Molecular microbial ecology /
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作 者：A. Mark Osborn (ed.) and Cindy J. Smith (ed.)
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ISBN：9781859962831
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简介
"Molecular Microbial Ecology gives a comprehensive guide to the recent revolution in the study of microorganisms in the environment. Details are given on molecular methods for isolating some of the previously uncultured and numerically dominant microbial groups. PCR-based approaches to studying prokaryotic systematics are described, including ribosomal RNA analysis and stable isotope probing. Later chapters cover DNA hybridization techniques (including fluorescent in situ hybridization), as well as genomic and metagenomic approaches to microbial ecology. Gathering together some of the world's leading experts, this book provides an invaluable introduction to the modern theory and molecular methods used in studying microbial ecology."--BOOK JACKET.
目录
Table Of Contents:
Contributors xi
Abbreviations xii
Preface xiii
1 Nucleic acid extraction from environmental samples 1(24)
Annett Milling, Newton C.M. Gomes, Miruna Oros-Sichler, Monika G枚tz and Kornelia Smalla
1.1 Introduction 1(1)
1.2 Recovery of cells from environmental matrices 2(1)
1.3 Cell lysis and DNA extraction protocols 3(2)
1.4 Immunochemical isolation of DNA from metabolically active cells 5(1)
1.5 RNA extraction from environmental matrices 6(5)
Protocol 1.1 11(6)
Protocol 1.2 17(4)
Protocol 1.3 21(4)
2 Prokaryotic systematics: PCR and sequence analysis of amplified 16S rRNA genes 25(40)
Wilfred F.M. R枚ling and Ian M. Head
2.1 Introduction 25(1)
2.2 Application of PCR in microbial ecology 26(8)
2.2.1 Good working practices in PCR: avoiding PCR inhibition and contamination 28(2)
2.2.2 Pitfalls of PCR: artifacts and differential amplification 30(2)
2.2.3 rRNA versus rDNA: indicative of active versus total microbial communities? 32(2)
2.3 Generation and analysis of clone libraries 34(4)
2.3.1 Generation of clone libraries 34(1)
2.3.2 Screening of clone libraries 35(3)
2.4 Quantitative analysis of clone libraries: coverage and diversity indices 38(3)
2.4.1 Coverage and sampling 38(2)
2.4.2 Diversity indices 40(1)
2.5 Phylogenetic inference 41(9)
2.5.1 Sequencing 41(1)
2.5.2 Phylogenetic analysis 42(2)
2.5.3 Parsimony 44(2)
2.5.4 Distance methods 46(1)
2.5.5 Maximum likelihood 47(3)
2.5.6 Bootstrap analysis 50(1)
2.6 Phylogenetic analysis compared to other molecular techniques 50(2)
2.6.1 Comparing large numbers of samples 51(1)
2.6.2 Quantification 51(1)
2.6.3 Relationship between phylogenetic information and function 52(1)
2.7 Concluding remarks 52(5)
Protocol 2.1 57(8)
3 DNA fingerprinting of microbial communities 65(32)
Andreas Felske and A. Mark Osborn
3.1 Introduction 65(9)
3.1.1 DGGE 66(2)
3.1.2 TGGE 68(1)
3.1.3 SSCP analysis 68(1)
3.1.4 T-RFLP analysis 69(1)
3.1.5 LH-PCR (length heterogeneity PCR) 70(1)
3.1.6 Comparison of the different methods 71(3)
3.2 Applications of microbial community fingerprinting 74(5)
3.2.1 Combination of cloning with fingerprinting 76(1)
3.2.2 Screening isolated strains next to fingerprints 76(1)
3.2.3 Southern blot hybridization and fingerprints 77(1)
