DOCSLIB.ORG

Escherichia Coli Cytochrome C Peroxidase Is a Respiratory Oxidase That Enables the Use of Hydrogen Peroxide As a Terminal Electron Acceptor

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Escherichia Coli Cytochrome C Peroxidase Is a Respiratory Oxidase That Enables the Use of Hydrogen Peroxide As a Terminal Electron Acceptor Escherichia coli cytochrome c peroxidase is a respiratory oxidase that enables the use of hydrogen peroxide as a terminal electron acceptor Maryam Khademiana and James A. Imlaya,1 aDepartment of Microbiology, University of Illinois, Urbana, IL 61801 Edited by Andreas J. Bäumler, University of California, Davis, CA, and accepted by Editorial Board Member Carl F. Nathan June 3, 2017 (received for review January 28, 2017) Microbial cytochrome c peroxidases (Ccp) have been studied for and the miniferritin Dps protects DNA from Fenton chemistry by 75 years, but their physiological roles are unclear. Ccps are located sequestering loose iron (18–20). When H2O2 levels later fall, glu- in the periplasms of bacteria and the mitochondrial intermem- taredoxins deactivate OxyR by reducing its disulfide bond (21). brane spaces of fungi. In this study, Ccp is demonstrated to be a Data indicate that ∼200 nM steady-state intracellular H2O2 is significant degrader of hydrogen peroxide in anoxic Escherichia sufficient to activate this response (10, 21). Because the cyto- coli ccp . Intriguingly, transcription requires both the presence of plasmic membrane is only semipermeable to H2O2 and the cyto- H2O2 and the absence of O2. Experiments show that Ccp lacks plasm contains robust peroxidase and catalase activities, the internal enough activity to shield the cytoplasm from exogenous H2O2. H2O2 concentration is 5- to 10-fold lower than the external H2O2 However, it receives electrons from the quinone pool, and its flux concentration (10). Accordingly, measurements indicate that rate approximates flow to other anaerobic electron acceptors. In- external H2O2 levels must exceed 2 μM to induce the intracellular deed, Ccp enabled E. coli to grow on a nonfermentable carbon OxyR response (21). Thus, one infers that despite dwelling in Salmonella source when H2O2 was supplied. behaved similarly. mostly hypoxic environments, E. coli must encounter micromolar ccp This role rationalizes repression in oxic environments. We spec- levels of external H2O2 at some stage in its normal lifestyle. ulate that micromolar H2O2 is created both biologically and abiot- Strikingly, even obligate anaerobes manifest H2O2-inducible ically at natural oxic/anoxic interfaces. The OxyR response appears defenses, mediated either by OxyR or by PerR (22–24). The to exploit this H O as a terminal oxidant while simultaneously 2 2 implication is that microbes may confront toxic doses of H2O2 defending the cell against its toxicity. even in low-oxygen environments. Such a conclusion might resolve the conundrum of cytochrome oxidative stress | anaerobic respiration | OxyR c peroxidases. The biochemical activity of these enzymes was first described in 1940 (25), and although their reaction mechanism has he facultative anaerobe Escherichia coli lives adjacent to the been elaborated in detail (26), their physiological role continues to Tepithelial layer of the mammalian gut, where it can respire by perplex. Notably, the bacterial enzymes are expressed only under scavenging trace oxygen that diffuses into the lumen from the anoxic conditions (27, 28). We now report that through YhjA, a epithelial cells. When oxygen levels decline, E. coli can ferment, cytochrome c peroxidase homolog, E. coli can exploit H2O2 as although it is less successful than the coresident obligate an- a terminal oxidant in a form of anaerobic respiration. This rep- aerobes. However, upon excretion, E. coli can thrive, whereas the resents a surprising positive use of H2O2 in metabolism. YhjA is obligate anaerobes enter a period of stasis. induced only when oxygen is scarce and H2O2 is present, implying In fully oxic habitats, E. coli must cope with reactive oxygen that micromolar H2O2 can be found at oxic–anoxic interfaces. We species that are generated internally through the adventitious − oxidation of redox enzymes (1). Superoxide (O2 ) and hydrogen Significance peroxide (H2O2) are potentially toxic, because they oxidatively inactivate cytoplasmic enzymes that use exposed [4Fe–4S] clus- – Hydrogen peroxide has been regarded exclusively