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Download (12Mb) A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/110352 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications THE BRITISH LIBRARY BRITISH THESIS SERVICE THE DISTRIBUTION OF PHENOTYPIC AND GENOTYPIC CHARACTERS WITHIN STREPTOMYCETES AND THEIR RELATIONSHIP TITLE . TO ANTIBIOTIC PRODUCTION. AUTHOR........ Lesley Phillips, DEGREE.................................................... AWARDING BODY _ TI. .. The University of Warwick, THESIS NUMBER THIS THESIS HAS BEEN MICROFILMED EXACTLY AS RECEIVED The quality of this reproduction is dependent upon the quality of the original thesis submitted for microfilming. Every effort has been made to ensure the highest quality of reproduction. Some pages may have indistinct print, especially if the original papers were poorly produced or if the awarding body sent an inferior copy. If pages are missing, please contact the awarding body which granted the degree. Previously copyrighted materials (journal articles, published texts, etc.) are not filmed. This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that Its copyright rests with its author and that no information derived from it may be published without the author's prior written consent. Reproduction of this thesis, other than as permitted under the United Kingdom Copyright Designs and Patents Act 1988, or under specific agreement with the copyright holder, is prohibited. 1 1 i T i j •7 Is 1 6 cms REDUCTION X j C A M ERA 3 No. of pages / THE DISTRIBUTION OF PHENOTYPIC AND GENOTYPIC CHARACTERS WITHIN STREPTOMYCETES AND THEIR RELATIONSHIP TO ANTIBIOTIC PRODUCTION. Lesley Phillips, Thesis submitted for the degree of Doctor of Philosophy (PhD), To The University of Warwick, Department of Biological Sciences, April, 1992. I Table of Contents, Page No. Chapter 1. Introduction 1 1.1. The Life-cycle and Ecology of the Streptomycetes. 1 1.2. The Taxonomy of Streptomycetes. 2 1.2.1. The Development of the genus Streptomyces. 2 1.2.2. Streptomycete Classifications Based on Chemotaxonomy. 4 1.2.3. Systematic Methods Used in Taxonomic Studies. 6 1.3. The Biosynthesis, Evolution and Natural Role of 10 Antibiotics in Streptomycetes. 1.3.1. Interactions Between Primary and Secondary 10 Metabolism in Streptomycetes. 1.3.2. The Diversity of Secondary Metabolites from 14 Streptomycetes. 1.3.3. The Evolution o f Antibiotics and its Relationship to 16 Antibiotic Function. 1.3.4. Theories Proposed For The Function of Secondary 18 Metabolites. 1.4. The Regulation and Control of Secondary Metabolism. 23 1.4.1. Carbon Source Regulation. 24 1.4.2. Nitrogen Source Regulation. 23 1.4.3. Regulation by Inorganic Phosphate (Pi). 26 1.4.4. Other Bioregulators. 27 1.4.3. Genetic Control and Organisation in Secondary 28 Metabolism. 1.4.6. Antibiotic Resistance Mechanisms in Streptomycetes. 32 1.4.7. The Relationship o f Antibiotic Resistance and 39 II Table of Contents, Page No. Biosynthesis to Phenotype and Genotype. l.S. The Exploitation of Antibiotics from Streptomycetes. 42 1.5.1. The History of Natural Product Screening. 42 1.5.2. Modern Antibiotic Discovery. 43 1.5.3. The Selection of Organisms for Industrial Screens. 44 1.5.4. The Design o f Media for Antibiotic Screening Programs. 45 1.5.5. Extraction Procedures and Chemical Identification Systems. 47 1.5.6. Target Directed Screening. 48 1.5.7. Screens used in the Agrochemical Industry. 49 1.5.8. Screens used in the Pharmacological Industry. 50 1.5.9. Beneficial Products Derived from the Pharmaceutical and 51 Agrochemical Industries. 1.5.10. Target Directed Isolation and Selection. 52 1.5.11. Other Approaches to Antibiotic Discovery. 53 1.6. Rationale and Aims for this Research. 55 Chapter 2. Materials and Methods. 56 2.1. Materials. 56 2.2. Strain Maintenance. 75 2.2.1. Routine Maintenance of Strains. 75 2.2.2. Culture Storage. 75 2.2.3. Strain Resuscitation. 75 2.2.4. Standardization of Streptomycete Inoculae. 76 2.3. Characterization of Streptomycetes. 76 2.3.1. Extraction of Bioactive Compounds. 76 2.3.2. Thin Layer Chromatography (TLC). 76 III Table of Contents, Page No. 2.3.3. Bioautography. 78 2.3.4. In vivo Agrochemical Screens. 78 2.3.3. Taxonomical Identification. 80 2.3.6. Scanning Electron Microscopy. 84 2.4. Fermentation Work. 86 2.4.1. Comparison of Antibiotic Production on Different Media. 86 2.4.2. Timecourses of Antibiotic Production by D133. 86 2.4.3. Oxygen Limitation Experiment. 87 2.4.4. Dry Weight Measurements. 87 2.4.5. Nutrient Gradients. 87 2.3. Fatty Acid Analysis. 88 2.5.1. Extraction Procedure. 88 2.5.2. Combined Capillary Gas Chromatography Mass. 89 Spectroscopy (GCMS). 