3.2.4 Multiple competitor RT-PCR/TGGE for rRNA 78(1)
3.2.5 Environmental fingerprints with protein-coding genes 79(1)
3.3 Analyzing microbial community fingerprints 79(6)
3.3.1 Cutting out fingerprint bands for sequencing 80(1)
3.3.2 The rRNA sequence heterogeneity 81(1)
3.3.3 Quantitative analysis of fingerprint signals 82(2)
3.3.4 Cluster analysis of DNA fingerprint signals 84(1)
3.3.5 Diversity and species richness 84(1)
3.3.6 The 'operational taxonomic unit' 85(1)
3.4 Miscellaneous advice 85(6)
Protocol 3.1 91(6)
4 Molecular typing of environmental isolates 97(38)
Jan L.W. Rademaker, Henk J.M. Aarts and Pablo Vinuesa
4.1 Introduction 97(1)
4.1.1 Role of PCR amplification-based typing for microbial ecology 98(1)
4.2 Microbial typing methods 98(15)
4.2.1 AFLP analysis 99(4)
4.2.2 rep-PCR fingerprinting 103(3)
4.2.3 Mini- and microsatellite fingerprinting 106(1)
4.2.4 Fingerprinting methods using random primers (RAPD, AP-PCR, DAF) 106(2)
4.2.5 Ribosomal RNA gene fingerprinting methods 108(5)
4.3 Computer-assisted analysis of PCR-generated fingerprint profiles 113(1)
4.4 Concluding remarks 114(13)
Protocol 4.1 127(4)
Protocol 4.2 131(4)
5 RT-PCR and mRNA expression analysis of functional genes 135(16)
Balbina Nogales
5.1 Introduction 135(1)
5.2 Advantages and limitations of the RT-PCR analysis of bacterial functional genes 136(1)
5.3 The RT-PCR reaction 137(3)
5.3.1 The RNA template 137(1)
5.3.2 Primers for reverse transcription 138(1)
5.3.3 Reverse transcriptases 138(1)
5.3.4 One-step and two-step RT-PCR protocols 139(1)
5.4 Quantitative RT-PCR 140(1)
5.5 Analysis of global gene expression 140(1)
5.6 Conclusions 141(4)
Protocol 5.1 145(1)
Protocol 5.2 146(5)
6 Quantitative real-time PCR 151(16)
Cindy J. Smith
6.1 Introduction 151(1)
6.2 Quantification 152(2)
6.3 qPCR chemistries 154(2)
6.3.1 Taq nuclease assay 154(2)
6.3.2 SYBR green 156(1)
6.4 Applications 156(5)
6.4.1 qPCR analysis of total microbial communities via analysis of amplified ribosomal RNA genes 156(1)
6.4.2 Functional ecology 157(4)
Protocol 6.1 161(6)
7 Stable-isotope probing 167(12)
Stefan Radajewski and I. Colin Murrell
7.1 Introduction 167(1)
7.2 Stable-isotope labeling of DNA 168(1)
7.3 Application of stable-isotope probing 169(1)
7.3.1 Methylotroph populations 169(1)
7.3.2 Ammonia-oxidizing populations 170(1)
7.4 Future prospects 170(3)
Protocol 7.1 173(6)
8 Applications of nucleic acid hybridization in microbial ecology 179(34)
A. Mark Osborn, Vivien Prior and Konstantinos Damianakis
8.1 Introduction 179(1)
8.2 Fundamentals of DNA hybridization 180(6)
8.2.1 Probe design 180(4)
8.2.2 Choice of nucleic acid template 184(2)
8.3 Hybridization applications in microbial ecology 186(12)
8.3.1 Hybridization analysis of cultured bacteria 186(1)
8.3.2 Hybridization analysis of total community DNA 187(2)
8.3.3 Reverse sample genome probing (RSGP) and microarray analysis of microbial communities 189(9)
Protocol 8.1 198(9)
Protocol 8.2 207(4)
Protocol 8.3 211(2)
9 Fluorescence in situ hybridization for the detection of prokaryotes 213(28)
Holger Daims, Kilian Stoecker and Michael Wagner
9.1 Introduction 213(1)
9.2 Fundamentals of rRNA FISH 214(3)
9.3 Structure-function analyses: combination of FISH with other techniques 217(6)
Protocol 9.1 223(18)
10 Lessons from the genomes: microbial ecology and genomics 241(20)