as a hazard ters or ferrous iron atoms as prosthetic cofactors (2 6). H2O2 for bacteria; its sources and concentrations in natural habitats also oxidizes the intracellular pool of unincorporated iron are uncertain. The cytochrome c peroxidase of Escherichia coli through the Fenton reaction, thereby generating hydroxyl radi- exhibits an expression pattern and flux rate that provides cals that can damage DNA (7). To protect itself from these oxi- surprising insights into these issues. This periplasmic enzyme is dants, E. coli—like virtually all organisms—routinely synthesizes − induced only when H2O2 is present and molecular oxygen is superoxide dismutases to keep O2 levels low and catalase and absent. Intriguingly, it was ineffective as a defensive enzyme, NADH peroxidase to minimize H2O2. As a consequence, wild- but through its linkage to the quinone pool it did enable E. coli type E. coli grows well in oxic environments. to respire using H2O2 as an anaerobic electron acceptor. We In addition to these basal protections, most bacteria have the suggest that both chemical and biotic processes generate mi- capacity to ramp up their defenses when they encounter extra- cromolar H2O2 at oxic/anoxic interfaces and that this scenario is cellular H2O2 (8, 9). Exogenous H2O2 moves freely through common enough that microbes have evolved strategies to porins into the periplasm, and because it is small and uncharged, productively exploit the H2O2. it can gradually diffuse across the cytoplasmic membrane into the cell interior, where it poses a hazard (10). When H2O2 levels Author contributions: M.K. and J.A.I. designed research; M.K. performed research; M.K. rise in E. coli, the H2O2 directly oxidizes the sensory cysteine and J.A.I. analyzed data; and M.K. and J.A.I. wrote the paper. residue of the OxyR transcription factor, triggering the forma- The authors declare no conflict of interest. tion of a disulfide bond that locks the protein into an activated This article is a PNAS Direct Submission. A.J.B. is a guest editor invited by the Editorial conformation (11, 12). OxyR then activates the expression of Board. defensive genes (13). Catalase and NADH peroxidase activities See Commentary on page 8678. are boosted more than 10-fold. Suf proteins improve the repair 1To whom correspondence should be addressed. Email: [email protected]. – of iron sulfur clusters (14, 15), the manganese importer MntH This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. allows Mn to replace ferrous iron in mononuclear enzymes (16, 17), 1073/pnas.1701587114/-/DCSupplemental. E6922–E6931 | PNAS | Published online July 10, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1701587114 Downloaded by guest on September 24, 2021 suggest that YhjA be renamed Ccp, and we continue to use Ccp PNAS PLUS throughout this paper. Results Cytochrome c Peroxidase Is a Fourth Enzyme That Scavenges H2O2. E. coli has three enzymes that are known to degrade H2O2 in vivo: the KatG and KatE catalases, plus the NADH peroxidase AhpCF. For convenience, mutants that lack all three enzymes − (katG katE ahpCF) are termed hydroperoxidase-deficient (Hpx ) − (29). When Hpx mutants are grown aerobically, they do not clear SEE COMMENTARY micromolar H2O2 from medium at a significant rate (Fig. 1). However, we were surprised to find that when the same strain was grown and exposed to H2O2 under anoxic conditions, it dis- played substantial ability to degrade H2O2 (Fig. 1, rightmost bar). The culture filtrate did not degrade H2O2 (Fig. S1), indicating that the scavenging activity resided within the cells. We considered the possibility that H2O2 might be eliminated through Fenton reactions with the cytoplasmic pool of unincorporated iron. However, our calculations indicated that the cell was unlikely to − − Fig. 2. Ccp scavenges H2O2 in anoxic Hpx cells. The Hpx parent strain and contain enough iron to support the observed rate of H2O2 clear- mutant derivatives were grown anaerobically, and the rate of H2O2 scav- ance (SI Materials and Methods), and indeed the scavenging ac- enging was measured. Cross-hatched bars: The cytochrome c maturation tivity persisted when cells were perfused with dipyridyl, a chelator machinery is required for Ccp activity. Spotted bar, far right: the Hpx− Δccp that fully blocks intracellular Fenton chemistry (7) (Fig. S1). mutant was genetically complemented using pACYC184-ccp under its own Therefore, we focused upon