DOCSLIB.ORG

Evidence of Cellulose Metabolism by the Giant Panda Gut Microbiome

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Evidence of Cellulose Metabolism by the Giant Panda Gut Microbiome Evidence of cellulose metabolism by the giant panda gut microbiome Lifeng Zhua,1,QiWua,1, Jiayin Daia, Shanning Zhangb, and Fuwen Weia,2 aKey Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China and bChina Wildlife Conservation Association, Beijing 100714, China Edited by Rita R. Colwell, University of Maryland, College Park, MD, and approved September 7, 2011 (received for review December 2, 2010) The giant panda genome codes for all necessary enzymes associ- cellulose digestion (10). Although the giant panda can use non- ated with a carnivorous digestive system but lacks genes for cellulosic material from the bamboo diet using enzymes coded in enzymes needed to digest cellulose, the principal component of its own genome, digestion of cellulose and hemicellulose is im- their bamboo diet. It has been posited that this iconic species must possible based on the panda’s genetic composition, and must be therefore possess microbial symbionts capable of metabolizing dependent on gut microbiome. However, previous research using cellulose, but these symbionts have remained undetected. Here we culture methods and small-scale sequencing identified three examined 5,522 prokaryotic ribosomal RNA gene sequences in wild predominant bacteria from the panda gut—Escherichia coli, and captive giant panda fecal samples. We found lower species Streptococcus, and Enterobacteriaceae—none of which aids in richness of the panda microbiome than of mammalian microbiomes cellulose digestion (11–13). Thus, an incomplete understanding for herbivores and nonherbivorous carnivores. We detected 13 of the gut microbial ecosystem in this interesting and high-profile operational taxonomic units closely related to Clostridium groups I species remains because of restrictions in methodology and past and XIVa, both of which contain taxa known to digest cellulose. reliance on studies of captive animals. Seven of these 13 operational taxonomic units were unique to pandas compared with other mammals. Metagenomic analysis us- Results and Discussion ing ∼37-Mbp contig sequences from gut microbes recovered puta- We undertook a large-scale analysis of 16S rRNA gene sequen- tive genes coding two cellulose-digesting enzymes and one ces to profile microbial flora inhabiting the digestive system of ECOLOGY β hemicellulose-digesting enzyme, cellulase, -glucosidase, and xy- giant pandas and used a metagenomic approach based on next- β Clostridium lan 1,4- -xylosidase, in group I. Comparing glycoside generation de novo sequencing to identify functional attributes fi hydrolase pro les of pandas with those of herbivores and omni- encoded in the gut microbiome. A total of 5,636 near–full-length vores, we found a moderate abundance of oligosaccharide-degrad- 16S rRNA gene segments were amplified from fecal samples of ing enzymes for pandas (36%), close to that for humans (37%), and seven wild and eight captive giant pandas. After exclusion of 74 the lowest abundance of cellulases and endohemicellulases (2%), putative chimeric and 30 chloroplast sequences, 5,522 sequences which may reflect low digestibility of cellulose and hemicellulose in were retained for analysis. Using a minimum identity of 97% as the panda’s unique bamboo diet. The presence of putative cellu- the threshold for any sequence pair, we identified 85 bacterial lose-digesting microbes, in combination with adaptations related operational taxonomic units (OTUs), 14 of which were pre- to feeding, physiology, and morphology, show that giant pandas viously undescribed (Fig. 1A and SI Appendix, Table S1). Cov- have evolved a number of traits to overcome the anatomical and physiological challenge of digesting a diet high in fibrous matter. erage of 99% was obtained across existing bacterial clone libraries, and we are confident that our dataset presents the most comprehensive assessment of gut microbes in this species based ccess to dietary resources shapes animal evolution (1). Early on the rarefaction method in DOTUR (14) (SI Appendix, Fig. Aon, animals lost the ability to synthesize many key com- S1A). The majority of microbes were members of the Firmicutes pounds, and instead this function is performed by symbionts (2). (62 OTUs, 4,633 sequences, 83.8% of the total of 5,522 For