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MOLECULAR MICROBIAL ECOLOGY MANUAL MOLECULAR MICROBIAL ECOLOGY MANUAL Edited by ANTOON D.L. AKKERMANS Department of Microbiology, Wageningen Agricultural University, The Netherlands JAN DIRK VAN ELSAS IPO-DLO, Wageningen, The Netherlands and FRANS J. DE BRUIJN MSU-DOE Plant Research Lab, NSF Center for Microbial Ecology and Department of Microbiology, Michigan State University, U.S.A. Springer-Science+Business Media, B.V. Library of Congress Cata1oging-in-Pub1ication Data Molecular licrc^^al ecology manual / edited by Antoon D.L. Akkermans, Jan Dirk V/an Elsas, and Frans J. De Bruijn. p . cm . ISBN 978-94-010-4156-0 ISBN 978-94-011-0351-0 (eBook) DOI 10.1007/978-94-011-0351-0 1. Molecular microbiology. 2. Microbial ecology. I. Akkermans, A. D. L. II. Elsas, J. D. van (Jan D.), 1951- III. De Bruijn, F. J. (Frans J. ) QR74.M64 1995b 576—dc20 95-31285 ISBN 978-94-010-4156-0 Neither Kluwer Academic Publishers nor any person acting on its behalf is responsible for the use which might be made of the information contained herein. Printed on acid-free paper All Rights Reserved © 1995 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 1995 Softcover reprint of the hardcover 1st edition 1995 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, or by any information storage and retrieval system, without written permission from the copyright owners. v Contents Preface SECTION 1: Isolation of microbial nucleic acids 1.1. Extraction of microbial DNA from aquatic sources 1.1.2. Extraction of microbial DNA from aquatic sources: Freshwater Roger W. Pickup, Glenn Rhodes and Jon R. Saunders 1.1.3. Extraction of microbial DNA from sewage and manure slurries K. Smalla 1.1.4. Methods for extracting DNA from microbial mats and cultivated micro-organisms: high molecular weight DNA from French press lysis Mary M. Bateson and David M. Ward 1.2. Extraction of microhial RNA from aquatic sources 1.2.3. Methods for extracting RNA or ribosomes from microbial mats and cultivated microorganisms David M. Ward, Alyson L. Ruff-Roberts and Roland Weller 1.3. Extraction ofmicrohial DNA from hulk soil 1.3.1. Cell extraction method Vigdis Torsvik 1.3.3. Extraction of microbial community DNA from soils J. D. van Elsas and K. Small a VI 1.3.4. Small scale extraction of DNA from soil with spun column cleanup Aimo Saano and Kristina Lindstrom 1.3.5. Gel purification of soil DNA extracts David D. Myrold, Kendall J. Martin and Nancy J. Ritchie 1.4. Extraction of DNA from phytosphere, rhizosphere, rhizoplane 1.4.2. Extraction and PCR amplification of DNA from the rhizoplane Penny A. Bramwell, Rita V. Barallon, Hilary J. Rogers and Mark J. Bailey 1.4.3. Extraction of microbial DNA from the phylloplane Penny A. Bramwell, Rita V. Barallon, Hilary J. Rogers and Mark J. Bailey 1.5. Extraction of RNA from bulk soil 1.5.1. Direct and simultaneous extraction of DNA and RNA from soil Sonja Selenska-Pobell 1.6. Nucleic acid extraction from cultures 1.6.2. Extraction of ribosomal RNA from microbial cultures Erko Stackebrandt and Naomi Ward SECTION 2: Detection of microbial nucleic acid sequences 2.2. Preparation of radioactive probes 2.2.1. Preparation of radioactive probes Martin Cunningham 2.3. Preparation of non-radioactive probes 2.3.1. Detection of nucleic acids by chemiluminescence Martin Cunningham, Bronwen Harvey and Martin Harris 2.6. Detection of microbial DNA sequences by colony hybridization 2.6.1. Detection of microbial DNA sequences by colony hybridization Penny R. Hirsch Vll 2.7. Detection and quantifIcation of microbial nucleic acid seque Polymerase Chain Reaction (PCR) 2.7.2. Polymerase chain reaction (PCR) analysis of soil microbial DNA J.D. van Elsas and A. Wolters 2.7.5. Detection of mRNA and rRNA via reverse transcription and PCR in soil Sonja Selenska-Pobell SECTION 3: Identification and classification of microbes usmg DNA and RNA sequences 3.1. Partial and complete 16S rDNA sequences, their use in generation oj" 16S rDNA phylogenetic trees and their implications in molecular eco logical studies 3.1.1. Partial and complete 16S rONA sequences, their use in generation of 16S rONA phylogenetic trees and their implications in molecular eco logical studies Erko Stackebrandt and Fred A. Rainey 3.3. Microbial identij"ication and design oj"phylogenetic trees based 3.3.1. Amplification of ribosomal RNA sequences Richard Devereux and Stephanie G. Willis 3.3.2 Bacterial community fingerprinting of amplified 16S and 16-23S ribosomal DNA gene sequences and restriction endonuclease analysis (ARDRA) Arturo A. Massol-Deya, David A. Odelson, Robert F. Hickey and James M. Tiedje 3.3.4. Investigation of fungal phylogeny on the basis of small ribosomal subunit RNA sequences Yves van de Peer and Rupert de Wachter 3.3.5. Sequence Databases Wolfgang Ludwig 3.3.6. In situ identification of micro-organisms by whole cell hybridization with rRNA-targeted nucleic acid probes Rudolf I. Amann Vlll SECTION 4: Detection, identification and classification of microbes using other methods 4.1. Detection, identification and