E. coli enzymes that have been promoter. Asterisks represent statistical significance (*P ≤ 0.05; **P ≤ 0.01; − reported to display peroxidase activities in vitro: thiol peroxidase ***P ≤ 0.001; ns, P > 0.05). The single mutants other than Hpx Δccp and − − Hpx Δccm were not significantly different from Hpx . None of the double (Tpx) (30), bacterioferritin comigratory protein (BCP) (31), a − − mutants were significantly different from Hpx Δccp. Strains used: Hpx glutathione peroxidase homolog (BtuE) (32), a predicted cyto- − − − (LC106), Hpx Δbcp (MK150), Hpx Δtpx (MK154), Hpx ΔbtuE (MK158), chrome c peroxidase (YhjA, or Ccp) (28), and osmotically in- − − − − Hpx Δccp (MK146), Hpx Δcyd Δcyo (SSK53), Hpx ΔappBC (MK180), Hpx ducible peroxiredoxin (OsmC) (33). The physiological roles of ΔosmC (MK208), Hpx− Δccp Δbcp (MK172), Hpx− Δccp Δtpx (MK174), Hpx− − − these enzymes are uncertain, and they have not been reported to Δccp ΔbtuE (MK176), Hpx Δccp ΔappBC (MK182), Hpx Δccp Δcyd − − − degrade
Load more

Recommended publications
  • Some English Terms Used in Microbiology 1
    Some English terms used in Microbiology 1 Shapes & Structures General terms Antibiotics and related Bacillus (pl. bacilli) Acid fast (acid fastness) ácido-alcohol resistente Acetylases Capsule Bacterial (adj.) Ampicillin Cell wall pared celular Bacterium (pl., bacteria): Beta-lactamase Coccus (cocci; and hence Staphylococcus, Bench: poyata Beta-lactamic Staphylococci) Biofilm Cephalosporin Core oligosaccharide núcleo oligosacarídico Burner (Bunsen Burner): mechero (Bunsen) Chloramphenicol Cortex Colony: colonia Colistin Fimbria (pl. fimbriae) Coverslip: (vidrio) cubreobjetos D-Cycloserine Flagellum (pl. flagella) Dye colorante DNA-gyrase Glycocalix Eukaryote or eucaryote Erythromycin Lipid A Incubator: estufa de incubación Ethambuthol Lipopolysaccharide Inoculating loop asa de siembra Fluoroquinolone Murein mureína Inoculum (inocula): Gentamicin (formerly gentamycin) Omp: outer membrane major protein proteína To flame: flamear Isoniazide de membrane externa Flask (Erlenmeyer flask): matraz Methicillin Outer membrane membrana externa Volumetric flask: matraz aforado Methylases PAMP (pathogen associated molecular pattern): Microorganism Nalidixic acid patrón molecular asociado a patógeno Motility movilidad Penicillin Peptidoglycan peptidoglucano Mycoplasm Penicillin binding protein (PBP) Periplasm periplasma Negative staining tinción negativa Phosphonomycin Periplasmic space espacio periplásmico Petri dish: placa de Petri Phosphorylases Permeability barrier barrera de permeabilidad Prokaryote or procaryote Polymyxin Pilus (pl. pili) Rack:
    [Show full text]
  • The Regulation of Arsenic Metabolism in Rhizobium Sp. NT-26
    The regulation of arsenic metabolism in Rhizobium sp. NT-26 Paula Corsini Madeira University College London Thesis submitted for the degree of Doctor of Philosophy 2016 I, Paula Corsini Madeira, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Date: Signed: II Abstract Arsenic (As) is a toxic metalloid and a major contaminant in terrestrial and aquatic environments. The two soluble forms, arsenite (AsIII) and arsenate (AsV) are toxic to most organisms. A range of phylogenetically distant bacteria are able to oxidize AsIII to the less toxic form, arsenate AsV using the periplasmic arsenite oxidase (AioBA). The two-component signal transduction system AioS/AioR and the AsIII-binding periplasmic protein AioX are required for AsIII oxidation and are involved in the transcriptional regulation of the aioBA operon. Most AsIII oxidisers can also reduce AsV to AsIII via the As (Ars) resistance system. The focus of this work was to understand the regulation of genes involved in AsIII oxidation and As resistance together with those involved in phosphate metabolism in the facultative chemolithoautotrophic AsIII oxidiser NT-26 grown under different conditions. Gene expression was studied by quantitative PCR in cells grown heterotrophically with and without AsIII or AsV in late-log and stationary phases. qPCR was optimised and suitable reference genes were chosen. The expression of genes involved in phosphate transport, sensing As and the genes aioX, aioS, aioR (AsIII-sensing and regulation) and aioB, aioA (AsIII oxidation) and cytC (cytochrome c) were also analysed in NT-26 grown heterotrophically in the presence or absence of AsIII or AsV at different growth stages (i.e., late-log and stationary phases).
