Molecular biological methods for Bacillus:
Saved in:
| Format: | Book |
|---|---|
| Language: | English |
| Published: |
Chichester u.a.
Wiley
1990
|
| Series: | Modern microbiological methods
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XXXV, 581 S. graph. Darst. |
| ISBN: | 0471923931 |
Staff View
MARC
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| 245 | 1 | 0 | |a Molecular biological methods for Bacillus |c ed. by Colin R. Harwood ... with contributions by R. Chambert ... |
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| 700 | 1 | |a Harwood, Colin R. |e Sonstige |4 oth | |
| 700 | 1 | |a Chambert, Régis |e Sonstige |4 oth | |
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Record in the Search Index
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| adam_text |
^Molecular Biological Methods
for Bacillus
Edited by
Colin R Harwood
Department of Microbiology
University of Newcastle upon Tyne, UK
and
Simon M Cutting
The Biological Laboratories
Harvard University, USA
•--- -with contributions by
R Chambert, A Galizzi, D Gaily, A D Gruss, P Haima,
J, A Hobot, S Holsappel, L Janniere, J Kok, G R Newman,
L Oskam, A Palva, M -F Petit-Glatron, F G Priest, W Quax,
R Sammons, J Vehmaanpera, M Young
A Wiley-Interscience Publication
JOHN WILEY amp; SONS
Chichester • New York • Brisbane • Toronto • Singapore
Contents
List of Methods by Chapter
Contributors
Series Preface
Preface
Acknowledgements
Warning Note
Abbreviations
Chapter 1: Growth, Maintenance and General Techniques
C R Harwood and A R Archibald
1 1 Introduction
1 2 The genus Bacillus
121 Bacillus subtilis
122 Strains of Bacillus subtilis
1 3 Growth of Bacillus
131 Growth temperature
132 Growth media
133 Growth in batch culture
134 Growth in continuous culture
1 4 Maintenance and shipping of strains
141 Glycerol cultures
142 Freeze-dried (lyophilised) cultures
143 Spores
144 Shipping strains
1 5 Cell permeablisation and breakage
151 Permeablisation of whole cells with toluene
152 Sonication
153 Mechanical breakage
154 Enzymic lysis
1 6 Radiolabelling of proteins
References
Methods - Chapter 1
xvii
xxiii
xxvii
xxix
xxxi
xxxii
xxxiii 9
! a
19 «
23 i
vi Contents
Chapter 2: Genetic Analysis
S M Cutting and P B VanderHorn
2 1 Introduction
2 2 Mutagenesis
221 Choice of mutagen
222 NTG mutagenesis
223 Mutagenesis by UV irradiation
224 Expression of the mutant phenotype
225 Localized mutagenesis using NTG
226 Mutagenesis in vitro
227 Introduction of in urtro-generated mutations back into
the chromosome
228 Purification of mutants
2 3 Transformation
231 Preparation of chromosomal DNA
232 Two-step transformation procedure
233 One-step transformation procedure
234 DNA concentrations
235 Congression
2 4 PBS1 Generalized transduction
241 Preparation of transducing lysates
242 Transduction with PBS1
243 Titration of PBS1
244 Isolation of motile host bacteria
2 5 Protoplast techniques
2 6 Selection and screening of recombinants
261 Prototrophic recombinants
262 Sporulation markers
263 Drug-resistance markers
2 7 Genetic mapping
271 The B subtilis mapping kit
272 Two-factor and three-factor crosses
273 Recombination indices
274 Mapping with integrational plasmids
2 8 Complementation analysis
281 Complementation at the amyE locus
282 Complementation analysis using SP/3
References
Methods - Chapter 2
Chapter 3: Plasmids
S Bron
3 1 Plasmids used in Bacillus
311 Introduction
58 a
61 '
75
Contents vii
312 Rolling-circle replication and structure of small Gram-
positive plasmids
313 Plasmids from Staphylococcus aureus
314 High-copy-number plasmid from Corynebacterium
xerosis
315 Small plasmids from Lactococcus lactis
316 Plasmids from Bacillus
317 Plasmids not replicating via single-stranded
intermediates
318E colilB subtilis shuttle plasmids
319 Integrational plasmids
3 1 10 Conclusions
Isolation of plasmid DNA from Bacillus
321 Specific problems with bacilli
322 Mini-scale purification of plasmid DNA
323 Large-scale purification of plasmid DNA
