Table Of ContentJ. T. Trevors J. D. van Elsas (Eds.)
Nucleic Acids
in the Environment
With 33 Figures
Springer
JACK. T. TREVORS
Department of Environmental Biology
University of Guelph
Lab. Microbial Techniques
NlG 2Wl Guelph, Ontario
Canada
1. DICK van ELSAS
DLO-Institute for Soil Fertility Research
c/o IPO-DLO
Binnenhaven12
P.O. Box 9060
6700 GW Wageningen
The Netherlands
ISBN-13:97S-3-540-5S069-0 e-ISBN-13:97S-3-642-79050-S
DOl: 10.1007/ 97S-3-642-79050-S
Library of Congress Cataloging-in-Publication Data. Nucleic acids in the enviromnent 1 J.T. Trevors, J.D. van
Elsas, eds. p. cm. - (Springer laboratory) Includes bibliographical references.
ISBN-13:978-3-540-58069-0 I. Nucleic
acids-Analysis-Laboratory manuals. 2. Enviromnental chemistry-Laboratory manuals. I. Trevors, J.T.
(Jack T.), 1953-. n. Elsas, J.D. van (Jan D.), 1951-. III. Series. QP620.N798 1994 574.87'328-<1c 20 94
-47364
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Preface
The continuing development and use of extraction, purification,
detection, quantification, and amplification methods for nucleic acids
in environmental samples have a significant role in understanding
complex soil and aquatic environments. Of various environmental
microbiology techniques, extraction and amplification of nucleic
acids sequencs are of prime importance. These techniques, together
with the conventional isolation of viable microbial cells, provide an
excellent combination for microbiologists and environmental
scientists to use their research and teaching programs. Direct
extraction of nucleic acids eliminates the need to isolate viable cells
from environmental samples for subsequent extraction of nucleic
acids. The direct extraction of the sample can yield sufficient
amounts of specific nucleic acid sequences to be detected by
hybridization analysis or can be amplified using the powerful
technology of the polymerase chain reaction (PCR).
This text includes both the theory and methods for extraction,
purification, quantification, and amplification of nucleic acids from
complex environmental samples of soil, sediment, and aquatic and
plant material. Specific applications have been provided to allow the
information to be used in both research and teaching programs.
The editors sincerely thank the contributing authors for their
excellent chapters, cooperation, and suggestion. We also thank the
publishers and especially Dr. J. Lindenborn from Springer-Verlag for
their cooperation and assistance during the preperation of this volume
IT. Trevors
Guelph, Ontario, Canada
J.D. van Elsas
Wageningen, The Netherlands
Contents
1 Introduction to Nucleic Acids in the Environment: Methods and
Applications
1.T.Trevors and 1.D.van Elsas
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1
1.2 Cell Lysis, Nucleic Acid Extraction
and Purification Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Analysis of Environmental Nucleic Acids ............... 3
1.4 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
References ............................................. 5
2 Recovery of Bacterial Cells from Soil
L.R. Bakken and V. Lindahl
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Strategies for Cell Extraction ........................ 12
2.3 Recommended Methods ............................ 20
2.4 Practical Experiences and Recommendations ........... 23
References ............................................ 26
3 Extraction, Purification, and Analysis of DNA from Soil Bacteria
V. Torsvik, F. L. Daae and 1. Goksoyr
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 29
3.2 Bacterial DNA .................................... 30
3.3 Direct Extraction of DNA from Soil ................... 40
3.4 Purity Control and Characterization
of Soil Bacterial DNA .............................. 41
References ............................................ 46
4 Extraction and Analysis of Microbial DNA from Soil
A. Saano, E. Tas, S. Pip pol a, K. Lindstrom and J. D. van Elsas
4.1 Introduction ...................................... 49
4.2 DNA Extraction Methods ........................... 50
4.3 Evaluation of DNA Extraction Methods ............... 55
4.4 Methods for Analysis of DNA from Soil ............... 61
4.5 Concluding Remarks ............................... 65
References ............................................ 65
5 Extraction and Amplification of DNA from the Rhizosphere and
Rhizoplane of Plants
K. Leung, J. T. Trevors and J. D. van Elsas
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
5.2 Sample Preparation ................................ 71
5.3 Extraction, Purification and Quantification of DNA
from the Rhizosphere .............................. 72
5.4 PCR Analysis of Bacterial DNA from the Rhizosphere
and Rhizoplane of Plants ........................... 82
5.5 Concluding Remarks ............................... 85
References ............................................ 86
6 Extraction of DNA from the Phyllosphere
MJ. Bailey
6.1 Introduction ...................................... 89
6.2 Materials, Methods and Protocols .................... 94
6.3 Concluding Remarks .............. : ............... 106