    [Show full text]
  • Activation of Microsomal Glutathione S-Transferase. in Tent-Butyl Hydroperoxide-Induced Oxidative Stress of Isolated Rat Liver
    Activation of Microsomal Glutathione S-Transferase. in tent-Butyl Hydroperoxide-Induced Oxidative Stress of Isolated Rat Liver Yoko Aniya'° 2 and Ai Daido' 'Laboratory of Physiology and Pharmacology , School of Health Sciences, 2Research Center of Comprehensive Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-01, Japan Received May 10, 1994 Accepted June 17, 1994 ABSTRACT-The activation of microsomal glutathione S-transferase in oxidative stress was investigated by perfusing isolated rat liver with 1 mM tert-butyl hydroperoxide (t-BuOOH). When the isolated liver was per fused with t-BuOOH for 7 min and 10 min, microsomal, but not cytosolic, glutathione S-transferase activ ity was increased 1.3-fold and 1.7-fold, respectively, with a concomitant decrease in glutathione content. A dimer protein of microsomal glutathione S-transferase was also detected in the t-BuOOH-perfused liver. The increased microsomal glutathione S-transferase activity after perfusion with t-BuOOH was reversed by dithiothreitol, and the dimer protein of the transferase was also abolished. When the rats were pretreated with the antioxidant a-tocopherol or the iron chelator deferoxamine, the increases in microsomal glutathione S-transferase activity and lipid peroxidation caused by t-BuOOH perfusion of the isolated liver was prevented. Furthermore, the activation of microsomal GSH S-transferase by t-BuOOH in vitro was also inhibited by incubation of microsomes with a-tocopherol or deferoxamine. Thus it was confirmed that liver microsomal glutathione S-transferase is activated in the oxidative stress caused by t-BuOOH via thiol oxidation of the enzyme. Keywords: Enzyme activation, Glutathione S-transferase, Liver perfusion, Oxidative stress, tert-Butyl hydroperoxide Glutathione (GSH) S-transferases (EC 2.
    [Show full text]
  • The Peroxiredoxin Tpx1 Is Essential As a H2O2 Scavenger During Aerobic Growth in Fission Yeast Mo´Nica Jara,* Ana P
    Molecular Biology of the Cell Vol. 18, 2288–2295, June 2007 The Peroxiredoxin Tpx1 Is Essential as a H2O2 Scavenger during Aerobic Growth in Fission Yeast Mo´nica Jara,* Ana P. Vivancos,* Isabel A. Calvo, Alberto Moldo´n, Miriam Sanso´, and Elena Hidalgo Departament de Cie`ncies Experimentals i de la Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain Submitted November 27, 2006; Revised March 16, 2007; Accepted March 26, 2007 Monitoring Editor: Thomas Fox Peroxiredoxins are known to interact with hydrogen peroxide (H2O2) and to participate in oxidant scavenging, redox signal transduction, and heat-shock responses. The two-cysteine peroxiredoxin Tpx1 of Schizosaccharomyces pombe has been characterized as the H2O2 sensor that transduces the redox signal to the transcription factor Pap1. Here, we show that Tpx1 is essential for aerobic, but not anaerobic, growth. We demonstrate that Tpx1 has an exquisite sensitivity for its substrate, which explains its participation in maintaining low steady-state levels of H2O2. We also show in vitro and in vivo that inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid is always preceded by a sulfinic acid form in a covalently linked dimer, which may be important for understanding the kinetics of Tpx1 inactivation. Furthermore, we provide evidence that a strain expressing Tpx1.C169S, lacking the resolving cysteine, can sustain aerobic growth, and we show that small reductants can modulate the activity of the mutant protein in vitro, probably by supplying a thiol group to substitute for cysteine 169. INTRODUCTION available to form a disulfide bond; the source of the reducing equivalents for regenerating this thiol is not known, al- Peroxiredoxins (Prxs) are a family of antioxidant enzymes though glutathione (GSH) has been proposed to serve as the that reduce hydrogen peroxide (H2O2) and/or alkyl hy- electron donor in this reaction (Kang et al., 1998b).
