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Molecular Approaches to Soil, Rhizosphere and

Plant Microorganism Analysis

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Molecular Approaches to Soil,

Rhizosphere and
Plant Microorganism Analysis

Edited by

J.E. Cooper and J.R. Rao

Department of Applied Plant Science, Queen's University Belfast,

Belfast, UK

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CABI is a trading name of CAB International

CABI Head Office CABI North American Office

Nosworthy Way 875 Massachusetts Avenue
Wallingford 7th Floor
Oxfordshire OX10 8DE Cambridge, MA 02139
UK USA

Tel: +44 (0)1491 832111 Tel: +1 617 395 4056

Fax: +44 (0)1491 833508 Fax: +1 617 354 6875
E-mail: [email protected] E-mail: [email protected]
Website: www.cabi.org

© CAB International 2006. All rights reserved. No part of this publication

may be reproduced in any form or by any means, electronically,
mechanically, by photocopying, recording or otherwise, without the
prior permission of the copyright owners.

A catalogue record for this book is available from the British Library,
London, UK.

A catalogue record for this book is available from the Library of Congress,
Washington, DC.

ISBN-10: 1 84593 062 2

ISBN-13: 978 1 84593 0622

Typeset by AMA DataSet Ltd, UK.

Printed and bound in the UK by Biddles Ltd, King’s Lynn.

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Contents

Contributors vii

Preface ix

1 Genomic Analyses of Microbial Processes in Biogeochemical Cycles 1

Shilpi Sharma, Heidrun Karl and Michael Schloter

2 Applications of Nucleic Acid Microarrays in Soil Microbial Ecology 18

Alexander Loy, Michael W. Taylor, Levente Bodrossy and Michael Wagner

3 Metagenomics for the Study of Soil Microbial Communities 42

Helen L. Steele and Wolfgang R. Streit

4 In Vivo Expression Technology (IVET) for Studying Niche-specific Gene

Expression by Plant- and Soil-colonizing Bacteria 55
Hans Rediers and René De Mot

5 Analysing Microbial Community Structure by Means of

Terminal Restriction Fragment Length Polymorphism (T-RFLP) 84
Christopher B. Blackwood

6 Characterization of Phylloplane and Rhizosphere Microbial

Populations Using PCR and Denaturing Gradient Gel Electrophoresis (DGGE) 99
Maureen O’Callaghan, Nicola Lorenz and Emily L. Gerard

7 Molecular Tools for Studying Plant Growth-promoting Rhizobacteria (PGPR) 116

Ashley Franks, Robert P. Ryan, Abdelhamid Abbas, G. Louise Mark and
Fergal O’Gara

8 Detection of Autotrophic Sulphur- and Iron-oxidizing Bacteria Using

Labelled Fatty Acid Methyl Esters (FAMEs) 132
André Lipski

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vi Contents

9 Molecular Analyses of Soil Denitrifying Bacteria 146

Laurent Philippot and Sara Hallin

10 Ecology of Streptomyces in Soil and Rhizosphere 166

Janice L. Strap and Don L. Crawford

11 Molecular Ecology of Ectomycorrhizal Fungal Communities: New Frontiers 183

Ian C. Anderson

12 Molecular Ecology of Arbuscular Mycorrhizal Fungi: a Review of

PCR-based Techniques 198
Dirk Redecker

13 Transcriptomics for Determining Gene Expression in Symbiotic

Root–Fungus Interactions 213
Philipp Franken and Franziska Krajinski

14 Differentiation of Nitrogen-fixing Legume Root Nodule Bacteria (Rhizobia) 236

Kristina Lindström, Paula Kokko-Gonzales, Zewdu Terefework and
Leena A. Räsänen

15 Molecular Markers for Studying the Ecology of Rhizobia 259

Angela Sessitsch

16 Molecular Characterization of Bacterial Plant Pathogens 272

Scott A. Godfrey and Robert W. Jackson

Index 293

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Contributors

Abdelhamid Abbas, The Biomerit Research Centre, Department of Microbiology, National