324 Plasmid plus chromosomal DNA from whole-cell
minilysates
325 Determination of plasmid DNA concentrations
Plasmid DNA transfer systems for Bacillus species
331 General
332 Transformation of competent cells
333 Transformation of protoplasts
334 Transformation by electroporation
(electrotransformation)
335 Transduction
336 Conjugation
337 Concluding remarks on plasmid DNA transfer systems
Plasmid replication in Bacillus
341 General
342 Identification of ssDNA plasmids
343 Analysis of replication
344 Determination of plasmid copy numbers
345 Replication of Gram-positive plasmids in vitro
Plasmid instability in B subtilis
351 Introduction
352 Methods for analysing segregational (in)stability
353 Methods for analysing structural (in)stability
354 Suggestions for improving plasmid stability
Efficient cloning systems for B subtilis based on pTA1060 and
pAM/Jl
361 General
362 Direct clone selection in B subtilis using /?-
galactosidase a-complementation
viii Contents
363 Marker rescue system for direct done selection and
transformation with plasmid monomers
364 Cloning in pAM/?l-derived vectors
References
Methods-Chapter 3
Chapter 4: Gene Cloning Techniques
/ Errington
4 1 Introduction
4 2 Cloning vectors
421 Phage vectors
422 Autonomous plasmid vectors
423 Integrative plasmid vectors
4 3 Cloning systems
431 Direct transfection
432 Prophage transformation
433 Cloning using integrative plasmids
434 Recovery of DNA adjacent to integrated plasmids or
transposons
435 Screening Bacillus genomic libraries made in E coli
4 4 Spedal-purpose cloning vectors
441 Promoter probe vectors
442 Expression vectors
4 5 Special-purpose hosts
451 Host strains cured of prophages
452 Restriction-defident hosts
453 Recombination-defident hosts
454 Protease-deficient hosts
References
Methods - Chapter 4
130 °
139 •
204 «
209 °
Chapter 5: Use of Transposons and Integrational Vectors for
Mutagenesis and Construction of Gene Fusions in
Bacillus Species 221
P Youngman
5 1 Introduction 221
5 2 Insertional mutagenesis with Tn92 7 221
521 The pTVl family of selection vectors 222
522A new generation of highly temperature-sensitive
selection vectors based on pE194Ts 222
523 Screening a library for insertional mutants 226
524 Making use of existing insertion libraries 227
5 3 Insertional mutagenesis with integrational vectors 228
Contents ix
531 The prindple of mutagenesis by integrational gene
disruption
532 Adapting integrational gene disruption to Bacillus
spedes that lack natural transformation competence
Using transposons or integrational vectors to tag spedfied
regions of the chromosome
541 Obtaining a Tn92 7 insertion linked by transformation
to a selectable marker
542 Targeting specified chromosomal regions for
mutagenesis
543 Obtaining chemically-induced mutations at random
locations linked to transposon insertions
Cloning of chromosomal sequences adjacent to transposon
insertions or plasmid integrations
551 Rescuing sequences within or adjacent to genes
identified by integrational vector-mediated
mutagenesis
552 Rescuing sequences adjacent to existing Tn92 7
insertions
553 Use of transposition-proficient derivatives of Tn927
that carry E coli cloning vectors
Construction of lacZ fusions using transposons, integrational
vectors and temperate phages
561 Construction of lacZ fusions by transposition
562 Constructing lacZ fusions from transposon insertions
by homologous recombination
563 Recombinational substitution of fusion-assodated
sequences
564 Construction of lacZ fusions using integrational vectors
565 Integration of lacZ fusions into temperate phage SP/J
566 Use of MUG as a plate indicator for lacZ fusion activity
567 Use of MUG for quantisation of /J-galactosidase
activity in liquid culture
Further information concerning vectors, strains and protocols
References
Methods - Chapter 5
Chapter 6: Measuring Gene Expression in Bacillus
C P Moran
Introduction
Transcription products
621 Isolation of RNA