example, microbial symbionts assist with extracting nutrients sequences) and Proteobacteria (12 OTUs, 871 sequences, 15.8% from food and key compounds from the environment, and also of the total sequences), with the remainder belonging to the synthesize necessary metabolic compounds (1). Gut microbiota phyla Actinobacteria, Bacteroidetes, Cyanobacteria, and Acid- share specialized relationships with their hosts, and advances in obacteria (Fig. 1 and SI Appendix, Fig. S2 and Table S1). Within genomics are revealing the dynamics of these relationships (3). the Firmicutes, 33 OTUs (60.8% of the total of 5,522 sequences) Recent developments in culture-independent methodologies were members of the class Clostridia and 29 OTUs (23.0% of based on large-scale comparative analyses of microbial small- total sequences) belonged to the class Bacilli. The high pro- subunit ribosomal RNA genes (16S ribosomal RNA) and met- portion of Firmicutes OTUs found in the gut of giant pandas agenomics have revealed the extent of microbial diversity and differs from the findings in previous studies attempting to char- – metabolic potential in greater detail (2 7). These techniques can acterize giant panda gut microbes (11–13) and is notably similar now be applied to animals that have acquired a profoundly new diet, presenting an opportunity to investigate host physiological and microbial systems in an evolutionary context. Author contributions: F.W. designed research; L.Z. and S.Z. performed research; L.Z., Q.W., The giant panda (Ailuropoda melanoleuca) is well known for J.D., S.Z., and F.W. analyzed data; and L.Z., Q.W. and F.W. wrote the paper. dietary oddities: a bamboo specialist within the mammalian or- The authors declare no conflict of interest. der Carnivora possessing a gastrointestinal tract typical of car- ∼ fi This article is a PNAS Direct Submission. nivores. It consumes 12.5 kg of this highly brous plant each Freely available online through the PNAS open access option. day (8), but because it lacks the long intestinal tract character- Data deposition: The sequences reported in this paper have been deposited in the Gen- istic of other herbivores, extensive fermentation is not possible Bank database. For a list of accession numbers, see SI Appendix. The metagenomic data (9). Giant pandas digest only ∼17% of dry matter consumed (8), project ID is 5936 in Integrated Microbial Genomes with Microbiome Samples (IMG/M). and have low digestion coefficients for bamboo hemicelluloses 1L.Z. and Q.W. contributed equally to this work. (27%) and celluloses (8%) (9). Indeed, the giant panda genome 2To whom correspondence should be addressed. E-mail: [email protected]. codes for all necessary enzymes associated with a carnivorous This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. digestive system, but lacks the enzyme homologs needed for 1073/pnas.1017956108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1017956108 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 Fig. 1. Microbial flora of giant pandas. (A) Neighbor-joining tree containing one representative of each of 85 OTUs. Colors represent different phyla: yellow, Clostridia class of Firmicutes; blue, Bacilli class of Firmicutes; purple, Proteobacteria; gray, Actinobacteria, Bacteroidetes, Cyanobacteria, and Acidobacteria. seqs, sequences. All bootstrap values >80% are shown in this tree. (B) Relative abundance of sequences from wild and captive giant panda fecal samples; other Firmicutes belong to the Bacilli class. (C) Percentage of sequences from each fecal sample assigned to different phyla. W1–W7, wild giant panda samples; C1–C8, captive giant panda samples. Numbers below the sample number are total sequences from each individual. to that found in herbivorous mammals (2, 6). However, the based algorithm implemented in MEGAN (24). The results species richness in the giant panda was lower than that seen in again showed that the majority of microbes (71%) belong to the previous work on mammalian herbivores and nonherbivorous phylum Firmicutes (Fig. 2A). We also compared the catalog with carnivores (SI Appendix, Fig. S1B), perhaps due to the panda’s the Kyoto Encyclopedia of Genes and Genomes (KEGG) da- unique bamboo diet and simple digestive system. tabase and the Clusters of Orthologous Groups (COG) data- The proportion of 16S rRNA gene sequences belonging to the bases to assess the functional capacities present in bacterial class Clostridia