Detailed Contents of Practical Streptomyces Genetics ISBN

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1 Detailed Contents of Practical Streptomyces Genetics ISBN Chapter 1 General introduction to actinomycete biology Taxonomy of Streptomyces... 2 The genera of actinomycetes... 2 The genus Streptomyces... 2 Ecology of Streptomyces... 5 Streptomycetes as pathogens... 6 Some physiological features of primary metabolism in streptomycetes... 7 Carbon sources... 7 Nitrogen sources... 8 Amino acid catabolism... 9 Biosynthesis... 9 Some physiological novelties Antibiotic production by Streptomyces Streptomycetes as antibiotic producers Antibiosis in soil Physiology and regulation of antibiotic production 16 Developmental biology of Streptomyces The Streptomyces chromosome and its genetic elements 18 DNA base composition The chromosome Plasmids Transposable elements Phages Restriction and modification of Streptomyces DNA Genetic studies with streptomycetes and their near relatives Actinomycetes used for genetical studies Genetics and strain improvement for antibiotic and enzyme production Safety guidelines for recombinant DNA experiments with Streptomyces Chapter 2 Growth and preservation of Streptomyces Selective isolation of streptomycetes Making a Streptomyces spore suspension Plating out a Streptomyces spore suspension Streptomyces cultures on agar Growth of mycelium on cellophane overlays, nitrocellulose filters or agar Growth of Streptomyces mycelium in liquid Growth of Streptomyces for physiological studies Conditions for spore germination and reproducible exponential growth and antibiotic production by Streptomyces coelicolor Germination of Streptomyces spores Procedure Procedure Preparation of aerial mycelium and spores separately. 54 Aerial mycelium Spores Sporulation of Streptomyces in submerged cultures Preparation of protoplasts from Streptomyces lividans 66 and S. coelicolor A3(2) Preservation of Streptomyces strains and phages Preparation of lyophils Alternative I Alternative II Growing a culture from the lyophil Chapter 3 Microscopical methods Light microscopy Cellophane cultures Coverslip or slide cultures Impression preparations Staining for light microscopy DAPI Staining of Streptomyces mycelium.. 65 Staining for respiratory activity Immunofluorescent staining of Streptomyces mycelium Electron microscopy Electron microscopy of thin sections Sectioning of Streptomyces colonies Staining thin sections of Streptomyces colonies for glycogen Spatial visualisation of gene expression Chapter 4 Specialised biochemical techniques Preparation of cell-free extracts from Streptomyces Cell-free extracts from mycelium Cell-free extracts from spores Small-scale cell-free extract from spores Large-scale cell-free extract from spores Preparation of a coupled transcription-translation system from S. lividans Two-dimensional polyacrylamide gel electrophoresis and its application to Streptomyces proteins.. 83 In-vivo radioactive labelling of Streptomyces proteins 90 Two-step liquid cultivation and radioactive labelling method for Streptomyces Growth and radioactive labelling of Streptomyces on zirconium-silica beads Detection and assay of S. coelicolor (and S. lividans) antibiotics... 93

2 The S. coelicolor antibiotics Actinorhodin Undecylprodigiosin CDA Methylenomycin S. lividans as an antibiotic producer Secondary metabolite production in S. lividans transformants Chapter 5 Mutagenesis of Streptomyces by irradiation or chemicals General remarks about mutagenesis The optimal amount of mutagenesis Expression of mutations Spores or mycelium? Choice of mutagen Precautions Mutagenesis of Streptomyces spores by ultraviolet light (UV) Mutagenesis of Streptomyces spores by N-methyl-N nitro-n-nitrosoguanidine (NTG) Isolation of specific classes of mutant Resistant mutants Mutants that have to be isolated by screening Auxotrophs Other classes of mutants screened by replica plating Mutants recognised by visual examination. 106 Chapter 6 Transposon mutagenesis in Streptomyces General points about transposon mutagenesis Discovery of Streptomyces transposable elements 110 Streptomyces transposons suitable for mutagenesis of Streptomyces Use of heterologous transposons for mutagenising Streptomyces genes Transposon delivery vectors for Streptomyces Isolation of independent mutant strains Cloning transposon-tagged Streptomyces DNA in E. coli Tn4556 from S. fradiae A. Transposition of Tn4560 from puc1169 to a chromosomal location B. Protocol for inserting Tn4560 into S. coelicolor NF strains C. Tn4560 mutagenesis of SCP2* plasmids in S. lividans D. Tn4560 mutagenesis of pock-forming SCP2* in S. coelicolor IS493 from S. lividans E. Sectoring method for delivering IS493 derivatives using temperature-sensitive plasmids IS6100 from Mycobacterium fortuitum F. Transposon mutagenesis of S. lividans using psit Tn5493 derived from Tn G. Mutagenising S. lividans using pjoe H. Alternative method for isolating S. lividans with random insertions of Tn Chapter 7 In vivo genetic analysis by conjugation and protoplast fusion The modes of gene exchange in Streptomyces Mating Protoplast fusion Transduction Chromosomal recombination by transformation 128 Electroporation The practicalities of making crosses Quantitative analysis of crosses Recombination frequency Frequency of plasmid transfer Linkage mapping Establishing a genetic map by the "four-on-four" procedure The "classical" way of analysing the results of a "four-on-four" cross Analysing the results of a "four-on-four" cross by minimizing multiple crossovers Mapping a new marker by a single selection Plate-crosses Detection of conjugative plasmids by "pock" formation by transconjugants Protoplast fusion Fusion of protoplasts of S. coelicolor or S. lividans Genetic analysis by protoplast fusion Chapter 8 Preparation and analysis of genomic and plasmid DNA Isolation of genomic DNA Discussion of individual steps A. Kirby mix procedure for the isolation of genomic DNA B. Salting out procedure for the isolation of genomic DNA C. CTAB procedure for the isolation of genomic DNA Isolation of CCC plasmid DNA Discussion of individual steps A. Plasmid isolation by neutral lysis B. Standard CsCl-ethidium bromide gradient centrifugation C. CsCl density gradient for removing polysaccharides from DNA D. Plasmid isolation by alkaline lysis and potassium acetate precipitation E. Plasmid isolation by alkaline lysis and phenol