2.5.3. Timecourses. 90 2.6. Metabolite Profiles. 90 2.7. Resistance Profiles. 90 2.7.1. The Use of Antibiotic Gradient Plates to Determine 90 Phenetic Resistance in Streptomyces Strains. 2.7.2. Determination of the Relationship Between Antibiotic 92 Concentrations Extrapolated from Gradient Plates and Corresponding Concentrations from Pour Plates. 2.8. DNA Isolation and Probing. 92 2.8.1. Small Scale Alkaline Lysis for Plasmid Preparation of 92 E.coli. 2.8.2. Maxi Preparation of Plasmid from E.coli. 93 2.8.3. Density Gradient Centrifugation. 93 IV Table of Contents. Page No. 2.8.4. Large Scale Isolation of Plasmid DNA from 94 Streptomycetes. 2.8.5. Isolation of Streptomyces " Total" DNA. 94 2.8.6. Phenol Extraction. 95 2.8.7. Quantitation of DNA. 95 2.8.8. Restriction Endonuclease Digestion of DNA. 95 2.8.9. Dot Blots. 96 2.8.12. Agarose Gel Electrophoresis. 96 2.8.13. Ethanol Precipitation. 96 2.8.14. Isopropanol Precipitation. 97 2.8.15. Electroelution. 97 2.8.16. Prehybridization. 97 2.8.17. Preparation of the Probe. 98 2.8.18. Removal of Unincorporated Nucleotides. 98 2.8.19. Hybridization. 99 2.8.20. Stringency. 99 2.8.21. Cerenkov Counting. 99 2.8.22. Detection of Radioactivity. 100 2.9. Cluster Analysis. 100 Chapter 3. The Distribution and Expression of Antibiotic 103 Production in Natural Streptomyces Isolates. 3.1. Introduction. 103 3.2. Bioactivity as Measured under Standard Conditions. 104 3.2.1. Antibacterial Activities. 104 3.2.2. Antifungal Activities. 107 V Table of Contents. Page No. 3.2.3. Specific Biological Activities. 109 3.2.4. Agrochemical Activities. 109 3.3. Factors Affecting the Expression of Bioactivity. 112 3.3.1. Choice of Medium. 112 3.3.2. Effects of Nutrient Gradients on Differentiation. 114 3.3.3. Effects of Nutrient Gradients on Antibiotic 118 Production in a Selection of Streptomycetes. 3.4. The Characterization of Antibiotic Production in D1S3. 121 3.4.1. Timecourses. 121 3.4.2. Repression of Nigericin Biosynthesis. 123 3.3. Discussion. 124 3.3.1. The Distribution of Antibiotic Production in 124 Streptomyces Isolates. 3.3.2. Factors Affecting Differentiation and Antibiotic 127 Production. Chapter 4. The Use of Fatty Add Profiles for Grouping 130 Streptomyces Species. 4.1. Introduction. 130 4.1.2. Choice and Characterization of Strains. 132 4.2. The Distributions of Fatty Acid Methyl Esters Within 133 Strep tomycetes. 4.2.1. The Distribution o f Individual FAMEs and FAME 133 Classes Within Representatives of the Genus Streptomyces. 4.2.2. The Diversity of FAME Patterns Within Streptomycetes. 137 4.3. Hierarchical Clustering to Determine Inter-relationships 140 VI Table of Contents, Page No. Between Streptomycetes Based on their Fatty Acid Profiles. 4.4. Distributions of FAMEs Within Sub-groups of the 145 Population. 4.4.1. The Distribution of FAMEs in Streptomyces Species 145 Groups. 4.4.2. The Distribution of FAMEs Within Producers of Specific 147 Classes of Antibiotics. 4.4.3. Distribution o f FAMEs Amongst Isolates from Specific 147 Geographical Locations and Isolation Procedures. 4.5. Alterations in the FAME Profile of D153 During Batch 151 Culture and its Relationship to Secondary Metabolite Biosynthesis. 4.5.1. Choice of Medium. 151 4.5.2. Timecourse o f Fatty Acid Biosynthesis Versus Antibiotic 154 Production by D1S3. 4.6. Discussion. 160 Chapter 5. The Relationship Between Chemical Profiles, 162 Taxonomic Status and Biological Activity. 5.1. Introduction. 162 5.1.1. Characteristics of Strains Chosen for this Study. 163 5.2. The Development of Methodology. 164 5.2.1. Scheme for the Numbering and Scoring of TLC Spots. 164 5.3. The Frequency of Occurrence of Different Spots. 166 5.3.1. Distribution of Metabolites. 166 VII Table o f Contents Page No. 5.4. The Estimation of Test Error Involved in Scoring 168 Metabolite Patterns. 5.4.1. The Effect of Operator Error in Scoring Metabolite 168 Patterns. 5.4.2. Reproducibility of Samples. 171 5.5. Cluster Analysis. 175 5.5.1. The Distribution of Streptomycetes Based on Chemical 175 Profiles, Using Hierarchical Methods. 5.5.2. Ordination of Streptomycete Chemical Profiles. 180 5.6. Recognising Interesting Strains using TLC. 184 5.7. Conclusions. 185 Chapter 6. Relationship Between Antibiotic Resistance 186 Phenotype and Biological Activities. 6.1.1. Introduction. 186 6.1.2. Choice of Strains. 188 6.2. The Distribution of Antibiotic Resistance in 190 Streptomycetes. 6.2.1. The Distribution of Antibiotic Resistance in Natural 190 Isolates. 6.2.2. The Diversity of Antibiotic Resistance Profiles. 193 6.2.3.
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