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Analysis of Secondary Metabolite Gene and Protein Expression Profiles in Streptomyces Coelicolor Grown Under Environmental Conditions

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Analysis of Secondary Metabolite Gene and Protein Expression Profiles in Streptomyces Coelicolor Grown Under Environmental Conditions _________________________________________________________________________Swansea University E-Theses Analysis of secondary metabolite gene and protein expression profiles in Streptomyces coelicolor grown under environmental conditions. Bell, Kathryn Laura How to cite: _________________________________________________________________________ Bell, Kathryn Laura (2012) Analysis of secondary metabolite gene and protein expression profiles in Streptomyces coelicolor grown under environmental conditions.. thesis, Swansea University. http://cronfa.swan.ac.uk/Record/cronfa42693 Use policy: _________________________________________________________________________ This item is brought to you by Swansea University. 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Please link to the metadata record in the Swansea University repository, Cronfa (link given in the citation reference above.) http://www.swansea.ac.uk/library/researchsupport/ris-support/ Swansea University Prifysgol Abertawe Analysis of Secondary Metabolite Gene and Protein Expression Profiles in Streptomyces coelicolor Grown Under Environmental Conditions Kathryn Laura Bell Submitted to Swansea University in fulfilment of the requirements for the Degree of Doctor of Philosophy 2012 ProQuest Number: 10807462 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10807462 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 For Mam and Dad Abstract Streptomycetes are Gram positive, soil dwelling filamentous bacteria, known for their production of secondary metabolites. Genome sequencing of Streptomyces coelicolor identified 26 known or predicted secondary metabolite gene clusters ranging from antibiotics to siderophores, lipids, pigments and lantibiotics. Most studies investigating secondary metabolite production in Streptomyces, as well as other bacteria, are undertaken in liquid or on solid media. There is little gene expression data available from in situ studies. This study determined expression in a totally different growth medium, soil, to gain insight into growth and adaptation under more 'normal' habitat conditions and the effect changing environmental factors has on the expression of secondary metabolite gene clusters. In order to do this, 5. coelicolor was grown in soil microcosms from which RNA was extracted and amplified using an optimised T7 polymerase-based RNA amplification protocol. The amplified RNA was used to determine gene expression profiles via endpoint RT-PCR and RT-qPCR. In a complementary approach, 5. coelicolor sand microcosms were subjected to a novel protein extraction procedure to determine protein expression profiles in soil. This study elucidated how carbon, nitrogen and metal availability, the environmentally bio­ active entomopathogenic fungus Metarhizium anisopliae and nematode Steinernema kraussei affect secondary metabolite gene expression in soil. In contrast to the consensus of secondary metabolism commencing after reduction or cessation of growth, this study revealed expression of secondary metabolite biosynthetic genes and proteins related to secondary metabolism before the onset of exponential growth. Some secondary metabolite genes/proteins were even expressed constitutively. Soil microcosms have been shown to be an important tool for gene expression analysis. The results of these novel transcriptomic and proteomic approaches therefore have given new insight into secondary metabolism and its role under natural habitat conditions. DECLARATIONS This work has not previously been accepted in substance for any degree and is not being concurrently submitted in candidature for any degree. Signed (candidate) STATEMENT 1 This thesis is the result of my own investigations, except where otherwise stated. Where correction services have been used, the extent and nature of the correction is clearly marked in a footnote(s). Other sources are acknowledged by footnotes