of Firmicutes were high for both wild (73% of the metagenome (Fig. 2 B–D and SI Appendix, Figs. S4 and S5). Our total of 3361 sequences) and captive (42% of the total of 2161 analysis revealed that half of these predicted genes coding for sequences) giant pandas (Fig. 1 B and C). In Clostridia, we cellulose and hemicellulose-digesting enzymes are found in detected 13 OTUs closely related to Clostridium group I (10 species within the Clostridium genus (SI Appendix, Table S7). For OTUs, 1,457 sequences, 26.4% of the total sequences) and XIVa example, 2 of 12 putative cellulase genes in this study are ho- (3 OTUs, 185 sequences, 3.3% of the total sequences) (SI Ap- mologous (E-value: 2.00E-115 and 6.00E-29, respectively) to pendix, Fig. S3), groups that contain
Load more

Recommended publications
  • Updating the Sequence-Based Classification of Glycosyl Hydrolases
    Article Updating the sequence-based classification of glycosyl hydrolases HENRISSAT, Bernard, BAIROCH, Amos Marc Reference HENRISSAT, Bernard, BAIROCH, Amos Marc. Updating the sequence-based classification of glycosyl hydrolases. Biochemical Journal, 1996, vol. 316 ( Pt 2), p. 695-6 PMID : 8687420 DOI : 10.1042/bj3160695 Available at: http://archive-ouverte.unige.ch/unige:36909 Disclaimer: layout of this document may differ from the published version. 1 / 1 Biochem. J. (1996) 316, 695–696 (Printed in Great Britain) 695 BIOCHEMICAL JOURNAL Updating the sequence-based classification of available. When the number of glycosyl hydrolase sequences reached C 480, ten additional families (designated 36–45) could glycosyl hydrolases be defined and were added to the classification [2]. There are at present over 950 sequences of glycosyl hydrolases in the data- A classification of glycosyl hydrolases based on amino-acid- banks (EMBL}GenBank and SWISS-PROT). Their analysis sequence similarities was proposed in this Journal a few years shows that the vast majority of the C 470 additional sequences ago [1]. This classification originated from the analysis of C 300 that have become available since the last update could be classified sequences and their grouping into 35 families designated 1–35. in the existing families. However, several sequences not fitting Because such a classification is necessarily sensitive to the sample, the existing families allow the definition of new families (desig- it was anticipated that it was incomplete and that new families nated 46–57) (Table 1). When the several present genome would be determined when additional sequences would become sequencing projects have reached completion, the number of Table 1 New families in the classification of glycosyl hydrolases Family Enzyme Organism SWISS-PROT EMBL/GenBank 46 Chitosanase Bacillus circulans MH-K1 P33673 D10624 46 Chitosanase Streptomyces sp.
    [Show full text]
  • Flavonoid Glucodiversification with Engineered Sucrose-Active Enzymes Yannick Malbert
    Flavonoid glucodiversification with engineered sucrose-active enzymes Yannick Malbert To cite this version: Yannick Malbert. Flavonoid glucodiversification with engineered sucrose-active enzymes. Biotechnol- ogy. INSA de Toulouse, 2014. English. NNT : 2014ISAT0038. tel-01219406 HAL Id: tel-01219406 https://tel.archives-ouvertes.fr/tel-01219406 Submitted on 22 Oct 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Last name: MALBERT First name: Yannick Title: Flavonoid glucodiversification with engineered sucrose-active enzymes Speciality: Ecological, Veterinary, Agronomic Sciences and Bioengineering, Field: Enzymatic and microbial engineering. Year: 2014 Number of pages: 257 Flavonoid glycosides are natural plant secondary metabolites exhibiting many physicochemical and biological properties. Glycosylation usually improves flavonoid solubility but access to flavonoid glycosides is limited by their low production levels in plants. In this thesis work, the focus was placed on the development of new glucodiversification routes of natural flavonoids by taking advantage of protein engineering. Two biochemically and structurally characterized recombinant transglucosylases, the amylosucrase from Neisseria polysaccharea and the α-(1→2) branching sucrase, a truncated form of the dextransucrase from L. Mesenteroides NRRL B-1299, were selected to attempt glucosylation of different flavonoids, synthesize new α-glucoside derivatives with original patterns of glucosylation and hopefully improved their water-solubility.