Andrew S. Whiteley, Mike Manefield, Sarah L. Turner and Mark J. Bailey
10.1 Introduction 241(1)
10.2 Structural genomics 242(1)
10.3 Comparative genomics 242(4)
10.3.1 Small genomes 243(1)
10.3.2 Large genomes 244(1)
10.3.3 The horizontal gene pool 244(1)
10.3.4 Insights into phylogeny 245(1)
10.4 Functional genomics in microbial ecology 246(3)
10.4.1 Characterizing microbial responses to environmental change 246(1)
10.4.2 Relating specific genes to specific functions 247(1)
10.4.3 Expression of genes from components of a genome 248(1)
10.5 The application of genomic tools in situ 249(5)
10.5.1 Organism diversity 249(1)
10.5.2 Functional genes 250(1)
10.5.3 Metagenomics 250(2)
10.5.4 Functional gene arrays 252(1)
10.5.5 Diversity, function and process 253(1)
10.6 Lessons learned and future perspectives 254(7)
11 Metagenomic libraries from uncultured microorganisms 261(20)
Doreen E. Gillespie, Michelle R. Rondon, Lynn L. Williamson and Jo Handelsman
11.1 Introduction 261(1)
11.2 Factors affecting experimental design 261(4)
11.2.1 Environmental source 262(2)
11.2.2 DNA isolation 264(1)
11.2.3 Vector selection 264(1)
11.2.4 Host cell selection 264(1)
11.3 Summary 265(4)
Protocol 11.1 269(12)
12 A molecular toolbox for bacterial ecologists: PCR primers for functional gene analysis 281(22)
Michael J. Larkin, A. Mark Osborn and Derek Fairley
12.1 Introduction 281(2)
12.2 Adopting a strategy to detect sequences from the environment 283(2)
12.3 Primer and probe design 285(1)
12.4 PCR primers to amplify 16S rRNA gene sequences 286(1)
12.5 PCR primers for detecting functional genes 287(16)
13 Molecular detection of fungal communities in soil 303(18)
Eric Smit, Francisco de Souza and Renske Landeweert
13.1 Introduction 303(1)
13.2 Molecular strategies for fungal community analyses 304(1)
13.3 DNA extraction 305(1)
13.4 Target genes 306(1)
13.5 Primers for fungal populations 307(3)
13.6 In silico analysis of 18S rRNA primers 310(1)
13.7 Specific application of molecular identification techniques for studying mycorrhizal fungi 311(2)
13.7.1 Analyzing afbuscular mycorrhizal fungal diversity 311(1)
13.7.2 Analyzing ectomycorrhizal fungal diversity 312(1)
13.8 Conclusions 313(4)
Protocol 13.1 317(4)
14 Environmental assessment: bioreporter systems 321(24)
Steven A. Ripp and Gary S. Sayler
14.1 Introduction 321(1)
14.2 Reporter systems 322(8)
14.2.1 β-Galactosidase (lacZ) 322(1)
14.2.2 Chloramphenicol acetyltransferase (CAT) 322(1)
14.2.3 Catechol 2,3-dioxygenase (xylE) 322(1)
14.2.4 β-Lactamase (bla) 323(1)
14.2.5 β-Glucorinidase (gusA, gurA, uidA) 324(1)
14.2.6 Ice nucleation (inaZ) 324(1)
14.2.7 Green fluorescent protein (GFP) 324(2)
14.2.8 Aequorin 326(1)
14.2.9 Uroporphyrinogen (Urogen) III methyltransferase (UMT) 326(1)
14.2.10 Luciferases 326(4)
14.3 Mini-transposons as genetic tools in biosensor construction 330(13)
Case study 343(2)
15 Bioinformatics and web resources for the microbial ecologist 345(28)
Wolfgang Ludwig
15.1 Introduction 345(2)
15.2 Databases 347(12)
15.2.1 European Bioinformatics Institute (EBI) databases 347(11)
15.2.2 GenBank 358(1)
15.3 Genome projects 359(1)
15.3.1 Munich Information Center for Protein Sequences (MIPS) 359(1)
15.3.2 Sanger Institute 359(1)