giving explicit references. A bibliography is appended. Signed., (candidate) Date STATEMENT 2 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter- library loan, and for the title and summary to be made available to outside organisations. Signed.. (candidate) Date 1 0 . Q d . II Contents Page Acknowledgments viii Abbreviations ix Chapter 1 Introduction 1.1 Actinomycetes 1 1.2 Streptomycetes 2 1.2.1 Regulation by bid genes 3 1.2.2 Streptomyces coelicolor 4 1.3.1 Primary metabolism 5 1.3.2 Secondary metabolism 8 1.3.2.1 Link with primary metabolism 8 1.3.2.2 Regulation of secondary metabolism 9 1.3.2.3 Secondary metabolite classes and biosynthesis 11 1.4.1 Streptomyces secondary metabolism 16 1.4.2.1 Polyketides 19 1.4.2.2 Fatty acids 20 1.4.2.4 Terpenoids 20 1.4.2.4 Peptides 21 1.4.2.5 Miscellaneous 23 1.5.1 Soil and soil ecology 24 1.5.2 Soil metals 26 1.5.3 Siderophores 29 1.5.4 Study of soil microbial ecology 31 1.5.5 Soil transcriptomics 32 1.5.6 S. coelicolor in soil studies 32 1.6 Proteomics 34 1.6.1 Soil Proteomics 35 1.7 Introduction to methods 36 1.7.1 RNA amplification 36 1.7.2 Reverse transcription polymerase chain reaction 38 1.7.3 Quantitative PCR 39 1.7.4 Separation of proteins 43 1.7.5 Peptide generation and cleanup 44 1.7.6.1 Mass spectrometry 45 1.7.6.2 LC ESI MS/MS 46 1.7.6.3 MS/MS Quadrupole-lon trap 46 iii 1.7.6.4 Protein identification by database searching 47 1.8 Aims and objectives 49 Chapter 2 Methods and materials 2.1 Soil 50 2.1.1 Soil preparation 50 2.1.2 Soil water content 50 2.1.3 Soil water saturation testing 50 2.1.4 Soil pH 51 2.1.5 WDPT 51 2.2 Buffers/media 51 2.2.1 Bradford reagent 51 2.2.2 DNA Loading dye 51 2.2.3 FA buffer 52 2.2.4 FA gel denaturation mix 52 2.2.5 FA running buffer 52 2.2.6 LB agar/broth 52 2.2.7 Liquefied phenol 52 2.2.8 Ringers solution 53 2.2.9 SDS PAGE solutions 53 2.2.10 Soya flour mannitol agar 54 2.2.11 Singleton et al protein extraction buffer 54 2.2.12 T10E1 (TE buffer) 54 2.2.13 Taq extraction buffers 54 2.2.14 TBE 55 2.2.15 YEME 55 2.3 Streptomyces methods 55 2.3.1 Spore Stock production 55 2.3.2 Spore concentration glycerol stocks 55 2.4 Microcosms methods 56 2.4.1 pH adjustments 56 2.4.2 Soil microcosms set up 56 2.4.3 Sand microcosms set up 57 2.4.4 Growth curve measurements of microcosms 57 2.4.5 Antibiotic assays 58 2.5 Molecular Biology methods 58 2.5.1 Genomic DNA (gDNA) extraction 58 2.5.2 RNA extraction from SFM-grown S. coelicolor 59 2.5.3 DNA/RNA quantitation: UV spectrophotometer and Nanodrop 60 2.5.4 DNA agarose gel electrophoresis 61 2.5.5 RNA formaldehyde (FA) gel electrophoresis 61 2.5.6 Endpoint PCR methods 61 2.5.6.1 Genes and primer design (web primer) 61 2.5.6.2 Taq polymerase 62 2.5.6.3 Endpoint PCR setup 62 2.5.7.1 Reverse transcription 63 2.5.7.2 Roche Transcriptor High Fidelity cDNA synthesis kit 63 2.5.8 qPCR 63 2.5.9 RNA amplification amended from Gao et al 2007 65 2.5.9.1 First strand synthesis 65 2.5.9.2 Second strand synthesis 65 2.5.9.3 in vitro transcription 66 2.5.10 RNA extractions from soil 66 2.5.10.1 PowerSoil Total RNA Isolation Kit (Mobio) 66 2.5.10.2 Nucleospin RNA from Plant Tissue (ABgene) 67 2.6 Proteomics Methods 68 2.6.1 Bradford assay 68 2.6.2 Protein extraction from soil: Benndorf et al. (Benndorf et al 2007) 68 2.6.3 Protein extraction from soil: Singleton et al. (Singleton et al 2003) 69 2.6.4 Protein extraction from soil: amended from Masciandaro et al 2008 69 2.6.5 SDS PAGE 70 2.6.6 Gel staining 71 2.6.6.1 Coomassie staining 71 2.6.6.2 Silver Staining 71 2.6.7 Band excision and treatment 72 2.6.8 Trypsin Digestion 72 2.6.9 Zip-Tip protein purification 73 2.6.10 LC-ESI MS/MS 73 2.6.11 Data processing and mining 74 2.6.12 Visual analysis for protein abundance 74 2.7 Statistical analysis 75 2.7.1 qPCR 75 2.7.2 Doubling time analysis 76 Chapter 3 Soil characterisation and optimisation of molecular techniques 3.1 Soil Sampling 77 v 3.2 Soil characterisation 78 3.3 S. coelicolor growth curves 82 3.4 Comparison of RNA extraction methods from soil 82 3.5.1 RNA amplification procedure optimisation 84 3.5.2 Troubleshooting and optimisation 85 3.6 PCR 90 3.6.1 Genes of interest (GOI) 90 3.6.2 Primer design 93 3.7 Chapter 3 discussion 97 3.7.1 Growth of 5.
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