    [Show full text]
  • 2010 Physical Biosciences Research Meeting
    2010 Physical Biosciences Research Meeting Sheraton Inner Harbor Hotel Baltimore, MD October 17-20, 2010 Office of Basic Energy Sciences Chemical Sciences, Geosciences & Biosciences Division 2010 Physical Biosciences Research Meeting Program and Abstracts Sheraton Inner Harbor Hotel Baltimore, MD October 17-20, 2010 Chemical Sciences, Geosciences, and Biosciences Division Office of Basic Energy Sciences Office of Science U.S. Department of Energy i Cover art is taken from the public domain and can be found at: http://commons.wikimedia.org/wiki/File:Blue_crab_on_market_in_Piraeus_-_Callinectes_sapidus_Rathbun_20020819- 317.jpg This document was produced under contract number DE-AC05-060R23100 between the U.S. Department of Energy and Oak Ridge Associated Universities. The research grants and contracts described in this document are, unless specifically labeled otherwise, supported by the U.S. DOE Office of Science, Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division. ii Foreword This volume provides a record of the 2nd biennial meeting of the Principal Investigators (PIs) funded by the Physical Biosciences program, and is sponsored by the Chemical Sciences, Geosciences, and Biosciences Division of the Office of Basic Energy Sciences (BES) in the U.S. Department of Energy (DOE). Within DOE-BES there are two programs that fund basic research in energy-relevant biological sciences, Physical Biosciences and Photosynthetic Systems. These two Biosciences programs, along with a strong program in Solar Photochemistry, comprise the current Photo- and Bio- Chemistry Team. This meeting specifically brings together under one roof all of the PIs funded by the Physical Biosciences program, along with Program Managers and staff not only from DOE-BES, but also other offices within DOE, the national labs, and even other federal funding agencies.
    [Show full text]
  • Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria Sacha A
    MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2006, p. 157–176 Vol. 70, No. 1 1092-2172/06/$08.00ϩ0 doi:10.1128/MMBR.70.1.157–176.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved. Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria Sacha A. F. T. van Hijum,1,2†* Slavko Kralj,1,2† Lukasz K. Ozimek,1,2 Lubbert Dijkhuizen,1,2 and Ineke G. H. van Geel-Schutten1,3 Centre for Carbohydrate Bioprocessing, TNO-University of Groningen,1 and Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,2 9750 AA Haren, The Netherlands, and Innovative Ingredients and Products Department, TNO Quality of Life, Utrechtseweg 48 3704 HE Zeist, The Netherlands3 INTRODUCTION .......................................................................................................................................................157 NOMENCLATURE AND CLASSIFICATION OF SUCRASE ENZYMES ........................................................158 GLUCANSUCRASES .................................................................................................................................................158 Reactions Catalyzed and Glucan Product Synthesis .........................................................................................161 Glucan synthesis .................................................................................................................................................161 Acceptor reaction ................................................................................................................................................161
    [Show full text]
  • THE ALPHA-GALACTOSIDASE SUPERFAMILY: SEQUENCE BASED CLASSIFICATION of ALPHA-GALACTOSIDASES and RELATED GLYCOSIDASES Naumoff D.G
    COMPUTATIONAL STRUCTURAL AND FUNCTIONAL PROTEOMICS THE ALPHA-GALACTOSIDASE SUPERFAMILY: SEQUENCE BASED CLASSIFICATION OF ALPHA-GALACTOSIDASES AND RELATED GLYCOSIDASES Naumoff D.G. State Institute for Genetics and Selection of Industrial Microorganisms, Moscow, Russia, e-mail: [email protected] Keywords: α-galactosidase, melibiase, glycoside hydrolase, GH-D clan, GH31 family, GHX family, COG1649, enzyme classification, protein family, protein phylogeny Summary Motivation: About 1 % of genes in genomes code enzymes with glycosidase activities. On the basis of sequence similarity all known glycosidases have been classified into 90 families. In many cases proteins of different families have common evolution origin. It makes necessary to combine the corresponding families into a superfamily. Results: Using of the PSI-BLAST program we found significant sequence similarity of several glycosidase families, two of which includes enzymes with the α galactosidase activity. Sequence homology, common catalytic mechanism, folding similarities, and composition of the active center allowed us to group three of these families – GH27, GH31, and GH36 – into the α-galactosidase superfamily. Phylogenetic analysis of this superfamily revealed polyphyletic origin of GH36 family, which could be divided into four families. Glycosidases of the α-galactosidase superfamily have a distant relationship with proteins belonging to families GH13, GH70, and GH77 of glycosidases, as well as with two families of predicted glycosidases. Introduction Glycoside hydrolases or glycosidases (EC 3.2.1.-) are a widespread group of enzymes, hydrolyzing the glycosidic bonds between two carbohydrates or between a carbohydrate and an aglycone moiety. A large multiplicity of these enzymes is a consequence of the extensive variety of their natural substrates: di-, oligo-, and polysaccharides.