    [Show full text]
  • Microbial Peroxidases and Their Applications
    International Journal of Scientific & Engineering Research Volume 12, Issue 2, February-2021 ISSN 2229-5518 474 Microbial Peroxidases and their applications Divya Ghosh, Saba Khan, Dr. Sharadamma N. Divya Ghosh is currently pursuing master’s program in Life Science in Mount Carmel College, Bangalore, India, Ph: 9774473747; E- mail: [email protected]; Saba Khan is currently pursuing master’s program in Life Science in Mount Carmel College, Bangalore, India, Ph: 9727450944; E-mail: [email protected]; Dr. Sharadamma N is an Assistant Professor in Department of Life Science, Mount Carmel College, Autonomous, Bangalore, Karnataka- 560052. E-mail: [email protected] Abstract: Peroxidases are oxidoreductases that can convert many compounds into their oxidized form by a free radical mechanism. This peroxidase enzyme is produced by microorganisms like bacteria and fungi. Peroxidase family includes many members in it, one such member is lignin peroxidase. Lignin peroxidase has the potential to degrade the lignin by oxidizing phenolic structures in it. The microbes that have shown efficient production of peroxidase are Bacillus sp., Providencia sp., Streptomyces, Pseudomonas sp. These microorganisms were optimized to produce peroxidase efficiently. These microbial strains were identified by 16S rDNA and rpoD gene sequences and Sanger DNA sequencing techniques. There are certain substrates on which Peroxidase acts are guaiacol, hydrogen peroxide, etc. The purification of peroxidase was done by salt precipitation, ion-exchange chromatography, dialysis, anion exchange, and molecular sieve chromatography method. The activity of the enzyme was evaluated with different parameters like enzyme activity, protein concentration, specific activity, total activity, the effect of heavy metals, etc.
    [Show full text]
  • Structural and Functional Studies of Rubrerythrin From
    STRUCTURAL AND FUNCTIONAL STUDIES OF RUBRERYTHRIN FROM DESULFOVIBRIO VULGARIS by SHI JIN (Under the direction of Dr. Donald M. Kurtz, Jr.) ABSTRACT Rubrerythrin (Rbr), found in anaerobic or microaerophilic bacteria and archaea, is a non-heme iron protein containing an oxo-bridged diiron site and a rubredoxin-like [Fe(SCys)4] site. Rbr has NADH peroxidase activity and it has been proposed as one of the key enzyme pair (Superoxide Reductase/Peroxidase) in the oxidative stress protection system of anaerobic microorganisms. In order to probe the mechanism of the electron pathway in Rbr peroxidase reaction, X-ray crystallography and rapid reaction techniques were used. Recently, the high-resolution crystal structures of reduced Rbr and its azide adduct were determined. Detailed information of the oxidation state changes of the irons at the metal-binding sites during the oxidation of Rbr by hydrogen peroxide was obtained using stopped-flow spectrophotometry and freeze quench EPR. The structures and activities of Rbrs with Zn(II) ions substituted for iron(III) ions at different metal-binding sites, which implicate the influence of positive divalent metal ions to the Rbr, are also investigated. The molecular mechanism for Rbr peroxidase reaction during the turnover based on results from X-ray crystallography and kinetic studies is proposed. INDEX WORDS: Rubrerythrin, Desulfovibrio vulgaris, NADH peroxidase, Alternative oxidative stress defense system, Crystal structures, Oxidized form, Reduced form, Azide adduct, Hydrogen peroxide, Internal electron transfer, Zinc ion derivatives STRUCTURAL AND FUNCTIONAL STUDIES OF RUBRERYTHRIN FROM DESULFOVIBRIO VULGARIS by SHI JIN B.S., Peking University, P. R. China, 1998 A Dissertation Submitted to the Graduate Faculty of The University Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2002 © 2002 Shi Jin All Rights Reserved STRUCTURAL AND FUNCTIONAL STUDIES OF RUBRERYTHRIN FROM DESULFOVIBRIO VULGARIS by SHI JIN Approved: Major Professor: Donald M.
