protein inGeobacter sulfurreducenswhich is required only for electron transfer to high-potential compounds, and we show thatG. sulfurreducenshas the ability to utilize different electron transfer pathways in response to the amount of energy available in a metal or electrode distant from the cell.

scholarly journals Transcriptome Analysis of Avian Pathogenic Escherichia coli O1 in Chicken Serum Reveals Adaptive Responses to Systemic Infection

2011 ◽  
Vol 79 (5) ◽  
pp. 1951-1960 ◽  
Author(s):  
Ganwu Li ◽  
Kelly A. Tivendale ◽  
Peng Liu ◽  
Yaping Feng ◽  
Yvonne Wannemuehler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Protein Transport ◽  
Mutational Analysis ◽  
Systemic Infection ◽  
Luria Bertani ◽  
Virulence Plasmid ◽  
Avian Host ◽  
Content Type ◽  
Chicken Serum

ABSTRACTInfections of avian pathogenicEscherichia coli(APEC) result in annual multimillion-dollar losses to the poultry industry. Despite this, little is known about the mechanisms by which APEC survives and grows in the bloodstream. Thus, the aim of this study was to identify molecular mechanisms enabling APEC to survive and grow in this critical host environment. To do so, we compared the transcriptome of APEC O1 during growth in Luria-Bertani broth and chicken serum. Several categories of genes, predicted to contribute to adaptation and growth in the avian host, were identified. These included several known virulence genes and genes involved in adaptive metabolism, protein transport, biosynthesis pathways, stress resistance, and virulence regulation. Several genes with unknown function, which were localized to pathogenicity islands or APEC O1's large virulence plasmid, pAPEC-O1-ColBM, were also identified, suggesting that they too contribute to survival in serum. The significantly upregulated genesdnaK,dnaJ,phoP, andybtAwere subsequently subjected to mutational analysis to confirm their role in conferring a competitive advantage during infection. This genome-wide analysis provides novel insight into processes that are important to the pathogenesis of APEC O1.

scholarly journals Significance of a Posttranslational Modification of the PilA Protein of Geobacter sulfurreducens for Surface Attachment, Biofilm Formation, and Growth on Insoluble Extracellular Electron Acceptors

2017 ◽  
Vol 199 (8) ◽  
Author(s):  
Lubna V. Richter ◽  
Ashley E. Franks ◽  
Robert M. Weis ◽  
Steven J. Sandler
Keyword(s):  
Electron Transfer ◽  
Amino Acid ◽  
Posttranslational Modification ◽  
Biofilm Formation ◽  
Electron Acceptors ◽  
Type Iv Pili ◽  
Geobacter Sulfurreducens ◽  
Surface Attachment ◽  
Content Type ◽  
Type Iv

ABSTRACT Geobacter sulfurreducens, an anaerobic metal-reducing bacterium, possesses type IV pili. These pili are intrinsic structural elements in biofilm formation and, together with a number of c-type cytochromes, are thought to serve as conductive nanowires enabling long-range electron transfer (ET) to metal oxides and graphite anodes. Here, we report that a posttranslational modification of a nonconserved amino acid residue within the PilA protein, the structural subunit of the type IV pili, is crucial for growth on insoluble extracellular electron acceptors. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry of the secreted PilA protein revealed a posttranslational modification of tyrosine-32 with a moiety of a mass consistent with a glycerophosphate group. Mutating this tyrosine into a phenylalanine inhibited cell growth with Fe(III) oxides as the sole electron acceptor. In addition, this amino acid substitution severely diminished biofilm formation on graphite surfaces and impaired current output in microbial fuel cells. These results demonstrate that the capability to attach to insoluble electron acceptors plays a crucial role for the cells' ability to utilize them. The work suggests that glycerophosphate modification of Y32 is a key factor contributing to the surface charge of type IV pili, influencing the adhesion of Geobacter to specific surfaces. IMPORTANCE Type IV pili are bacterial appendages that function in cell adhesion, virulence, twitching motility, and long-range electron transfer (ET) from bacterial cells to insoluble extracellular electron acceptors. The mechanism and role of type IV pili for ET in Geobacter sulfurreducens is still a subject of research. In this study, we identified a posttranslational modification of the major G. sulfurreducens type IV pilin, suggested to be a glycerophosphate moiety. We show that a mutant in which the glycerophosphate-modified tyrosine-32 is replaced with a phenylalanine has reduced abilities for ET and biofilm formation compared with those of the wild type. The results show the importance of the glycerophosphate-modified tyrosine for surface attachment and electron transfer in electrode- or Fe(III)-respiring G. sulfurreducens cells.

scholarly journals Genetic Analysis Reveals the Identity of the Photoreceptor for Phototaxis in Hormogonium Filaments of Nostoc punctiforme

2014 ◽  
Vol 197 (4) ◽  
pp. 782-791 ◽  
Author(s):  
Elsie L. Campbell ◽  
Kari D. Hagen ◽  
Rui Chen ◽  
Douglas D. Risser ◽  
Daniela P. Ferreira ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Gene Clusters ◽  
Gliding Motility ◽  
Transcriptional Analysis ◽  
Nostoc Punctiforme ◽  
Recognition Sequence ◽  
Content Type ◽  
Wide Range ◽  
Genes Encoding

In cyanobacterialNostocspecies, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm thatNostoc punctiformehormogonia are positively phototactic to white light over a wide range of intensities.N. punctiformecontains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome–methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis inN. punctiformehormogonia; here these genes are designatedptx(forphototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of theptxgenes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpAandgvpC), which also are expressed only in the hormogonium filaments ofN. punctiforme.

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