    [Show full text]
  • Highly Thermostable and Alkaline Α-Amylase from a Halotolerant- Alkaliphilic Bacillus Sp. Ab68
    Brazilian Journal of Microbiology (2008) 39:547-553 ISSN 1517-8382 HIGHLY THERMOSTABLE AND ALKALINE α-AMYLASE FROM A HALOTOLERANT- ALKALIPHILIC BACILLUS SP. AB68 Ashabil Aygan1*; Burhan Arikan2; Hatice Korkmaz2; Sadik Dinçer2; Ömer Çolak2 1Kahramanmaras Sutcu Imam University, Faculty of Science and Letters, Department of Biology, K. Maras, Turkey; 2Cukurova University, Faculty of Science and Letters, Department of Biology, Molecular Biology Laboratory, Adana, Turkey Submitted: August 13, 2007; Returned to authors for corrections: October 22, 2007; Approved: July 16, 2008. ABSTRACT An alkaliphilic and highly thermostable α-amylase producing Bacillus sp. was isolated from Van soda lake. Enzyme synthesis occurred at temperatures between 25ºC and 40ºC. Analysis of the enzyme by SDS-PAGE revealed a single band which was estimated to be 66 kDa. The enzyme was active in a broad temperature range, between 20ºC and 90ºC, with an optimum at 50ºC; and maximum activity was at pH 10.5. The enzyme was almost completely stable up to 80ºC with a remaining activity over 90% after 30 min pre-incubation. Thermostability was not increased in the presence of Ca2+. An average of 75% and 60ºC of remaining activity was observed when the enzyme was incubated between pH 5 and 9 for 1 h and for 2 h, respectively. The activity of the enzyme was inhibited by SDS and EDTA by 38% and 34%, respectively. Key words: Bacillus sp., α-amylase, Alkaliphilic, Thermostable, Enzyme. INTRODUCTION unique, buffered haloalkaline habitat appropriate for a stable development of obligately (halo)alkaliphilic microorganisms Amylases are one of the most important industrial enzymes. growing optimally at pH around 10 (39).
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 9,689,046 B2 Mayall Et Al
    USOO9689046B2 (12) United States Patent (10) Patent No.: US 9,689,046 B2 Mayall et al. (45) Date of Patent: Jun. 27, 2017 (54) SYSTEM AND METHODS FOR THE FOREIGN PATENT DOCUMENTS DETECTION OF MULTIPLE CHEMICAL WO O125472 A1 4/2001 COMPOUNDS WO O169245 A2 9, 2001 (71) Applicants: Robert Matthew Mayall, Calgary (CA); Emily Candice Hicks, Calgary OTHER PUBLICATIONS (CA); Margaret Mary-Flora Bebeselea, A. et al., “Electrochemical Degradation and Determina Renaud-Young, Calgary (CA); David tion of 4-Nitrophenol Using Multiple Pulsed Amperometry at Christopher Lloyd, Calgary (CA); Lisa Graphite Based Electrodes', Chem. Bull. “Politehnica” Univ. Kara Oberding, Calgary (CA); Iain (Timisoara), vol. 53(67), 1-2, 2008. Fraser Scotney George, Calgary (CA) Ben-Yoav. H. et al., “A whole cell electrochemical biosensor for water genotoxicity bio-detection”. Electrochimica Acta, 2009, 54(25), 6113-6118. (72) Inventors: Robert Matthew Mayall, Calgary Biran, I. et al., “On-line monitoring of gene expression'. Microbi (CA); Emily Candice Hicks, Calgary ology (Reading, England), 1999, 145 (Pt 8), 2129-2133. (CA); Margaret Mary-Flora Da Silva, P.S. et al., “Electrochemical Behavior of Hydroquinone Renaud-Young, Calgary (CA); David and Catechol at a Silsesquioxane-Modified Carbon Paste Elec trode'. J. Braz. Chem. Soc., vol. 24, No. 4, 695-699, 2013. Christopher Lloyd, Calgary (CA); Lisa Enache, T. A. & Oliveira-Brett, A. M., "Phenol and Para-Substituted Kara Oberding, Calgary (CA); Iain Phenols Electrochemical Oxidation Pathways”, Journal of Fraser Scotney George, Calgary (CA) Electroanalytical Chemistry, 2011, 1-35. Etesami, M. et al., “Electrooxidation of hydroquinone on simply prepared Au-Pt bimetallic nanoparticles'. Science China, Chem (73) Assignee: FREDSENSE TECHNOLOGIES istry, vol.