15.3.3 The Institute for Genomic Research (TIGR) 359(1)
15.3.4 Mobile genetic elements (MGE) databases 360(1)
15.4 Ribosomal RNA databases 360(3)
15.4.1 Ribosomal Database Project 360(2)
15.4.2 European Ribosomal RNA database 362(1)
15.5 rRNA-targeted probes 363(1)
15.5.1 ProbeBase 363(1)
15.5.2 Roscoff database 363(1)
15.5.3 PRIMROSE 363(1)
15.6 ARB project 363(6)
15.6.1 ARB databases 364(2)
15.6.2 Phylogenetic treeing 366(3)
15.6.3 Future developments 369(1)
15.7 Concluding remarks 369(4)
Index 373
Contributors xi
Abbreviations xii
Preface xiii
1 Nucleic acid extraction from environmental samples 1(24)
Annett Milling, Newton C.M. Gomes, Miruna Oros-Sichler, Monika G枚tz and Kornelia Smalla
1.1 Introduction 1(1)
1.2 Recovery of cells from environmental matrices 2(1)
1.3 Cell lysis and DNA extraction protocols 3(2)
1.4 Immunochemical isolation of DNA from metabolically active cells 5(1)
1.5 RNA extraction from environmental matrices 6(5)
Protocol 1.1 11(6)
Protocol 1.2 17(4)
Protocol 1.3 21(4)
2 Prokaryotic systematics: PCR and sequence analysis of amplified 16S rRNA genes 25(40)
Wilfred F.M. R枚ling and Ian M. Head
2.1 Introduction 25(1)
2.2 Application of PCR in microbial ecology 26(8)
2.2.1 Good working practices in PCR: avoiding PCR inhibition and contamination 28(2)
2.2.2 Pitfalls of PCR: artifacts and differential amplification 30(2)
2.2.3 rRNA versus rDNA: indicative of active versus total microbial communities? 32(2)
2.3 Generation and analysis of clone libraries 34(4)
2.3.1 Generation of clone libraries 34(1)
2.3.2 Screening of clone libraries 35(3)
2.4 Quantitative analysis of clone libraries: coverage and diversity indices 38(3)
2.4.1 Coverage and sampling 38(2)
2.4.2 Diversity indices 40(1)
2.5 Phylogenetic inference 41(9)
2.5.1 Sequencing 41(1)
2.5.2 Phylogenetic analysis 42(2)
2.5.3 Parsimony 44(2)
2.5.4 Distance methods 46(1)
2.5.5 Maximum likelihood 47(3)
2.5.6 Bootstrap analysis 50(1)
2.6 Phylogenetic analysis compared to other molecular techniques 50(2)
2.6.1 Comparing large numbers of samples 51(1)
2.6.2 Quantification 51(1)
2.6.3 Relationship between phylogenetic information and function 52(1)
2.7 Concluding remarks 52(5)
Protocol 2.1 57(8)
3 DNA fingerprinting of microbial communities 65(32)
Andreas Felske and A. Mark Osborn
3.1 Introduction 65(9)
3.1.1 DGGE 66(2)
3.1.2 TGGE 68(1)
3.1.3 SSCP analysis 68(1)
3.1.4 T-RFLP analysis 69(1)
3.1.5 LH-PCR (length heterogeneity PCR) 70(1)
3.1.6 Comparison of the different methods 71(3)
3.2 Applications of microbial community fingerprinting 74(5)
3.2.1 Combination of cloning with fingerprinting 76(1)
3.2.2 Screening isolated strains next to fingerprints 76(1)
3.2.3 Southern blot hybridization and fingerprints 77(1)
3.2.4 Multiple competitor RT-PCR/TGGE for rRNA 78(1)
3.2.5 Environmental fingerprints with protein-coding genes 79(1)
3.3 Analyzing microbial community fingerprints 79(6)
3.3.1 Cutting out fingerprint bands for sequencing 80(1)
3.3.2 The rRNA sequence heterogeneity 81(1)
3.3.3 Quantitative analysis of fingerprint signals 82(2)
3.3.4 Cluster analysis of DNA fingerprint signals 84(1)
3.3.5 Diversity and species richness 84(1)
3.3.6 The 'operational taxonomic unit' 85(1)
3.4 Miscellaneous advice 85(6)
Protocol 3.1 91(6)
4 Molecular typing of environmental isolates 97(38)
