Induction of secondary metabolism across actinobacterial genera A thesis submitted for the award Doctor of Philosophy at Flinders University of South Australia Rio Risandiansyah Department of Medical Biotechnology Faculty of Medicine, Nursing and Health Sciences Flinders University 2016 TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................ ii TABLE OF FIGURES ............................................................................................. viii LIST OF TABLES .................................................................................................... xii SUMMARY ......................................................................................................... xiii DECLARATION ...................................................................................................... xv ACKNOWLEDGEMENTS ...................................................................................... xvi Chapter 1. Literature review ................................................................................. 1 1.1 Actinobacteria as a source of novel bioactive compounds ......................... 1 1.1.1 Natural product discovery from actinobacteria .................................... 1 1.1.2 The need for new antibiotics ............................................................... 3 1.1.3 Secondary metabolite biosynthetic pathways in actinobacteria ........... 4 1.1.4 Streptomyces genetic potential: cryptic/silent genes ........................... 7 1.2 Actinobacteria life cycle and secondary metabolite production ................. 10 1.2.1 Actinobacteria taxonomy, morphology and life cycle ......................... 10 1.2.2 A-factor regulates sporulation and secondary metabolite production 11 1.3 Actinobacteria in microorganism communities ......................................... 14 1.3.1 Actinobacteria isolation from microorganism communities ................ 14 1.3.2 Pseudonocardia: a case study on interactivity................................... 15 1.4 Co-culture for secondary metabolite production ....................................... 17 1.4.1 Co-culture increases unique metabolite production ........................... 17 1.4.2 General theories in the mechanism of co-culture .............................. 21 1.4.3 Physical contact in induction of secondary metabolites ..................... 21 1.4.4 Small molecules in induction of secondary metabolite ...................... 22 1.4.5 Conclusion ........................................................................................ 24 Chapter 2. Putative species identification using 16S rRNA sequencing of actinobacteria used in this study ................................................................................................... 25 2.1 Introduction .............................................................................................. 25 2.2 Method ..................................................................................................... 26 2.2.1 Spore suspension ............................................................................. 26 2.2.2 Comparison of morphology in different medium ................................ 26 2.2.3 DNA extraction .................................................................................. 26 2.2.4 16S rRNA gene sequencing .............................................................. 27 2.2.5 Phylogenetic analysis ....................................................................... 27 2.3 Results and Discussion ............................................................................ 28 2.3.1 Pseudonocardia species show two or more different variants ........... 28 ii 2.3.2 Putative species identification using 16S rRNA gene sequencing ..... 30 2.3.2.1 Pseudonocardia species ............................................................... 30 2.3.2.2 Streptomyces species ................................................................... 31 2.3.2.3 Other species ................................................................................ 35 2.3.3 Potential symbiosis capability of Pseudonocardia ............................. 39 2.3.4 Conclusion ........................................................................................ 41 Chapter 3. Screening for interaction between various Streptomyces and uncommon actinobacteria ........................................................................................................ 43 3.1 Introduction .............................................................................................. 43 3.2 Method ..................................................................................................... 44 3.2.1 Glycerol stock and spore suspension of cultures .............................. 44 3.2.2 Preliminary screening for interaction ................................................. 45 3.2.3 Secondary screening for interaction .................................................. 46 3.2.4 Pseudonocardia sp. and non-Streptomyces with a two to one interaction testing ......................................................................................................... 46 3.2.5 Streptomyces and non-Streptomyces screening for interaction ......... 47 3.2.6 Screening of Pseudonocardia sp. PIP 161 and other Streptomyces species with the ‘Ueda’ configuration .................................................................................. 47 3.2.7 Medium preparation for antibiotic activity measurement .................... 47 3.2.8 “Flip-plate” assay for antibiotic activity measurement ........................ 48 3.2.9 Statistical analysis ............................................................................ 48 3.3 Results and Discussion ............................................................................ 49 3.3.1 Preliminary screening method used reduced plate number ............... 49 3.3.2 Preliminary screening suggests that Micromonospora sp. and Streptomyces sp. were most susceptible to induction.................................................................. 52 3.3.3 Co-culture screening shows that most interaction in clusters can be replicated in binary interactions ........................................................................................... 57 3.3.4 Morphologically similar Pseudonocardia spp. induce morphological changes in different species of actinobacteria ................................................................... 59 3.3.5 Sporulation was induced in Streptomyces sp. EUM 76 when co-cultured with other Streptomyces species ............................................................................ 60 3.3.6 Amycolatopsis sp. PIP 207 also inhibits several Streptomyces species…….... ......................................................................................................... 62 3.3.7 Micromonospora sp. EUC 38 was also inhibited by Streptomyces sp. and other genera 63 3.3.8 Pseudonocardia sp. PIP 161 was able to induce changes in multiple Streptomyces species ..................................................................................... 64 iii 3.3.9 Pseudonocardia sp. PIP 161 alters antibiotic production in various Streptomyces species. 66 3.3.10 Interaction map showing interactions between actinobacteria ........... 67 3.3.11 Current concepts on the mechanism of interactions between actinobacteria……. 69 3.4 Conclusion ............................................................................................... 71 Chapter 4. The interaction between Streptomyces sp. EUC 63 with Pseudonocardia sp. PIP 161 in the induction of antibiotics ........................................................................... 73 4.1 Introduction .............................................................................................. 73 4.2 Methodology ............................................................................................ 74 4.2.1 Spore suspension ............................................................................. 74 4.2.2 Liquid cultivation of monocultures and co-cultures ............................ 75 4.2.3 Metabolite extraction from liquid cultures .......................................... 75 4.2.4 Thin layer chromatography (TLC) and Scale-up TLC ........................ 76 4.2.5 Bioautogram ..................................................................................... 76 4.2.6 High Performance Liquid Chromatography (HPLC) conditions .......... 77 4.2.7 “Preconditioning” of media ................................................................ 77 4.2.7.1 Preconditioning in solid medium .................................................... 77 4.2.7.2 Preconditioning in liquid medium ................................................... 78 4.2.8 Colony selection methodology .......................................................... 78 4.2.9 Flip plate antibiotic assay .................................................................. 79 4.2.10 Statistical analysis ............................................................................ 79 4.3 Results and Discussion ............................................................................ 79 4.3.1 Co-culture increased antibiotic activity of Streptomyces sp. EUC 63 or Streptomyces sp. PIP 146 with Pseudonocardia sp. PIP 161 in liquid cultivation……….. ......................................................................................................... 79 4.3.2 Co-culture increases multiple antibiotic