    [Show full text]
  • Glucans with Potential Health Benefits Through Controlled Glucose Release in the Human
    University of Groningen Synthesis of novel α-glucans with potential health benefits through controlled glucose release in the human gastrointestinal tract Gangoiti, Joana; Corwin, Sarah F; Lamothe, Lisa M; Vafiadi, Christina; Hamaker, Bruce R; Dijkhuizen, Lubbert Published in: Critical Reviews in Food Science and Nutrition DOI: 10.1080/10408398.2018.1516621 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2020 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Gangoiti, J., Corwin, S. F., Lamothe, L. M., Vafiadi, C., Hamaker, B. R., & Dijkhuizen, L. (2020). Synthesis of novel α-glucans with potential health benefits through controlled glucose release in the human gastrointestinal tract. Critical Reviews in Food Science and Nutrition, 60(1), 123-146. https://doi.org/10.1080/10408398.2018.1516621 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
    [Show full text]
  • Studies Into the Structural Basis of the DNA Uridine Endonuclease Activity of Exonuclease III Homolog Mth212
    Studies into the structural basis of the DNA uridine endonuclease activity of exonuclease III homolog Mth212 Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August Universität zu Göttingen Vorgelegt von Khaliun Tseden aus Greifswald, Deutschland Göttingen 2011 D7 Referent: Prof. Dr. Hans-Joachim Fritz Korreferent: PD Dr. Wilfried Kramer Tag der mündlichen Prüfung: 02. Mai 2011 TABLE OF CONTENTS Table of Contents 1 Introduction ………………………………………………………………….. 1 1.1. Background to the study ………………………………………………… 1 1.1.1. Necessity of mutation avoidance………………………………… 1 1.1.2. Mutations arising in DNA during replication …………………… 1 1.1.3. Exogenous sources of DNA damage …………………………….. 2 1.1.4. Endogenous sources of DNA damage …………………………… 2 1.1.4.1. Hydrolytic DNA deamination ........……………………… 4 1.1.5. Repair of uracil in DNA …………………………………………. 5 1.1.5.1. Uracil-initiated base excision repair …………………...... 5 1.1.5.2. Uracil-initiated nucleotide incision repair ……………… 7 1.2. Objective and methodology of the study ………………………………… 9 1.2.1. Objective of the study.…………………………………………… 9 1.2.2. Methodology of the study ………………………………………. 9 1.2.2.1. Necessity of screening or selection methodology in 11 directed evolution of enzymes …………………………………………… 1.2.2.2. Selection of a protein with acquired DNA uridine 11 endonuclease activity ………………………………………………….... 2. Materials and Methods ………………………………………………………. 13 2.1. Materials …………………………………………………………………. 13 2.1.1. Bacterial strains ………………………………………………….. 13 2.1.1.1. Escherichia coli …………………………………………. 13 2.1.1.2. Bacillus subtilis ………………………………………… 14 2.1.2. Bacteriophage strains …………………………………………….. 14 2.1.3. Plasmid vectors ………………………………………………… 15 2.1.4. 2’ Desoxyriboseoligonucleotides ………………………………. 17 2.1.5. Molecular ladders and markers ………………………………….. 21 2.1.6.