    [Show full text]
  • Cell Structure and Function in the Bacteria and Archaea
    4 Chapter Preview and Key Concepts 4.1 1.1 DiversityThe Beginnings among theof Microbiology Bacteria and Archaea 1.1. •The BacteriaThe are discovery classified of microorganismsinto several Cell Structure wasmajor dependent phyla. on observations made with 2. theThe microscope Archaea are currently classified into two 2. •major phyla.The emergence of experimental 4.2 Cellscience Shapes provided and Arrangements a means to test long held and Function beliefs and resolve controversies 3. Many bacterial cells have a rod, spherical, or 3. MicroInquiryspiral shape and1: Experimentation are organized into and a specific Scientificellular c arrangement. Inquiry in the Bacteria 4.31.2 AnMicroorganisms Overview to Bacterialand Disease and Transmission Archaeal 4.Cell • StructureEarly epidemiology studies suggested how diseases could be spread and 4. Bacterial and archaeal cells are organized at be controlled the cellular and molecular levels. 5. • Resistance to a disease can come and Archaea 4.4 External Cell Structures from exposure to and recovery from a mild 5.form Pili allowof (or cells a very to attach similar) to surfacesdisease or other cells. 1.3 The Classical Golden Age of Microbiology 6. Flagella provide motility. Our planet has always been in the “Age of Bacteria,” ever since the first 6. (1854-1914) 7. A glycocalyx protects against desiccation, fossils—bacteria of course—were entombed in rocks more than 3 billion 7. • The germ theory was based on the attaches cells to surfaces, and helps observations that different microorganisms years ago. On any possible, reasonable criterion, bacteria are—and always pathogens evade the immune system. have been—the dominant forms of life on Earth.
    [Show full text]
  • New Automatically Built Profiles for a Better Understanding of the Peroxidase Superfamily Evolution
    University of Geneva Practical training report submitted for the Master Degree in Proteomics and Bioinformatics New automatically built profiles for a better understanding of the peroxidase superfamily evolution presented by Dominique Koua Supervisors: Dr Christophe DUNAND Dr Nicolas HULO Laboratory of Plant Physiology, Dr Christian J.A. SIGRIST University of Geneva Swiss Institute of Bioinformatics PROSITE group. Geneva, April, 18th 2008 Abstract Motivation: Peroxidases (EC 1.11.1.x), which are encoded by small or large multigenic families, are involved in several important physiological and developmental processes. These proteins are extremely widespread and present in almost all living organisms. An important number of haem and non-haem peroxidase sequences are annotated and classified in the peroxidase database PeroxiBase (http://peroxibase.isb-sib.ch). PeroxiBase contains about 5800 peroxidase sequences classified as haem peroxidases and non-haem peroxidases and distributed between thirteen superfamilies and fifty subfamilies, (Passardi et al., 2007). However, only a few classification tools are available for the characterisation of peroxidase sequences: InterPro motifs, PRINTS and specifically designed PROSITE profiles. However, these PROSITE profiles are very global and do not allow the differenciation between very close subfamily sequences nor do they allow the prediction of specific cellular localisations. Due to the rapid growth in the number of available sequences, there is a need for continual updates and corrections of peroxidase protein sequences as well as for new tools that facilitate acquisition and classification of existing and new sequences. Currently, the PROSITE generalised profile building manner and their usage do not allow the differentiation of sequences from subfamilies showing a high degree of similarity.