622 Primer extension analysis of RNA transcripts
256 »
Contents
623 SI nudease analysis 271
624 Northern blots 272
625 Pulse-labeling RNA 272
6 3 Translation products 273
631 Western blots 273
632 Minicell analysis of plasmid-encoded proteins 273
6 4 Transcription in vitro 274
641 Phase-partitioning procedure for isolation of RNA
polymerase 274
642 Heparin-agarose chromatography 276
643 High-pressure liquid chromatography 276
644 In vitro transcription assays 276
References 277
Methods-Chapter 6 279
Chapter 7: DNA Repair and DNA Replication in Bacillus subtilis 295
R E Yasbin, W Firshein, J Laffan and R G Wake
7 1 Introduction 295
7 2 DNA repair mechanisms 295
721 Photoreactivation 296
722 Nucleotide excision repair (NER) 296
723 Base exdsion repair (BER) 296
724 Adaptive response 297
725 Postreplication repair 297
726 Translesion DNA synthesis 298
7 3 DNA repair in Bacillus 299
731 Bacterial strains and growth conditions 300
732 Ultraviolet (UV) irradiation 302
733 UV mutagenesis 302
734 Exposure of cells to chemical agents 303
735 Bacteriophage lysates 303
736 Weigle reactivation 304
737 Prophage induction 304
738 Phenotypic testing 305
7 4 DNA replication 305
741 Bacterial strains and growth conditions used to study
DNA replication in B subtilis 306
742 Extraction and purification of a DNA-membrane
complex 306
743 Storage conditions for the DNA-membrane complex 307
744 DNA synthesis 307
745 Identification, isolation and analysis of DNA
replication forks in B subtilis 311
Contents xi
746 Identification of replication forks in restriction enzyme
digests of B subtilis DNA 312
747 Isolation of forked DNA molecules 313
748 Breakdown of forked DNA molecules 313
749 The osmolysate extraction procedure for minimizing
the breakdown of forked DNA molecules 315
7 4 10 Analysis of forked DNA molecules 315
References 318
Methods - Chapter 7 321
Chapter 8: The Bacillus Cell Envelope and Secretion 327
C R Harwood, R D Coxon and I C Hancock
8 1 Structure and function of the cell envelope 327
811 The protoplast membrane 328
812 The Bacillus cell wall 330
8 2 Synthesis and maturation of the Bacillus cell wall 337
821 Peptidoglycan 338
822 Teichoic add 338
823 Teichuronic add 339
824 Assay of synthesis and assembly of cell wall polymers
in permeablised cells 339
825 Coordinated cell wall assembly and maturation 339
8 3 Protein secretion 341
831 Proteins secreted from Bacillus 342
832 Assay of polysaccharide-hydrolysing enzymes 344
833 Assaying for protease activity 346
834 Radiolabelling Bacillus exoproteins 347
835 Analysis of Bacillus exoproteins by SDS-PAGE 348
836 Translocation and processing of Bacillus exoenzymes
during secretion 348
837 The physicochemical influence of cell wall on protein
export 350
8 4 Electron microscopy for bacterial cells 352
841 Modern developments in sample preparation 352
842 Efficacy of the acrylic system 353
843 Choice of embedding technique 354
8 5 Electron microscope methodologies 354
851 Cell harvesting, preparation and fixation 354
852 Complete dehydration protocol with resin infiltration 355
853 Partial dehydration protocol with resin infiltration 356
854 Resin polymerisation 357
855 Sectioning/staining of L R White sections 358
856 Immunolabelling studies 358
xii Contents
857 Low-temperature methods 358
858 Practical approach 361
References 362
Methods-Chapter 8 371
Chapter 9: Sporulation, Germination and Outgrowth 391
W L Nicholson and P Setlow
9 1 Introduction 391
9 2 Induction of sporulation 393
921 Solid media 393
922 Liquid media 394
9 3 Sporulation-specific marker events 399
931 Assay of stage-O-specific enzymes 400
932 Assay of a stage-II-specific enzyme 401
933 Assay of a stage-III-specific enzyme 401
934 Assay of a stage-V-spedfic gene product 401
935 Determination of spore resistance properties 401
9 4 Isolation and analysis of small, acid-soluble spore proteins
(SASPs) 402
9 5 Extraction and analysis of spore coat proteins 405
951 Isolation of spore coat proteins 407