References ........................................... 106
7 Specific DNA Sequences for Detection of Soil Bacteria
X. Nesme, C. Picard and P. Simonet
7.1 Introduction ..................................... III
7.2 Objectives and Limitations ......................... 112
7.3 Target DNA, DNA Extraction and Detection in Soil ..... 114
7.4 Detailed Procedures .............................. 122
7.5 Application of Methodology ........................ 129
References ........................................... 13 6
8 PCR Amplification of DNA from Root Nodules
1. L. Pepper and S.D. Pillai
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 141
8.2 Methods for Detection of Rhizobia .................. 141
8.3 Detection of Root Nodule Bacteria by PCR ............ 143
8.4 Conclusions ..................................... 149
References ........................................... 149
9 Extraction of DNA and RNA from Aquatic Environments
J. H. Paul and S. L. Pichard
9.1 Introduction ..................................... 153
9.2 DNA as a Biomass and Ecological Indicator ........... 153
9.3 RNA as a Biomass and Ecological Indicator ........... 155
9.4 Biomass and Activity Measurements of Aquatic
Microorganisms using RNA and RNA / DNA Ratios .... 156
9.5 Extraction of Nucleic Acids
for Molecular Biological Studies .................... 158
9.6 Molecular Methods for Studying RNA
in Aquatic Environments .......................... 163
9.7 Extraction of DNA from Aquatic Microbial Populations . 166
9.8 Extraction of RNA from Aquatic Microbial Populations . 171
References ........................................... 173
10 PCR Amplification of DNA Recovered from the Aquatic
Environment
A. K. Bej
10. 1 Introduction..................................... 179
10.2 Concentration of Water Samples
from Aquatic Envirolm1ents ........................ 182
10.3 Sample Preparation for PCR Amplification ............ 186
10.4 Processing of Dissolved DNA
in the Aquatic Environment ........................ 187
10.5 Processing of Nucleic Acids from Biofilms ., .......... 188
10.6 PCRAmplification ............................... 189
10.7 Identification and Analysis of the Amplified DNA ...... 193
10.8 PCR Contamination Control ........................ 197
10.9 Enhancing the Specificity of the PCR Reaction ........ 198
10.10 Use of Arbitrarily Primed PCR (Ap-PCR)
in Environmental Microbiology ..................... 198
10.11 In situ PCR Amplification ......................... 201
10.12 The Use ofPCR in Aquatic Environments ............. 204
10.13 Detection of Viruses in Water ...................... 211
10.14 Discussion .................. '" ...... " ......... 212
References ........................................... 213
11 Extraction and Amplification of 16S rRNA Genes from Deep Marine
Sediments and Seawater to Assess Bacterial Community Diversity
P. A. Rochelle, J. A. K. Will, J. C. Fry, G. J. S. Jenkins, R. J. Parkes,
C. M. Turley and A. J. Weightman
11.1 Introduction..................................... 219
11.2 DNA Extraction ................................. 220
11.3 Amplification of 16S Ribosomal RNA Gene Sequences .. 228
11.4 Cloning and Sequencing ........................... 233
11.5 Sequences Obtained from Marine Sediments
and Coastal Waters ............................... 235
References ........................................... 237
12 Application of the PCR for Detection of Antibiotic Resistance Genes
in Enviromnental Samples
K. Smalla and J. D.van Elsas
12.1 Introduction ..................................... 241
12.2 Selection ofPCR Primers and Gene Probes
for Specific Detection of sat and nptII Genes .......... 243
12.3 Detection of Antibiotic Resistance Genes
in Bacterial Isolates Using PCR ..................... 243
12.4 Detection of Antibiotic Resistance Genes Using DNA
Extraction and PCR .............................. 245
12.5 Detection of Antibiotic Resistance Genes in Total
COn1l11Wlity DNA Extracts from Enviromnental Habitats. 247
12.6 Conclusions ..................................... 252
References ........................................... 253
List of Contributors
Bailey, M. J. Lindahl, V.
Molecular Microbial Ecology, Department of Biotechnological Sciences
Natural Environment Research Council Agricultural University of Norway
Institute of Virology and Environmental PO Box 5040 N-1432 As Norway
Microbiology
Mansfield Road, Oxford, OXI 3SR, UK Lindstrom, K.
Department of Applied Chemistry and
Bakken, L. R. Microbiology, Division of Microbiology
Dept Soil and Water Sciences POB 27, FlN-00014 University of Helsinki,
POBox 5028 Finland
N-1432 Aas, Norway
Nesme, X.
Bej, A.K. Ecologie Microbienne du Sol,
Department of Biology URA CNRS 1450,
University of Alabama at Birmingham Universite Claude Benard Lyon 1
UAB Station, Birmingham, 43 bd du 11 novembre 1918
AL 35294-1170, USA 69622, Villeurbanne cedex, France
Daae, F. L. Parkes, R. J.
Department of Microbiology and Plant Department of Geology, University of
Physiology Bristol, Wills Memorial Building
University of Bergen, Jahnebakken 5 Queens Road, Bristol, UK, BS8 IRJ
N-5020 Bergen, Norway
Paul, J. H.