classification of microbes using other methods 4.1.3. Immunofluorescence colony-staining (IFC) J.W.L. van Vuurde and J.M. van der Wolf 4.1.8. Fluorescent staining of microbes for total direct counts Jaap Bloem 4.1.9. Slide immunoenzymatic assay (SIA) Everly Conway de Macario and Alberto J.L. Macario SECTION 5: Detection of gene transfer in the environment 5.1. Gene transfer by transformation 5.1.1. Natural transformation in aquatic environments John H. Paul and Haydn G. Williams 5.2. Gene tramier by conjugation 5.2.3. Detection of gene transfer in the environment: Conjugation in soil Eric Smit and Jan Dirk van Elsas 5.3. Gene transfer hy transduction 5.3.2. Phage ecology and genetic exchange in soil Paul R. Herron SECTION 6: Tracking of specific microbes in the environment 6.1. Marker genes 6.1. 7. Heavy metal resistances in microbial ecosystems M. Mergeay 6.1.8. Biodegradation genes as marker genes in microbial ecosystems Bruce M. Applegate, Udayakumar Matrubutham, John Sanseverino and Gary S. Sayler lX 6.2. Designing field and microcosm experiments with GEM's 6.2.1. Design of microcosms to provide data reflectin field trials of GEMS Mary A. Hood and R.J. Seidler 6.2.3. Designing release experiments with GEM's in foods: Risk assessment of the use of genetically modified Lactococcus lactis strains in fermented milk products; a case study Nicolette Klijn, Anton H. Weerkamp and W.M. de Vos Xl Preface For a long time microbial ecology has been developed as a distinct field with in Ecology. In spite of the important role of microorganisms in the environ ment, this group of 'invisible' organisms remained unaccessable to other ecologists. Detection and identification of microorganisms remain largely dependent on isolation techniques and characterisation of pure cultures. We now realise that only a minor fraction of the microbial community can be cultivated. As a result of the introduction of molecular methods, microbes can now be detected and identified at the DNA/RNA level in their natural environment. This has opened a new field in ecology: Molecular Microbial Ecology. In the present manual we aim to introduce the microbial ecologist to a selected number of current molecular techniques that are relevant in micro bial ecology. The first edition of the manual contains 33 chapters and an equal number of additional chapters will be added this year. Since the field of molecular ecology is in a continuous progress, we aim to update and extend the Manual regularly and will invite anyone to deposit their new protocols in full detail in the next edition of this Manual. We hope this book finds its place where it was born: at the lab bench! Antoon D.L. Akkermans, Jan Dirk van Elsas and Frans J. de Bruijn March 1995 MolcClilur Microhiul Ecologl' Manual 1.1.2: I-II. 19'15 (" 19\)5 Klul\'(,f Academic Puht;shers. Printed in the Nethf'rland, Extraction of microbial DNA from aquatic sources: Freshwater ROGER W. PICKUpl, GLENN RHODES I and JON R. SAUNDERS2 J Institute o{ Freslll\"ater Ecology, Windermere Laboratory, The Ferry House, Far SanTe!', Ambleside, Cumbria, LA220LP, UK; ] Department of Genetics and Microbiology, Unil'ersitl' of'Liverpool, Merseyside, L69 3BX, UK Introduction Although this chapter is principally concerned with DNA extraction other considerations related to sampling and sample preparation are discussed. Freshwater lake environment Most deep lakes in temperate regions experience a seasonal cycle of thermal (or density) stratification and destratification and this is a major factor in controlling bacterial activity [8, 9]. Throughout winter and into spring the water column is isothermal and well oxygenated. Increased solar radiation raises the temperature of the surface water and the related changes in density give rise to distinct zones. The surface water (the epilimnion) is warm and well mixed whilst the deep water (the hypolimnion) remains cool. These two layers are separated by a water mass (the metalimnion) which is characterized by steep temperature gradients. The temperature difference, and associated changes in density, ensure that mixing between the epilimnion and hypolimnion is minimal and the latter can be treated essentially as a closed system [8]. The air-water interface has been recognized as an important and active environment which after application of specialized sampling procedures can be treated like other water samples. The surface sediment layers in contact with these zoned water masses exhibit similar temperature cycles and can be broadly defined as shallow (littoral) or deep (profundal) deposits. A lake can be more or less productive depending on its trophic status (a descriptive term for the nutrient loading of the system with eutrophic and oligotrophic as the extremes). Its status has two components, the cultural (enrichment from the catchment and man's activities) and the morphometric (the volume/area relationship of the lake basin). Generally a large deep lake is less likely to be eutrophic than a shallow lake simply due to the dilution of incoming nutrients and the greater volume of MMEM-1.1.2/1