    [Show full text]
  • Dynafit—A Software Package for Enzymology
    Author's personal copy CHAPTER TEN DynaFit—A Software Package for Enzymology Petr Kuzmicˇ Contents 1. Introduction 248 2. Equilibrium Binding Studies 250 2.1. Experiments involving intensive physical quantities 250 2.2. Independent binding sites and statistical factors 252 3. Initial Rates of Enzyme Reactions 255 3.1. Thermodynamic cycles in initial rate models 255 4. Time Course of Enzyme Reactions 260 4.1. Invariant concentrations of reactants 261 5. General Methods and Algorithms 262 5.1. Initial estimates of model parameters 263 5.2. Uncertainty of model parameters 269 5.3. Model-discrimination analysis 273 6. Concluding Remarks 275 6.1. Model discrimination analysis 275 6.2. Optimal design of experiments 276 Acknowledgments 276 References 276 Abstract Since its original publication, the DynaFit software package [Kuzmicˇ,P.(1996). Program DYNAFIT for the analysis of enzyme kinetic data: Application to HIV proteinase. Anal. Biochem. 237, 260–273] has been used in more than 500 published studies. Most applications have been in biochemistry, especially in enzyme kinetics. This paper describes a number of recently added features and capabilities, in the hope that the tool will continue to be useful to the enzymo- logical community. Fully functional DynaFit continues to be freely available to all academic researchers from http://www.biokin.com. BioKin Ltd., Watertown, Massachusetts, USA Methods in Enzymology, Volume 467 # 2009 Elsevier Inc. ISSN 0076-6879, DOI: 10.1016/S0076-6879(09)67010-5 All rights reserved. 247 Author's personal copy
    [Show full text]
  • How Nature Can Exploit Nonspecific Catalytic and Carbohydrate Binding
    How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity Fiona Cuskina,b,1, James E. Flinta,1, Tracey M. Glosterc,1,2, Carl Morlanda, Arnaud Basléa, Bernard Henrissatd, Pedro M. Coutinhod, Andrea Strazzullie, Alexandra S. Solovyovaa, Gideon J. Daviesc, and Harry J. Gilberta,b,3 aInstitute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; bThe Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602; cStructural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom; dArchitecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, 13288 Marseille Cedex 9, France; and eInstitute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, 80131 Naples, Italy Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved October 30, 2012 (received for review July 16, 2012) Noncatalytic carbohydrate binding modules (CBMs) are components CBMs enhance the activity of their cognate enzymes, and though of glycoside hydrolases that attack generally inaccessible substrates. the mechanism(s) by which this occurs remains uncertain, CBMs CBMs mediate a two- to fivefold elevation in the activity of endo- most likely fulfill a targeting function by increasing the effective acting enzymes, likely through increasing the concentration of the concentration of the appended enzymes, in the vicinity of the appended enzymes in the vicinity of the substrate. The function of substrate, thereby enhancing catalytic efficiency (8, 9). CBMs appended to exo-acting glycoside hydrolases is unclear Fructans, such as inulin and levan (polymers of predominantly because their typical endo-binding mode would not fulfill a target- β-2,1– or β-2,6–linked fructose units, respectively) are common ing role.
    [Show full text]
  • 154581A0.Pdf
    No. 3914, NOVEMBER 4, 1944 NATURE 581 Alleged Role of Fructofuranose in the ture of fructofuranoses and fructopyranoses. The recent demonstration that glucose-1-phosphate (Cori Synthesis of Levan ester) and fructose form a dynamic equilibrium with THE view was long widely entertained that cells sucrose and phosphoric acid in the presence of a. synthesize macromolecules of polysaccharides and specific enzyme8 corroborates this view. Addition of proteins by a reversion .of the process of hydrolysis. fructose to sucrose does not inhibit levan production It has been suggested accordingly that the synthesis from the latter, yet fructose itself, although it pre­ of the polyfructoside levan specifically from aldo­ sumably contains ready fructofuranose, is not con­ side< >fructofuranosides (sucrose, raffinose) involves verted into levan by levansucrase7 • Similarly, levan­ two distinct steps : first, hydrolysis of the substrate ; sucrase fails to form levan from reaction mixtures secondly, polymerization of fructofuranose b;r a con­ in which fructofuranose is sustainedly liberated in densation involving removal of water1• Bacteria statu nascendi, for example, in reaction mixtures of which form levan from sucrose do so also from methyl gamma fructoside + yeast invertase, and of raffinose•. This polymerative type of sucrose de­ inulin inulase. gradation is concurrent with an ordinary hydrolytic (c) Extracts of an Aerobacter, although they pro­ inversions·'· The same banteria ferment !evans. duce levan from sucrose and hydro'yse the latter as Investigators might be tempted by these correlations well, do not hydrolyse levan. Thus they contain to consider the enzyme system, levansucrase, to be levansucrase and invertase but no polyfructosidase but a mixture of invertase and polyfructosidase.
    [Show full text]