Fry, J. C. Department of Marine Science
School of Pure and Applied Biology, University of South Florida
University of Wales Cardiff 140 Seventh Ave. S.
P. o. Box 915, Cardiff, UK, CFI 3TL St. Petersburg, FL USA 33701
Goksoyr, J. Pepper, l. L.
Department of Microbiology and Plant Department of Soil and Water Science
Physiology 429 Shantz Building, University of Arizona
University of Bergen, Jahnebakken 5 Tucson, AZ, USA 85721
N-5020 Bergen, Norway
Picard, C.
Jenkins, G. J. S. Ecologie Microbienne du Sol,
School of Pure and Applied Biology, URA CNRS 1450
University of Wales Cardiff Universite Claude Benard Lyon 1
P. o. Box 915, Cardiff, UK, CFI 3TL 43 bd du II novembre 1918
69622, Villeurbanne cedex, France
Leung, K.
Department of Environmental Biology Pichard, S. L.
University of Guelph, Guelph, Ontario Department of Marine Science
CanadaNIG2Wl University of South Florida
140 Seventh Ave. S.
St. Petersburg, FL USA 33701
Pippo/a, S. Tas, E.
Department of Applied Chemistry and Department of Applied Chemistry and
Microbiology, Division of Microbiology Microbiology, Division of Microbiology
POB 27, FIN-00014 University of Helsinki, POB 27, FIN-OOO 14 University of Helsinki
Finland Finland
Pillai, S. D. Torsvik, V.
Agricultural Research and Extension Department of Microbiology and Plant
Center Physiology
The Texas A & M University System University of Bergen, Jahnebakken 5
1380 A & M Circle, EI Paso, Texas N-5020 Bergen, Norway
USA 79927
Trevors, J. T.
Rochelle, P. A. Department of Environmental Biology
School of Pure and Applied Biology, University of Guelph, Guelph, Ontario
University of Wales Cardiff Canada NI G 2Wl
P. O. Box 915, Cardiff, UK, CFl 3TL
van E/sas, J. D.
Saano, A. Institute for Soil Fertility Research
Department of Applied Chemistry and Binnenhaven 5
Microbiology, Division of Microbiology 6700 GW Wageningen
POB 27, FIN-00014 University of Helsinki, The Netherlands
Finland
Weightman, A. J.
Simonet, P. School of Pure and Applied Biology,
Ecologie Microbienne du Sol, University of Wales Cardiff
URACNRS 1450 P. O. Box 915, Cardiff, UK, CFl 3TL
Universite Claude Benard Lyon 1
43 bd du 11 novembre 1918 Will, J. A. K.
69622, Villeurbanne cedex, France School of Pure and Applied Biology,
University of Wales Cardiff
Smalla, K. P. O. Box 915, Cardiff, UK, CFl 3TL
Biologische Bundesanstalt, Inst. Biochem.
and Plant Viro!.
Messeweg 11112
W-3000 Braunschweig
Germany
1 Introduction to Nucleic Acids in the
Environment: Methods and Applications
IT.TREVORS AND lD.VAN ELSAS
1.1 Introduction
Presently and in the future, research in environmental microbiology, microbial
ecology, soil science and plant pathology will increasingly involve extraction,
purification, amplification, detection and molecular analysis of both
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from microorganisms,
plants and enviromnental samples such as soil, water and sediment. Most of the
current research in this field is being conducted by scientists interested in
developing and applying rapid, sensitive and reproducible methods to extract
and detect specific nucleic acid sequences in complex environmental samples.
Viable microbial cells can only be partially recovered from complex
enviromnental samples by traditional plating methods. This is due firstly to the
fact that classical cell extraction, e.g. from soils, only recovers a fraction, i.e.,
34% (Holben et al. 1988), of the cells present. Secondly, a large fraction, often
up to 90%-99%, of microbial cells present in environmental samples is not
culturable and will therefore escape detection if cultivation-based methods are
applied. Since a large percentage of such microbial cells in environmental
samples may be viable, yet are not culturable, their detection via molecular
techniques becomes important. Until about a decade ago, the only application of
molecular detection techniques was in hybridization analysis of isolated colonies
(Sayler et al. 1985); however, it was soon realized that extractive techniques
based on cellular "fingerprint" macromolecules such as nucleic acids were
needed in order to study the non-culturable microbial majority populations in the
enviromnent.
Such techniques, pioneered by Torsvik and Goksoyr (1978) and Torsvik
(1980), provided an excellent foundation for future methods of DNA extraction
from sediment (Ogram et al. 1987) and sediment and soil (Holben et al. 1988;
Steffan et al. 1988).
The further development and application of nucleic acid-based techniques in
the enviromnental sciences received thrust from the biosafety issues of
genetically engineered microorganisms (GEMs) in the late 1980s. Sensitive
detection techniques were thought to be essential in assessing putative risks of
GEMs (Sussman et al. 1988). The techniques now available also are of great