3 precipitation F. Plasmid purification using QIAGEN anion exchange column chromatography G. Plasmid isolation by the boiling method H. Alkaline denaturation of partially purified DNA samples I. Purification of samples using ethidium bromide J. Acid phenol (ph4) extraction K. Jurassic preps plasmid purification using guanidine thiocyanate Standard agarose gel electrophoresis Pulsed-field gel electrophoresis General considerations Standard procedure for preparing Streptomyces chromosomal DNA for PFGE Phenol wash procedure for preparation of actinomycete DNA for PFGE Digestion of DNA with restriction endonucleases in agarose blocks for PFGE Loading gels Running conditions for PFGE Removal of small DNA fragments Solutions Ammonium acetate EDTA ph Lithium chloride Sodium chloride Sodium hydroxide Potassium acetate and sodium acetate, 3M Tris-HCl buffer Chapter 9 General considerations about gene cloning in Streptomyces Features of Streptomyces genes Restriction-modification and other host factors Preparation of vector and target DNA How many clones are required for making a representative gene library? Choice and use of restriction endonucleases for making gene libraries Choice of cloning vector Vector host-range Size of the target DNA Integrating vectors Low copy number plasmid vectors High copy number plasmid vectors Plasmid versus phage vectors Positive selection vectors E. coli vectors containing Streptomyces selection markers Using bifunctional vectors that replicate in E. coli and Streptomyces Selective markers Antibiotic resistance Counterselectable markers Ligation conditions Transformation Finding the desired clone Sib-Selection Antibiotic biosynthetic genes Confirmation of clones Does the phenotype depend on the cloned DNA? 224 Is the cloned DNA rearranged? Is the promoter for a cloned gene present on the cloned DNA? What if the desired gene cannot be cloned? Assessing the quality of Streptomyces gene libraries. 226 Storing gene libraries Chapter 10 Introduction of DNA into Streptomyces Methods available Restriction barriers Use of single-stranded DNA Transformation and transfection in Streptomyces Polyethylene glycol (PEG) Transformation and transfection frequencies PEG-assisted transformation of Streptomyces protoplasts with plasmid DNA Standard procedure Rapid small-scale procedure Use of denatured DNA for protoplast transformation Spot-transformation Cosmid transformation Electroporation of mycelium Recognition, selection and screening of Streptomyces transformants Lethal zygosis reaction (pocks) Fertility Resistance markers Selection of antibiotic-resistant transformants by overlaying or flooding Soft agar overlays Flooding Detection of melanin-producing colonies Screening for plasmid DNA Colony hybridisation Using nitrocellulose filters Using Whatman 541 paper Preparation of colony replicas Hybridisation end labelling of oligonucleotide probes Using Whatman 540 paper Use of the Polymerase Chain Reaction (PCR) to identify transformants Conjugation from E. coli

