CONTENTS 1. Introduction 2. Review of Literature 7 2. 1. Characteristics of myxoba c teria 7 2. 1.1. Morphol ogy 7 2. 1. 2. Nutrition 10 2. 1.3. Metabolism 12 2 .1.4. Genetics 1 3 2.2. Habitats of myxobacteria 14 2 .3. Enrichment, isolation and preservation of myxobacteria 16 2.3.1. Enrichment of bacteriolytic species 16 2.3.1.1. Incubation of natural materials 16 2.3.1.2. Soil baiting 17 2.3.1.3. Singh plates 17 2.3.1.4. Water samples 17 2.3.2. Enrichment of cellulolytic species 18 2.3.3. Isolation of pure cultures 18 2.3.4. Preservation 21 2.4. Classification of myxobacteria 23 2.5. Antibiotics from myxobacteria 28 2.5.1. Screening 29 2.5.2. Chemical structures 30 2.5.3. Mechanism of action 36 2.5.4. Biosynthesis 38 2.5.5. Fermentation 39 2.6. Practical interest 41 3. (il) Materials and Methods 43 3 . 1 . 3.2 . 3 . 3. Culture media used 4 3 Reagents used 52 Experimental 5S 3 . 3 . 1. Collection of soil sampl es 5S 3.3 . 2 . Determination of pH o f soil s amples 5S 3.3.3 . Enrichment, isolation and maintenance of myxobacteria 55 3.3.4 . Characterization of myxobacterial isolates 3.3.4.1. General morphology 57 3.3.4.1.1. Fruiting body 57 3.3.4.1.2. Swarm 57 3.3.4.1.3. Vegetative cell 58 3.3.4.1.4. Microcyst/myxospore 3.3.4.2. Gram staining 58 3.3.4.3. Motility 59 3.3.4.4. Ultraviolet fluorescence 59 3.3.4.5. Adsorption of Congo red 59 3.3.4.6. Production of catalase 59 3.3.4.7. Production of oxidase 59 3.3.4.8. Hydrolysis of starch 60 3.3.4.9. Hydrolysis of fat 60 3.3.4.10. Hydrolysis of gelatin 60 3.3.4.11. Hydrolysis of casein 60 3.3.4.12. Hydroysis of esculin 60 3.3.4.13. Bacteriolytic activity 61 3.3.4.14. Cellulolytic activity 61 3~3~ 4.15. Anaerobic growth 61 57 58 ( i ii) 3.3.4.16. Produc tion of indole 61 3.3.4.17. Voges-Prosk.auer r~action 61 3.3.4.18. Reduction of nitrate 62 3.3.4.19. Production of urease 62 3.3.4.20. Production of acid and gas from carbohydrates o/. 3.3.4.21. DNA base composition 62 3.3.4.22. Antibiotic sensitivity 63 3.3.5. Identification of myxobacterial isolates 63 3.3.6. Screening for antimicrobial activity 63 3.3.6.1. Primary Screening 63 3.3.&.2. Secondary screening 64 3.3.7. Optimization of culture conditions for antibiotic proauction by Myxococcus fulvus SF126 (ATCC 49305) 65 3.3.7.1. Effect of growth media and time of fermentation 65 3.3.7.2. Effect of aeration and time of fermentation 65 3.3.7.3. Effect of initial pH 65 3.3.7.4. Effect of carbon source supplement 3.3.7.5. Effect of nitrogen source 66 3.3.7.6. Effect of temperature 66 3.3.7.7. Effec t of inocubm size 67 3.3.7.8. Effect of yeast extract 67 66 ( i v) 3 . 3 . 8 . Iso l atio n o f t he a n tibiotic s ubs tance produced by Myxococcus f ulvus SF126 (ATCC 493 0 5 ) h7 3 . 3 . 9 . Homogeneity of t h e isolated a n tibi o ti c 69 3 . 3 . 9 .1. Bioaut ography 69 3 . 3 . 9 . 2 . Thin layer c hromatog raphy 69 3 . 3 .1 0 . Ph ys i c ochemi c a l proper tie s of the i so l a t ed a nti bio ti c 70 3 . 3 .1 0 .1. So lubilit y 70 3 .3.1 0 . 2. Me lting point 70 3.3.1 0 . 3 . Res po nse t o s pec ific c hemical reage n t s 70 3.3.10.4. Pr esence of hal oge n 70 3.3.10. 5 . Ultraviolet and visibl e spec t rosco py 7 1 3.3.10.6. Infrared spectroscopy 7 1 3.3.10.7. Nuclear magnetic resonance s pectroscopy 71 3.3.10. 8 . Mas s spe ctrometry 71 3.3. 11 . Biological properties of the isolat e d a n ti bio t ic 7 1 3.3.11.1. Antimicrobial spectrum 71 3.3.12. Statistical analysis 72 4. Results 73 4.1. Source of soil fo r isolation of myxobacteria 73 4.2. Enrichment and isolation of myxobacteria 73 4.3. Characterization and identification of the myxobacterial iso l a te s 73 4.4. Distribution of myxobacteria in Indian soils 102 4.5. Screening of myxobacterial isolates for antimicrobial a cti vi ti es 10~ ( v) 4.6. Optimization of culture conditions for antibiotic production by Myx ococcus f ulvus SF126 (ATCC 4930 5 ) 107 4.6. 1. Effect of media and time o f fermentati on 107 4 .6. 2 . Effect of aeration a nd time of fermentation 114 4.6. 3 . Effect of initial pH 11 4 4.6. 4. Effect of carbon sourc e s upplement 11 4 4.6. 5 . Effect of nitroge n source 114 4.6.6. Effect of temperature 122 4.6.7. Effect of in oc ulum size 122 4.6.8. Effect of yeast ex tract 122 4.7. Isolation of a ntibiotic substance produced by Myxococcus fulvus SF1 2C ( ATCC 4 9305) 127 4.8. Homogeneity of the isolated antibiotic 127 4.8.1. Bioautography 127 4.8.2. Thin layer chromatography 130 4.9. Physicochemical prope rtie s of the isolated antibiotic 130 4.9.1. Physical properties 130 4.9.2. Solubility 130 4.9.3. Melting point 130 4.9.4. Response to specific chemical reagents 130 4.9.5. Presence of halogen 130 4.9.6. Ultraviolet and visible spectroscopy 13 0 4.9.7. Infrared spectroscopy 132 4.9.8. Nuclear magnetic resonance spectroscopy 132 4.9.9. Mass spectroscopy 13 2 (vi) 4.10. Biological properties of the isolated antibioti c 132 4.10.1. Antimicrobial spectrum 13 2 5. Discussion 134 5 .1. Myxobacteria from Indian s oil 134 5 . 2. Antimicrobial activities o f the myxobact e rial i s olate s 136 5 . 2.1. Screening o f the i so lates 136 5 . 2 . 2 . Optimization of culture condition for antibiotic production by Myxococ cus fulvus SF126 137 5.2.3. Isolation and purification of the a ntibiotic produced by Myxococcus fulvus SF126 141 5.2.4. Phys icochemica l properties of the isolated antibiotic 141 5.2.5. Antimicrobial spectrum of the isolated antibiotic 143 6. Summary 145 7. Bibliography 148