University of Ireland (UCC), Cork, Ireland.
Ian C. Anderson, The Macaulay Institute, Craigiebuckler, Aberdeen AB15 8QH, Scotland,
UK.
Christopher, B. Blackwood, Department of Biological Sciences, Kent State University, Kent
OH 44242, USA
Levente Bodrossy, Department of Bioresources/Microbiology, ARC Seibersdorf research
GmbH, A-2444 Seibersdorf, Austria.
J.E. Cooper, Department of Applied Plant Science, Queen’s University Belfast, Newforge
Lane, Belfast BT9 5PX, Northern Ireland, UK
Don L. Crawford, Director, Environmental Science Program, 216 Morrill Hall, University of
Idaho, Moscow, Idaho 83844 3006, USA.
René De Mot, Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven,
Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium.
Philipp Franken, Institute for Vegetables and Ornamental Crops, Theodor-Echtermeyer-
Weg, D-14979 Grossbeeren, Germany.
Ashley Franks, The Biomerit Research Centre, Department of Microbiology, National
University of Ireland (UCC), Cork, Ireland.
Emily M. Gerard, AgResearch, Biocontrol and Biosecurity Group, PO Box 60, Lincoln,
Canterbury, New Zealand.
Scott A. Godfrey, School of Biological and Molecular Sciences, Oxford Brookes University,
Oxford OX3 0BP, UK.
Sara Hallin, Department of Microbiology, Swedish University of Agricultural Sciences,
Box 7025, SE 750 07 Uppsala, Sweden.
Robert W. Jackson, Department of Biology and Biochemistry, University of Bath, Bath BA2
7AY, UK.
Heidrun Karl, Institute of Soil Ecology, GSF-National Research Center for Environment
and Health, PO Box 1129, D-85764 Neuherberg, Germany.
Paula Kokko-Gonzales, Department of Applied Chemistry and Microbiology, Biocenter 1,
PO Box 56, FIN-00014 University of Helsinki, Finland.
Franziska Krajinski, Department of Molecular Genetics, University of Hannover,
Herrenhäuser Strasse 2, D-30419 Hannover, Germany.

vii

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viii Contributors

Kristina Lindström, Department of Applied Chemistry and Microbiology, Biocenter 1,

PO Box 56, FIN-00014 University of Helsinki, Finland.
André Lipski, Universität Osnabrück, Abteilung Mikrobiologie, Fachbereich Biologie/
Chemie, D-49069 Osnabrück, Germany.
Nicola Lorenz, The Ohio State University, School of Environment and Natural Resources,
2021 Coffey Road, Columbus, OH 43210, USA.
Alexander Loy, Department of Microbial Ecology, University of Vienna, A-1090 Vienna,
Austria.
G. Louise Mark, The Biomerit Research Centre, Department of Microbiology, National
University of Ireland (UCC), Cork, Ireland.
Maureen O’Callaghan, AgResearch, Biocontrol and Biosecurity Group, PO Box 60, Lincoln,
Canterbury, New Zealand.
Fergal O’Gara, The Biomerit Research Centre, Department of Microbiology, National
University of Ireland (UCC), Cork, Ireland.
Laurent Philippot, UMR Microbiologie et Géochimie des Sols, INRA-Université de
Bourgogne, CMSE, 17, rue Sully, B.V. 86510, 21065 Dijon Cedex, France.
J.R. Rao, Department of Applied Plant Science, Queen’s University Belfast, Newforge Lane,
Belfast BT9 5PX, Northern Ireland, UK
Leena A. Räsänen, Department of Applied Chemistry and Microbiology, Biocenter 1,
PO Box 56, FIN-00014 University of Helsinki, Finland.
Dirk Redecker, Institute of Botany, University of Basel, Hebelstrasse 11, 4056 Basel,
Switzerland.
Hans Rediers, Hogeschool voor Wetenschap & Kunst – De Nayer Instituut, Jan De
Nayerlaan 5, B-2860 Sint-Katelijne-Waver, Belgium.
Robert P. Ryan, The Biomerit Research Centre, Department of Microbiology, National
University of Ireland (UCC), Cork, Ireland.
Michael Schloter, Institute of Soil Ecology, GSF-National Research Center for Environment
and Health, PO Box 1129, D-85764 Neuherberg, Germany.
Angela Sessitsch, ARC Seibersdorf research GmbH, Department of Bioresources, A-2444
Seibersdorf, Austria.
Shilpi Sharma, Institute of Soil Ecology, GSF-National Research Center for Environment
and Health, PO Box 1129, D-85764 Neuherberg, Germany.
Helen L. Steele, Molecular Enzyme Technology, Biofilm Centre, Universität Duisburg-
Essen, Lotharstrasse 1, D-47057 Duisburg, Germany.
Janice L. Strap, Environmental Biotechnology Institute, Food Research Center 103,
University of Idaho, Moscow, Idaho 83844 1052, USA.
Wolfgang R. Streit, Molecular Enzyme Technology, Biofilm Centre, Universität
Duisburg-Essen, Lotharstrasse 1, D-47057 Duisburg, Germany.
Michael W. Taylor, Department of Microbial Ecology, University of Vienna, A-1090
Vienna, Austria.
Zewdu Terefework, Department of Applied Chemistry and Microbiology, Biocenter 1,
PO Box 56, FIN-00014 University of Helsinki, Finland.
Michael Wagner, Department of Microbial Ecology, University of Vienna, A-1090 Vienna,
Austria.