4 Chapter 11 Plasmids and their use for gene cloning General properties of Streptomyces plasmids and their use for gene cloning Wild-type plasmids that have been used extensively to construct cloning vectors pij pjv psg SCP2* Higher copy number derivatives of SCP2*. 263 SCP2* vectors as delivery systems for gene disruptions SLP1 and psam Other integrating vectors List of special purpose vectors Bifunctional E. coli-streptomyces plasmids orit (RK2) vectors for conjugation between E. coli and Streptomyces cosmid vectors Expression vectors Vectors with promoterless reporter genes Positive selection vectors Integrating vectors Unstable and temperature-sensitive plasmids useful for gene replacement and transposon delivery Vectors without the tsr gene Vectors with resistance markers other than the common ones Vectors with blue/white selection (lacz ) in E. coli Chapter 12 Streptomyces phages The relevance of phages to Streptomyces genetics Occurrence, isolation and storage of Streptomyces phages Lytic and temperate phages Phages from soil Phages from lysogens Phages as industrial contaminants Streptomyces phage genetics In vivo physiological and genetic studies Deletion mutants and DNA packaging limitation 273 Phage DNA Uses of wild-type phages in the study of their hosts. 274 Transduction Localised mutagenesis using generalised transduction Restriction-modification systems Storage of Streptomyces phages Plaque assay of Streptomyces phages Single-plaque isolation of Streptomyces phages Preparation of high-titre Streptomyces phage stocks. 278 Isolation of new Streptomyces phages Isolation procedure I (direct method) Isolation procedure II (specific enrichment) Selection of potential transducing phages by pyrophosphate resistance Generalised transduction of S. venezuelae using SV1 phage Large-scale preparation of Streptomyces phage DNA 282 Small-scale preparation of Streptomyces phage DNA 285 Chapter 13 Cloning with phage vectors General features of C31 and its vector derivatives. 290 Shotgun cloning with C31 vectors Choice of C31 vectors for mutational cloning. 291 Choice of C31 vectors for screening by complementation of mutants or acquisition of new capabilities Ligation conditions Maximising and estimating insert frequency Construction and stability of lysogens Homogenotisation Application of C31 to gene fusions General features of the C31::xylE vectors KC862: a xyle-containing C31 derivative that gives yellow plaques only when carrying inserts with active promoters 296 A single copy number promoter-probe vector, KC Vectors for in situ fusions of xyle to chromo somally located transcription units Other phage-based cloning systems Prophage transformation with phage SAt-1 of S. azureus Phage-mediated transduction of plasmids Vectors based on other Streptomyces phages Use of integration functions of Streptomyces phages Transfection Transfer of Streptomyces phage DNA onto nitrocellulose filters for plaque hybridisation. 303 Preparation of C31 lysogens Procedure A Procedure B Low-tech method for detecting C31 derivatives containing resistance genes Use of glka counterselection to select deletions from, or loss of, C31 prophages

5 Chapter 14 Gene disruption and gene replacement Creating null mutants Method (a). Insertional inactivation via a single crossover Method (b). Insertional inactivation via double crossing over Method (c). Insertional inactivation using an inframe deletion Other factors affecting the choice of approach Polar effects The size of intervals used Mutation stability Method of delivery Non-replicating E. coli plasmids Temperature-sensitive replicons Phage vectors Unstable replicons Dealing with essential genes Introduction of point mutations and other subtle changes Counterselection of the delivery vector "Heterologous" disruptions and replacements Choice of resistance markers Selecting for single crossover intermediates during gene replacement A practical example of gene disruption Homogenotisation Problems arising from unintended homogenotisation events Cloning mutant alleles by homogenotisation Gene replacements involving whole gene clusters Mutational analysis of transcription units Chapter 15 Reporter systems Introduction The problems with lacz Choice of vector: plasmid, phage and transposon systems Antibiotic resistance genes as reporters: neo and cat. 342 The tyrosinase-encoding operon of S. glaucescens as a reporter system The whie (spore pigment) major operon as a reporter system The catechol 2,3-dioxygenase determinant, xyle, a readily quantified reporter gene for Streptomyces Detection of xyle expression in situ in colonies. 344 Detection of xyle expression in C31 plaques. 344 Assay of catechol 2,3-dioxygenase in cell-free extracts Use of the Vibrio harveyi luxab genes as a reporter system Reporter systems based on EGFP A reporter gene encoding a thermostable malate dehydrogenase Assay of thermostable malate dehydrogenase in cell-free extracts The ampc ( -lactamase) gene as a reporter The lac (secreted -galactosidase) gene of S. lividans as a reporter for transcription and secretion The redd gene of S. coelicolor as an easily scorable reporter of transcription in S. coelicolor and S. lividans Chapter 16 RNA methods General precautions when working with RNA Harvesting Streptomyces cultures for RNA isolation. 354 Isolation of RNA Isolation of RNA using modified Kirby mix, phenol/chloroform extraction and DNase I treatment Isolation of RNA using CsCl gradients Isolation of RNA using SDS and hot phenol Storage of RNA Assessing the quantity and quality of RNA preparations Spectrophotometry Agarose gel electrophoresis High resolution S1 nuclease mapping General strategies for making probes for high resolution S1 nuclease mapping Notes of caution in probe construction Specific activity of the probe How much probe to add to each hybridisation reaction High resolution S1 nuclease mapping of the 3 ends of transcripts Hybridisation solution The practicalities of high resolution S1 nuclease mapping Controls Generating sequencing ladders for high resolution S1 nuclease mapping Interpretation of results Low resolution S1 nuclease mapping Primer extension mapping Northern blotting In vitro transcription Streptomyces RNA polymerase purification Standard purification of Streptomyces RNA polymerase Purification of histidine-tagged RNA polymerase