Jan L.W. Rademaker, Henk J.M. Aarts and Pablo Vinuesa
4.1 Introduction 97(1)
4.1.1 Role of PCR amplification-based typing for microbial ecology 98(1)
4.2 Microbial typing methods 98(15)
4.2.1 AFLP analysis 99(4)
4.2.2 rep-PCR fingerprinting 103(3)
4.2.3 Mini- and microsatellite fingerprinting 106(1)
4.2.4 Fingerprinting methods using random primers (RAPD, AP-PCR, DAF) 106(2)
4.2.5 Ribosomal RNA gene fingerprinting methods 108(5)
4.3 Computer-assisted analysis of PCR-generated fingerprint profiles 113(1)
4.4 Concluding remarks 114(13)
Protocol 4.1 127(4)
Protocol 4.2 131(4)
5 RT-PCR and mRNA expression analysis of functional genes 135(16)
Balbina Nogales
5.1 Introduction 135(1)
5.2 Advantages and limitations of the RT-PCR analysis of bacterial functional genes 136(1)
5.3 The RT-PCR reaction 137(3)
5.3.1 The RNA template 137(1)
5.3.2 Primers for reverse transcription 138(1)
5.3.3 Reverse transcriptases 138(1)
5.3.4 One-step and two-step RT-PCR protocols 139(1)
5.4 Quantitative RT-PCR 140(1)
5.5 Analysis of global gene expression 140(1)
5.6 Conclusions 141(4)
Protocol 5.1 145(1)
Protocol 5.2 146(5)
6 Quantitative real-time PCR 151(16)
Cindy J. Smith
6.1 Introduction 151(1)
6.2 Quantification 152(2)
6.3 qPCR chemistries 154(2)
6.3.1 Taq nuclease assay 154(2)
6.3.2 SYBR green 156(1)
6.4 Applications 156(5)
6.4.1 qPCR analysis of total microbial communities via analysis of amplified ribosomal RNA genes 156(1)
6.4.2 Functional ecology 157(4)
Protocol 6.1 161(6)
7 Stable-isotope probing 167(12)
Stefan Radajewski and I. Colin Murrell
7.1 Introduction 167(1)
7.2 Stable-isotope labeling of DNA 168(1)
7.3 Application of stable-isotope probing 169(1)
7.3.1 Methylotroph populations 169(1)
7.3.2 Ammonia-oxidizing populations 170(1)
7.4 Future prospects 170(3)
Protocol 7.1 173(6)
8 Applications of nucleic acid hybridization in microbial ecology 179(34)
A. Mark Osborn, Vivien Prior and Konstantinos Damianakis
8.1 Introduction 179(1)
8.2 Fundamentals of DNA hybridization 180(6)
8.2.1 Probe design 180(4)
8.2.2 Choice of nucleic acid template 184(2)
8.3 Hybridization applications in microbial ecology 186(12)
8.3.1 Hybridization analysis of cultured bacteria 186(1)
8.3.2 Hybridization analysis of total community DNA 187(2)
8.3.3 Reverse sample genome probing (RSGP) and microarray analysis of microbial communities 189(9)
Protocol 8.1 198(9)
Protocol 8.2 207(4)
Protocol 8.3 211(2)
9 Fluorescence in situ hybridization for the detection of prokaryotes 213(28)
Holger Daims, Kilian Stoecker and Michael Wagner
9.1 Introduction 213(1)
9.2 Fundamentals of rRNA FISH 214(3)
9.3 Structure-function analyses: combination of FISH with other techniques 217(6)
Protocol 9.1 223(18)
10 Lessons from the genomes: microbial ecology and genomics 241(20)
Andrew S. Whiteley, Mike Manefield, Sarah L. Turner and Mark J. Bailey
10.1 Introduction 241(1)
10.2 Structural genomics 242(1)
10.3 Comparative genomics 242(4)
10.3.1 Small genomes 243(1)
10.3.2 Large genomes 244(1)
10.3.3 The horizontal gene pool 244(1)
10.3.4 Insights into phylogeny 245(1)
10.4 Functional genomics in microbial ecology 246(3)
10.4.1 Characterizing microbial responses to environmental change 246(1)
10.4.2 Relating specific genes to specific functions 247(1)
10.4.3 Expression of genes from components of a genome 248(1)
