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Chapter Titles (Titles link to detailed contents)
1. General introduction to actinomycete biology
2. Growth and preservation of Streptomyces
4. Specialised biochemical techniques
5. Mutagenesis of Streptomyces by irradiation or chemicals
6. Transposon mutagenesis in Streptomyces
7. In vitro genetic analysis by conjugation and protoplast fusion
8. Preparation and analysis of genomic and plasmid DNA
9. General considerations about gene cloning in Streptomyces
10. Introduction of DNA into Streptomyces
11. Plasmids and their use for gene cloning
13. Cloning with phage vectors
14. Gene disruption and gene replacement
15. Reporter systems
16. RNA methods
17. Production and secretion of proteins by Streptomyces
18. Analysing Streptomyces DNA
19. Media, buffers and suppliers
20. Genome maps and genetically marked strains
22. Maps of plasmids, transposons and phage genomes
Index (3000 entries)
References Each chapter has a comprehensive list of references
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Detailed Contents
Chapter 1 General introduction to actinomycete biology
Taxonomy of Streptomyces
The genera of actinomycetes
The genus Streptomyces
Ecology of Streptomyces
Streptomycetes as pathogens
Some physiological features of primary metabolism in streptomycetes
Carbon sources
Nitrogen sources
Amino acid catabolism
Biosynthesis
Some physiological novelties
Antibiotic production by Streptomyces
Streptomycetes as antibiotic producers
Antibiosis in soil
Physiology and regulation of antibiotic production
Developmental biology of Streptomyces
The Streptomyces chromosome and its genetic elements
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
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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 1
Procedure 2
Preparation of aerial mycelium and spores separately
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
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Chapter 3 Microscopical methods
Light microscopy
Cellophane cultures
Coverslip or slide cultures
Impression preparations
Staining for light microscopy
DAPI Staining of Streptomyces mycelium
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
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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
In-vivo radioactive labelling of Streptomyces proteins
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
The S. coelicolor antibiotics
Actinorhodin
Undecylprodigiosin
CDA
Methylenomycin
S. lividans as an antibiotic producer
Secondary metabolite production in S. lividans transformants
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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
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Chapter 6 Transposon mutagenesis in Streptomyces
General points about transposon mutagenesis
Discovery of Streptomyces transposable elements
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 66
E. Sectoring method for delivering IS493 derivatives using temperature-sensitive plasmids
IS6100 from Mycobacterium fortuitum
F. Transposon mutagenesis of S. lividans using pSIT151
Tn5493 derived from Tn5
G. Mutagenising S. lividans using pJOE2577
H. Alternative method for isolating S. lividans with random insertions of Tn5493
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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
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
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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 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 pH8
Lithium chloride
Sodium chloride
Sodium hydroxide
Potassium acetate and sodium acetate, 3M
Tris-HCl buffer
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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?
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
Storing gene libraries
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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
5 end labelling of oligonucleotide probes
Using Whatman 540 paper
Use of the Polymerase Chain Reaction (PCR) to identify transformants
Conjugation from E. coli
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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
pIJ101
pJV1
pSG5
SCP2
Higher copy number derivatives of SCP2*
SCP2* vectors as delivery systems for gene disruptions
SLP1 and pSAM2
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
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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
Phage DNA
Uses of wild-type phages in the study of their hosts
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
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
Small-scale preparation of Streptomyces phage DNA
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Chapter 13 Cloning with phage vectors
General features of C31 and its vector derivatives
Shotgun cloning with C31 vectors
Choice of C31 vectors for mutational cloning
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
A single copy number promoter-probe vector, KC859
Vectors for in situ fusions of xylE to chromosomally 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"
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
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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 in-frame 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 replacemen
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
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Introduction
The problems with lacZ
Choice of vector: plasmid, phage and transposon systems
Antibiotic resistance genes as reporters: neo and cat
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
Detection of xylE expression in C31 plaques
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
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General precautions when working with RNA
Harvesting Streptomyces cultures for RNA isolation
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
Preparation of DNA-cellulose
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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 over-express streptomycete genes
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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
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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)
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)
Minimal liquid medium (NMMP)
Labelling medium for Streptomyces
2 X YT medium
Growth factor supplements
Buffers
P (protoplast) Buffer
T (transformation) buffer
L (lysis) buffer
TE Buffer
SM Buffer
120 × SSC
Addresses of suppliers
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Chapter 20 Genome maps and genetically marked strains
S. coelicolor A3(2)
Genetic/physical map
Genetically marked strains
Genome sequencing project
S. lividans 66
Genetic/physical map
Genetically marked strains
Genetic differences between S. coelicolor A3(2) and S. lividans 66
S. griseus
S. ambofaciens
S. rimosus
Plasmids SCP1 and SLP2
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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
Gyrase inhibitors, novobiocin, ciprofloxacin
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
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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)
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
Tn5 derivative
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