6 Preparation of DNA-cellulose Chapter 17 Production and secretion of proteins by Streptomyces Transcription initiation Translation initiation Signal peptides Codon usage Regulated expression systems Culture conditions Levels of expression A selection of plasmids suitable for intracellular expression Plasmids suitable for secretion Optimising expression of Streptomyces genes in E. coli Changing the codon usage at the 5 end of a coding region E. coli vectors that have been used to overexpress streptomycete genes Chapter 18 Analysing Streptomyces DNA DNA sequencing Alternative and additional Maxam and Gilbert base-specific reactions for sequencing end-labelled DNA Sequence analysis Identifying protein coding regions FRAME analysis Codon preference Hidden Markov model Codon usage tables Accessing Streptomyces (actinomycete) sequences in the databases Design of oligonucleotides for use as probes and PCR primers PCR conditions Chapter 19 Media, buffers and suppliers Agar Media Minimal medium (MM) Complete medium (CM) Hickey-Tresner agar (HT agar) R2 Medium R2YE Medium R5 Medium Mannitol soya flour medium (MS) Supplemented minimal medium, solid (SMMS) 410 MMT Difco nutrient agar (DNA) Oxoid nutrient agar (ONA) Soft nutrient agar (SNA) L agar Liquid media Yeast extract-malt extract medium (YEME) Tryptone soya broth (TSB) Difco nutrient broth (DNB) L broth (LB) Supplemented liquid minimal medium (SMM). 413 Minimal liquid medium (NMMP) Labelling medium for Streptomyces X YT medium Growth factor supplements Buffers P (protoplast) Buffer T (transformation) buffer L (lysis) buffer TE Buffer SM Buffer SSC Addresses of suppliers Chapter 20 Genome maps and genetically marked strains S. coelicolor A3(2) Genetic/physical map Genetically marked strains Genome sequencing project S. lividans Genetic/physical map Genetically marked strains Genetic differences between S. coelicolor A3(2) and S. lividans S. griseus S. ambofaciens S. rimosus Plasmids SCP1 and SLP Chapter 21 Maps of DNA fragments Conventions used for the restriction maps List of restriction endonucleases, recognition sites and isoschizomers (Table) Lists of genes Alphabetical list of resistance and indicator genes, and other DNA fragments (Table) List of genes grouped according to function Resistance genes Counterselectable markers Indicator genes Other DNA fragments Resistance genes grouped according to antibiotic class Aminoglycosides Bialaphos, phosphinothricin Bleomycin, phleomycin Chloramphenicol

7 Gyrase inhibitors, novobiocin, ciprofloxacin 450 Hygromycin Macrotetrolides, nonactin, tetranactin MLS (macrolide, lincosamide and streptogramin B) resistance Puromycin Spectinomycin, streptomycin Streptothricins Tetracyclines Thiostrepton and analogues Viomycin, capreomycin B Other resistances Antibiotics, antimetabolites, and suppliers (Table) Maps of DNA fragments Resistance genes Counterselectable markers Indicator genes Other DNA fragments Integrating plasmids derived from C31 and phage VWB (Table) Plasmid maps grouped according to the Streptomyces replicon (Table) E. coli plasmids (Table) Transposable elements (Table) Restriction maps Phage C31 and its derivatives Integrating plasmids derived from C31 and phage VWB Plasmid maps grouped according to the Streptomyces replicon pij101 derivatives pres1 derivatives pjv1 derivatives psg5 derivatives SCP2* derivatives psam2 and SLP1 derivatives E. coli plasmids Transposable elements IS117 derivatives IS493 derivatives Tn4556 derivatives Chapter 22 Maps of plasmids, transposons and phage genomes Conventions used for the restriction maps Lists of all restriction maps Phage C31 and its derivatives (Table) Tn5 derivative Index with 3000 entries

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