    [Show full text]
  • Oxidative Polymerization of Heterocyclic Aromatics Using Soybean Peroxidase for Treatment of Wastewater
    University of Windsor Scholarship at UWindsor Electronic Theses and Dissertations Theses, Dissertations, and Major Papers 3-10-2019 Oxidative Polymerization of Heterocyclic Aromatics Using Soybean Peroxidase for Treatment of Wastewater Neda Mashhadi University of Windsor Follow this and additional works at: https://scholar.uwindsor.ca/etd Recommended Citation Mashhadi, Neda, "Oxidative Polymerization of Heterocyclic Aromatics Using Soybean Peroxidase for Treatment of Wastewater" (2019). Electronic Theses and Dissertations. 7646. https://scholar.uwindsor.ca/etd/7646 This online database contains the full-text of PhD dissertations and Masters’ theses of University of Windsor students from 1954 forward. These documents are made available for personal study and research purposes only, in accordance with the Canadian Copyright Act and the Creative Commons license—CC BY-NC-ND (Attribution, Non-Commercial, No Derivative Works). Under this license, works must always be attributed to the copyright holder (original author), cannot be used for any commercial purposes, and may not be altered. Any other use would require the permission of the copyright holder. Students may inquire about withdrawing their dissertation and/or thesis from this database. For additional inquiries, please contact the repository administrator via email ([email protected]) or by telephone at 519-253-3000ext. 3208. Oxidative Polymerization of Heterocyclic Aromatics Using Soybean Peroxidase for Treatment of Wastewater By Neda Mashhadi A Dissertation Submitted to the Faculty of Graduate Studies through the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Windsor Windsor, Ontario, Canada 2019 © 2019 Neda Mashhadi Oxidative Polymerization of Heterocyclic Aromatics Using Soybean Peroxidase for Treatment of Wastewater by Neda Mashhadi APPROVED BY: _____________________________ A.
    [Show full text]
  • Gram Staining Staining & Acid Fast & Acid Fast Staining
    GRAM STAINING && AACCIIDD FFAASSTT SSTTAAIINNIINNGG Dr. R. Haritha Lecturer in Biotechnology Visakha Government Degree College for Women Visakhapatnam Staining Stains Principle Types Staining methods Smear Preparation Smear- Distribution of Bacterial cells on a slide Objective-To kill the microorganism & fix bacteria Method- Air Dry, Heat Fixation GRAM STAINING Gram staining is most widely Hans Christian Joachim Gram used differential staining in Microbiology. It classifies bacteria into two major groups: Gram positive Gram negative Appears violet Appears red after Gram’s stain after Gram’s stain REAGENTS 1. CRYSTAL VIOLET Primary stain Violet colored, stains all micro-organism 2. GRAM IODINE Mordant Forms Crystal violet iodine complexes 3. DECOLORIZER Acetone + Methanol Removes Crystal violet iodine complex from thin peptidoglycan layers 4. GRAM SAFRANINE Counter stain Red colored Step 1: Crystal Violet Step 2: Gram’’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4: Safranin Red 7 PRINCIPLE : Composition of the cell wall Gram-positive bacteria Cell wall has a thick peptidoglycan layer The Crystal Violet stain gets trapped into this layer and the bacteria turned purple. Retains the color of the primary stain (crystal violet) after decolorization with alcohol. Gram-negative bacteria Cell wall has a thin peptidoglycan layer that does not retain crystal violet stain. Cell wall has a thick lipid layer which dissolves easily upon decoulorization with Aceton-Alcohol. Therefore, cells will be counterstained with safranin and turned red. Gram’s +ve Bacteria Gram’s -ve Bacteria 9 RESULT Bacteria that manage to keep the original purple are called Gram positive. Bacteria that lose the original purple dye and can therefore take up the second red dye are called Gram negative APPLICATIONS 1.Rapid preliminary diagnosis of diseases such as Bacterial meningitis.