952 SDS-PAGE of spore coat proteins 408
9 6 Sequential gene expression 408
961 General procedure 409
962 Assay of/7-galactosidase activity in spores 410
963 Potential problems 410
9 7 Compartmentalization 411
971 Determination of compartment-spedfic expression by
transformation of a Spo~ strain 412
972 Forespore isolation 412
973 Differential extraction of mother cell and forespore 413
974 Other approaches to determine compartmentalization
of gene expression 413
9 8 Purification of spores 414
981 Spore purification with water washing 415
982 Spore purification by lysozyme treatment and salt and
detergent washes 415
983 Release of mature spores from sporangia by using a
French press 416
984 Spore purification on urografin gradients 416
985 Potential problems 416
9 9 Storage of purified spores 417
991 Storage of lyophilized spores 417
Contents xiii
992 Storage of spores in water 417
993 Potential problems 417
9 10 Isolation and characterization of germination mutants 418
9 10 1 Germination media 419
9 10 2 Isolation of germination mutants 421
9 10 3 Preparation of spores for germination studies 422
9 10 4 Activation of spores for germination 423
9 10 5 Characterization of germination mutants 423
9 10 6 Detection of germination at low germinant
concentrations 423
9 11 Isolation of spore outgrowth mutants 424
9 11 1 Mutagenesis and sporulation 425
9 11 2 Penicillin enrichment of outgrowth mutants 425
9 11 3 Density gradient enrichment of outgrowth mutants 425
9 12 Isolation of sporulation mutants 426
References 426 o
Methods-Chapter 9 431 o
Chapter 10: Bacteriophages 451
H E Hemphill
10 1 Introduction 451
10 1 1 Generalized transducing phages 451
10 1 2 Spedalized transdudng phages 452
10 1 3 Cloning vectors 452
10 1 4 Physiological studies of phage development 453
10 1 5 Defective phages 453
10 2 Host bacteria for growing B subtilis phages 453
10 2 1 Considerations in choosing host bacteria 453
10 2 2 Preferred hosts 454
10 3 Media for culturing and maintaining B subtilis phages 455
10 3 1 Complex nutritional media for propagation of
I bacteriophages 455
\ 10 3 2 Synthetic media for propagation of bacteriophages 456
j 10 3 3 Antibiotic-containing media 456
! 10 3 4 Cation requirements for adsorption of phage to
I bacteria 457
| 10 4 Culturing and maintaining bacteriophage stocks 457
10 4 1 Recovering lytic phages after shipment 457
10 4 2 Recovering lysogens 458
: 10 4 3 Lysates of temperate phages 459
- 10 4 4 Storage of bacteriophage lysates 459
10 4 5 Preservation of phage in B subtilis spores 460
10 5 Titering B subtilis phages 461
xiv Contents
10 5 1 Preparation of media 461
10 5 2 Phage dilutions 462
10 5 3 Plating bacteriophages 462
10 5 4 Alternative plating procedure 463
10 5 5 Incubation 463
10 6 Batch culturing of bacteriophages 463
10 6 1 Growing lytic bacteriophages 463
10 6 2 Growing temperate phages 464
10 6 3 Notes on indudng lysogens 464
10 7 Lysogens of temperate B subtilis bacteriophages 465
10 7 1 Construction of new lysogens 465
10 7 2 Problems in identifying lysogenic bacteria 465
10 7 3 Construction of phages containing transposon Tn927 466
10 7 4 Selection of new lysogens of temperate phages
containing Tn92 7 466
10 7 5 Suggestions for construction of SP/?::Tn927 lysogens 467
10 7 6 Selection of lysogens of 03T and pll onthymine
auxotrophs 467
10 7 7 Curing lysogenic bacteria of prophages 468
10 7 8 Inactivation of prophages 469
10 8 Phage isolation and purification 470
10 9* Isolation of bacteriophage DNA 471
10 10 Bacteriophage mutants 472
10 10 1 Heat-sensitive and suppressible mutants 472
10 10 2 Potential problems in obtaining mutants of temperate
phages 473
10 10 3 Deletion mutants 473
10 11 Radioactive labelling of phage proteins and nucleic adds 474
10 11 1 Radioactive labelling of nucleic adds during phage
infection 476
10 11 2 Radioactive labelling of phage proteins 476
10 12 Metabolic activity in phage-infected cells 477
References 477
Methods - Chapter 10 479