10.5 The application of genomic tools in situ 249(5)
10.5.1 Organism diversity 249(1)
10.5.2 Functional genes 250(1)
10.5.3 Metagenomics 250(2)
10.5.4 Functional gene arrays 252(1)
10.5.5 Diversity, function and process 253(1)
10.6 Lessons learned and future perspectives 254(7)
11 Metagenomic libraries from uncultured microorganisms 261(20)
Doreen E. Gillespie, Michelle R. Rondon, Lynn L. Williamson and Jo Handelsman
11.1 Introduction 261(1)
11.2 Factors affecting experimental design 261(4)
11.2.1 Environmental source 262(2)
11.2.2 DNA isolation 264(1)
11.2.3 Vector selection 264(1)
11.2.4 Host cell selection 264(1)
11.3 Summary 265(4)
Protocol 11.1 269(12)
12 A molecular toolbox for bacterial ecologists: PCR primers for functional gene analysis 281(22)
Michael J. Larkin, A. Mark Osborn and Derek Fairley
12.1 Introduction 281(2)
12.2 Adopting a strategy to detect sequences from the environment 283(2)
12.3 Primer and probe design 285(1)
12.4 PCR primers to amplify 16S rRNA gene sequences 286(1)
12.5 PCR primers for detecting functional genes 287(16)
13 Molecular detection of fungal communities in soil 303(18)
Eric Smit, Francisco de Souza and Renske Landeweert
13.1 Introduction 303(1)
13.2 Molecular strategies for fungal community analyses 304(1)
13.3 DNA extraction 305(1)
13.4 Target genes 306(1)
13.5 Primers for fungal populations 307(3)
13.6 In silico analysis of 18S rRNA primers 310(1)
13.7 Specific application of molecular identification techniques for studying mycorrhizal fungi 311(2)
13.7.1 Analyzing afbuscular mycorrhizal fungal diversity 311(1)
13.7.2 Analyzing ectomycorrhizal fungal diversity 312(1)
13.8 Conclusions 313(4)
Protocol 13.1 317(4)
14 Environmental assessment: bioreporter systems 321(24)
Steven A. Ripp and Gary S. Sayler
14.1 Introduction 321(1)
14.2 Reporter systems 322(8)
14.2.1 β-Galactosidase (lacZ) 322(1)
14.2.2 Chloramphenicol acetyltransferase (CAT) 322(1)
14.2.3 Catechol 2,3-dioxygenase (xylE) 322(1)
14.2.4 β-Lactamase (bla) 323(1)
14.2.5 β-Glucorinidase (gusA, gurA, uidA) 324(1)
14.2.6 Ice nucleation (inaZ) 324(1)
14.2.7 Green fluorescent protein (GFP) 324(2)
14.2.8 Aequorin 326(1)
14.2.9 Uroporphyrinogen (Urogen) III methyltransferase (UMT) 326(1)
14.2.10 Luciferases 326(4)
14.3 Mini-transposons as genetic tools in biosensor construction 330(13)
Case study 343(2)
15 Bioinformatics and web resources for the microbial ecologist 345(28)
Wolfgang Ludwig
15.1 Introduction 345(2)
15.2 Databases 347(12)
15.2.1 European Bioinformatics Institute (EBI) databases 347(11)
15.2.2 GenBank 358(1)
15.3 Genome projects 359(1)
15.3.1 Munich Information Center for Protein Sequences (MIPS) 359(1)
15.3.2 Sanger Institute 359(1)
15.3.3 The Institute for Genomic Research (TIGR) 359(1)
15.3.4 Mobile genetic elements (MGE) databases 360(1)
15.4 Ribosomal RNA databases 360(3)
15.4.1 Ribosomal Database Project 360(2)
15.4.2 European Ribosomal RNA database 362(1)
15.5 rRNA-targeted probes 363(1)
15.5.1 ProbeBase 363(1)
15.5.2 Roscoff database 363(1)
15.5.3 PRIMROSE 363(1)
15.6 ARB project 363(6)
15.6.1 ARB databases 364(2)
15.6.2 Phylogenetic treeing 366(3)
15.6.3 Future developments 369(1)
15.7 Concluding remarks 369(4)
Index 373
Molecular microbial ecology /
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