    [Show full text]
  • Metabolic Flexibility of Enigmatic SAR324 Revealed Through
    bs_bs_banner Environmental Microbiology (2014) 16(1), 304–317 doi:10.1111/1462-2920.12165 Metabolic flexibility of enigmatic SAR324 revealed through metagenomics and metatranscriptomics Cody S. Sheik,1 Sunit Jain1 and Gregory J. Dick1,2,3* deep ocean (ArÍstegui et al., 2009) is thought to constrain 1Department of Earth and Environmental Sciences and heterotrophic microbial activity. However, recent identifi- 2Center for Computational Medicine and Bioinformatics cation of carbon fixation genes in the dark ocean (> 200 m and 3Department of Ecology and Evolutionary Biology, below surface) (Swan et al., 2011) underscores auto- University of Michigan, Ann Arbor, MI 48109, USA. trophy as a metabolic strategy for many dark ocean- associated micro-organisms (Karl et al., 1984). Because autotrophy is energetically taxing (Hügler and Sievert, Summary 2011), organisms must couple carbon fixation to an Chemolithotrophy is a pervasive metabolic lifestyle electron-donating reaction such as the oxidation of for microorganisms in the dark ocean. The SAR324 ammonia, nitrite, sulfur, iron, methane, hydrogen or group of Deltaproteobacteria is ubiquitous in the formate. Yet such electron donors required to power ocean and has been implicated in sulfur oxidation and carbon fixation in the deep ocean are scarce (Reinthaler carbon fixation, but also contains genomic signatures and Van, 2010), suggesting that mixotrophy (Sorokin, of C1 utilization and heterotrophy. Here, we recon- 2003; Moran et al., 2004; Dick et al., 2008) may be nec- structed the metagenome and metatranscriptome of a essary for micro-organisms to persist until preferred population of SAR324 from a hydrothermal plume and energy sources are encountered. surrounding waters in the deep Gulf of California to Within the deep ocean, hydrothermal vents are crucial gain insight into the genetic capability and transcrip- sources of reduced compounds such as sulfur, ammonia, tional dynamics of this enigmatic group.
    [Show full text]
  • NADH PEROXIDASE Ph
    Enzymatic Assay of NADH PEROXIDASE (EC 1.11.1.1) PRINCIPLE: NADH Peroxidase ß-NADH + H2O2 > ß-NAD + 2H2O Abbreviations used: ß-NADH = ß-Nicotinamide Adenine Dinucleotide, Reduced Form ß-NAD = ß-Nicotinamide Adenine Dinucleotide, Oxidized Form CONDITIONS: T = 25°C, pH 6.0, A365nm, Light path = 1 cm METHOD: Continuous Spectrophotometric Rate Determination REAGENTS: A. 200 mM Tris Acetate Buffer, pH 6.0 at 25°C (Prepare 100 ml in deionized water using Trizma Acetate, Sigma Prod. No. T-1258. Adjust to pH 6.0 at 25°C with 1 M HCl.) B. 0.30% (w/w) Hydrogen Peroxide Solution (H2O2) (Prepare 10 ml in deionized water using Hydrogen Peroxide 30% (w/w) Solution, Sigma Prod. No. H-1009.) C. 12 mM ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Solution (NADH) (Dissolve the contents of one 10 mg vial of ß-Nicotinamide Adenine Dinucleotide, Reduced Form, Sodium Salt, Sigma Stock No. 340-110, in the appropriate volume of deionized water.) D. NADH Peroxidase Enzyme Solution (Immediately before use, prepare a solution containing 0.2 - 0.4 unit of NADH Peroxidase in cold deionized water.) SPHYDR04 Page 1 of 3 Revised: 01/17/97 Enzymatic Assay of NADH PEROXIDASE (EC 1.11.1.1) PROCEDURE: Pipette (in milliliters) the following reagents into suitable cuvettes: Test Blank Reagent A (Buffer) 3.00 3.00 Reagent C (NADH) 0.10 0.10 Reagent D (Enzyme Solution) 0.10 ------ Deionized Water ------ 0.10 Mix by inversion and equilibrate to 25°C. Monitor the baseline at A365nm for 5 minutes in order to determine any NADH oxidase activity which may be present.1 Then add: Reagent B (H2O2) 0.10 0.10 Immediately mix by inversion and monitor the decrease in A365nm for approximately 5 minutes.
    [Show full text]