Chapter 11: Genetic and Physical Maps of Bacillus subtilis 168 493
P J Piggot, M Amjad, J -] Wu, H Sandoval and J Castro
11 1 The genetic map 493
11 2 The physical map 533
References 540
Methods-Chapter 11 541
Appendix 1: Media 545
Al l Nutrient media 545
Contents xv
Al 1 1 BHIB (brain heart infusion broth) 545
Al 1 2 BST (Bacillus stearothermovhilus) medium 545
Al 1 3 GM1/GM2 media 545
Al 14J medium 546
Al 1 5 LB (Luria-Bertani) medium/agar 546
Al 16M medium/agar 546
Al 1 7 Modified M broth/agar (MMB/MMA) 547
Al 1 8 MB medium/agar 547
Al 1 9 Nutrient broth/nutrient agar 547
Al 1 10 PAB(Penassay broth) 547
Al 1 11 SMM + CAS 547
Al 1 12 TBAB 547
A1 2 Chemically defined growth media (CDGM) and supplements 548
Al 2 1 M9 minimal medium 548
Al 2 2 SMS/SMM (Spizizeh's minimal salts/medium) 548
Al 2 3 Trace elements solution 549
A1 3 Spedal-purpose media 549
Al 3 1 Schaeffer's sporulation medium/agar 549
Al 3 2 Non-carbon repressing medium 550
Al 3 3 Phosphate starvation medium 550
Appendix 2: Buffers/Solutions 551
A2 1 Chloroform/isoamyl alcohol 551
A2 2 Denhardfs solution 551
A2 3 Phenol 551
A2 4 Phenol/chloroform 551
A2 5 SSC (saline sodium dtrate) 552
A2 6 TBE (electrophoresis buffer) 552
A2 7 TE buffer 552
A2 8 TSE buffer 552
Appendix 3: The Bacillus Genetic Stock Center 553
D R: Zeigler
A3 1 History and organization of the BGSC 553
A3 2 The BGSC collection 554
A3 3 BGSC services 554
A3 4 Computerization of BGSC strain data 555
Appendix 4: Protein-Coding Genes: DNA Sequence Database and
Codon Usage 557
P M Sharp, D G Higgins, D C Shields and K M Devine
A4 1 Database availability 557
A4 2 Contents of Table A4 1 558
A4 2 1 Gene/ORF and map 558
xvi Contents
A4 2 2 CAI (codon adaptation index) 558
A4 2 3 AA (amino adds), product/function and molecular
mass 565
A4 2 4 Reference 565
A4 2 5 Ace No (accession number) 565
A4 3 Codon usage 566
A4 3 1 Codon usage bias and gene expression 566
A4 3 2 Base composition in reading frames 568
References 569
Index 571 |
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| record_format | marc |
| series2 | Modern microbiological methods |
| spelling | Molecular biological methods for Bacillus ed. by Colin R. Harwood ... with contributions by R. Chambert ... Chichester u.a. Wiley 1990 XXXV, 581 S. graph. Darst. txt rdacontent n rdamedia nc rdacarrier Modern microbiological methods Molekularbiologie (DE-588)4039983-7 gnd rswk-swf Bacillus (DE-588)4143852-8 gnd rswk-swf (DE-588)4143413-4 Aufsatzsammlung gnd-content Bacillus (DE-588)4143852-8 s Molekularbiologie (DE-588)4039983-7 s DE-604 Harwood, Colin R. Sonstige oth Chambert, Régis Sonstige oth HEBIS Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=002904097&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Molecular biological methods for Bacillus Molekularbiologie (DE-588)4039983-7 gnd Bacillus (DE-588)4143852-8 gnd |
| subject_GND | (DE-588)4039983-7 (DE-588)4143852-8 (DE-588)4143413-4 |
| title | Molecular biological methods for Bacillus |
| title_auth | Molecular biological methods for Bacillus |
| title_exact_search | Molecular biological methods for Bacillus |
| title_full | Molecular biological methods for Bacillus ed. by Colin R. Harwood ... with contributions by R. Chambert ... |
| title_fullStr | Molecular biological methods for Bacillus ed. by Colin R. Harwood ... with contributions by R. Chambert ... |
| title_full_unstemmed | Molecular biological methods for Bacillus ed. by Colin R. Harwood ... with contributions by R. Chambert ... |
| title_short | Molecular biological methods for Bacillus |
| title_sort | molecular biological methods for bacillus |
| topic | Molekularbiologie (DE-588)4039983-7 gnd Bacillus (DE-588)4143852-8 gnd |
| topic_facet | Molekularbiologie Bacillus Aufsatzsammlung |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=002904097&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT harwoodcolinr molecularbiologicalmethodsforbacillus AT chambertregis molecularbiologicalmethodsforbacillus |