University of Warwick institutional repository: http://go.warwick.ac.uk/wrap A Thesis Submitted for the Degree of PhD at the University of Warwick http://go.warwick.ac.uk/wrap/2773 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. Evolution of the streptomycin and viomycin biosynthetic clusters and resistance genes Paris Laskaris, B.Sc. (Hons.) A thesis submitted to the University of Warwick for the degree of Doctor of Philosophy. Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL September 2009 Contents List of Figures ........................................................................................................................ vi List of Tables ....................................................................................................................... xvi Abbreviations ........................................................................................................................ xx Acknowledgements .............................................................................................................. xxi Declaration .......................................................................................................................... xxii Abstract .............................................................................................................................. xxiii 1 Introduction ..................................................................................................................... 1 1.1 Antibiotic function and resistance ........................................................................... 1 1.2 Streptomycin ........................................................................................................... 3 1.2.1 Streptomycin function ..................................................................................... 4 1.2.2 Streptomycin production ................................................................................. 6 1.2.3 Streptomycin resistance .................................................................................. 7 1.3 Viomycin............................................................................................................... 11 1.3.1 Viomycin function ........................................................................................ 12 1.3.2 Viomycin production .................................................................................... 14 1.3.3 Viomycin resistance ...................................................................................... 15 1.4 Streptomyces genus ............................................................................................... 16 1.4.1 Streptomyces morphology and genetics........................................................ 16 1.4.2 Streptomycete ecology .................................................................................. 18 1.4.3 Streptomycete phylogeny .............................................................................. 20 1.5 Aims and objectives .............................................................................................. 23 2 Materials and Methods .................................................................................................. 24 2.1 Media and Buffers ................................................................................................. 24 2.2 Growth, Storage and DNA acquisition from Streptomyces strains ....................... 26 2.2.1 Preparation and storage of spore suspensions ............................................... 26 2.2.2 Determination of antibiotic resistance level .................................................. 26 2.2.3 Determination of phenotype .......................................................................... 27 2.2.4 Growth and extraction of DNA ..................................................................... 27 2.2.5 Agarose Gel Electrophoresis ......................................................................... 28 2.3 Gene amplification and sequencing ...................................................................... 28 2.3.1 Polymerase Chain Reaction (PCR) ............................................................... 28 2.3.2 Purification of PCR products ........................................................................ 28 2.3.3 Cloning of PCR products .............................................................................. 29 ii 2.3.4 DNA Sequencing and Analysis ..................................................................... 29 2.4 Fosmid library construction and screening ........................................................... 30 2.4.1 Isolation of high molecular weight Streptomyces DNA................................ 30 2.4.2 Ligation into vector and transformation ........................................................ 30 2.4.3 Screening of library and sequencing ............................................................. 31 2.4.4 Creation and screening of orgy prep ............................................................. 31 2.5 Antibiotic production assay .................................................................................. 32 2.6 Gene detection from soil samples ......................................................................... 32 2.6.1 Estimation of water content .......................................................................... 32 2.6.2 Preparation of soil microcosms ..................................................................... 33 2.6.3 Preparation of seeded soil microcosms ......................................................... 33 2.6.4 DNA extraction from soil ............................................................................. 33 2.6.5 Amplification and cloning of target gene ...................................................... 34 2.7 RT real-time PCR ................................................................................................. 34 2.7.1 Preparation of high-density spore suspension ............................................... 34 2.7.2 Growth and sampling .................................................................................... 34 2.7.3 RNA extraction and DNA removal ............................................................... 35 2.7.4 Reverse transcription..................................................................................... 36 2.7.5 Real-time PCR .............................................................................................. 36 2.7.6 Data analysis ................................................................................................. 37 3 Distribution of the streptomycin cluster, strA resistance gene and other secondary metabolite genes in the genus Streptomyces ......................................................................... 39 3.1 Introduction ........................................................................................................... 39 3.2 Aims ...................................................................................................................... 40 3.3 Results ................................................................................................................... 40 3.3.1 Screening of eight previously isolated strains containing strA for other streptomycin genes ........................................................................................................ 40 3.3.2 Screening of other streptomycete isolates for streptomycin resistance ......... 43 3.3.3 Screening of streptomycete isolates for streptomycin cluster genes ............. 44 3.3.4 Analysis of the phylogeny of partial strA nucleotide sequences ................... 49 3.3.5 Analysis of the phylogeny of partial strA translated nucleotide sequences .. 51 3.3.6 Analysis of the amino acid sequence of StrA ............................................... 53 3.3.7 Phylogeny of the strW biosynthetic gene ...................................................... 56 3.3.8 Screening for alternate resistance mechanisms ............................................. 57 3.3.9 Screening for viomycin biosynthetic and resistance genes ........................... 60 3.3.10 Screening for other secondary metabolite genes ........................................... 66 iii 3.4 Discussion ............................................................................................................. 75 4 Investigation of the chromosomal location of the streptomycin cluster and strA gene in soil isolates ............................................................................................................................ 83 4.1 Introduction ........................................................................................................... 83 4.2 Aims ...................................................................................................................... 84 4.3 Results ................................................................................................................... 84 4.3.1 AR23 flanking regions .................................................................................. 84 4.3.2 CR50 flanking regions .................................................................................. 86 4.3.3 CR50 streptomycin cluster ...........................................................................