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Preface

The profusion of molecular techniques now available to microbial ecologists allows pre-
viously unobtainable information to be gathered on the microflora of soils, plants and their
rhizospheres. Most of these methods target nucleic acids and a high proportion are in some
way dependent on the polymerase chain reaction (PCR). A researcher embarking on a new
project is faced with the problem of choosing the most appropriate techniques from what
appears to be a bewildering range of options. It is the purpose of this book to provide
critiques, written by internationally recognized experts, of many of the molecular
techniques that are currently employed for studying soil and plant microorganisms at the
community, population, taxonomic and functional group levels. In so doing, it supplies the
critical and comparative bases upon which an informed choice of technique can be made.
The book is not intended as a bench manual but technical information on, for example,
PCR primer sequences and experimental protocols is to be found in a number of chapters
and the extensive reference lists will lead the reader to relevant articles for each topic.

J.E. Cooper
J.R. Rao
Belfast

ix

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1 Genomic Analyses of Microbial Processes

in Biogeochemical Cycles

Shilpi Sharma,* Heidrun Karl and Michael Schloter

Institute of Soil Ecology, GSF-National Research Center for Environment and Health,
PO Box 1129, D-85764 Neuherberg, Germany

Introduction Even though the amount of nitrogen in

the atmosphere is > 70%, it cannot be used
Without the cycling of elements, continua- directly by the majority of life forms. Bac-
tion of life on earth would be impossible, teria are the only organisms capable of the
since essential nutrients would rapidly be biological fixation of nitrogen. Fixed nitro-
taken up by organisms and locked in a form gen may be obtained through the death and
that cannot be used by others. The reactions lysis of free-living nitrogen-fixing bacteria.
involved in elemental cycling are often Nitrogen-fixing bacteria, however, frequently
chemical in nature, but biochemical reac- form close associations with plants and, in
tions also play an important role. Microbes the case of rhizobia, can supply the plant
are of prime significance in this process. with all of its fixed nitrogen demands. In
Microbial and biochemical characteristics return, they receive a supply of organic car-
are used as potential indicators of soil qual- bon compounds. Fixation aside, cycling of
ity because of their central role in cycling of nitrogen involves interconversions among
carbon and nitrogen and their sensitivity to inorganic nitrogen compounds as well as
change (Nannipieri et al., 2003). conversions from inorganic to organic nitro-
In the cycling of carbon, photosynthetic gen, and vice versa. Many bacteria reduce
plants and microbes are the primary pro- nitrates to nitrites and some further reduce
ducers of organic carbon compounds and nitrites to ammonia. Ammonium salts may
these provide nutrients for other organisms, then be incorporated into organic polymers
which act as consumers of organic carbon by the process of assimilatory nitrate reduc-
and break down organic material in the pro- tion. Nitrates may be used by some bacteria
cesses of respiration and fermentation. Cer- instead of oxygen for a type of respiration
tain bacteria are also capable of anaerobic referred to as dissimilatory nitrate reduc-
carbon cycling. Methane can itself act as tion. Bacteria of the genus Pseudomonas,
a carbon and energy source for methane- micrococci and Thiobacillus species can
oxidizing bacteria, which can generate reduce nitrates to liberate nitrogen gas into
sugars and amino acids from methane found the environment. Bacteria that can generate
in their environments, again contributing nitrogen gas from the reduction of nitrates
to the cycling of carbon compounds. are commonly found in organically rich

*Corresponding author; Phone: +49 89 3187 3054, Fax: +49 89 3187 3376, E-mail: [email protected]
©CAB International 2006. Molecular Approaches to Soil, Rhizosphere and
Plant Microorganism Analysis (Cooper and Rao) 1

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2 S. Sharma et al.

soils, compost heaps and in sewage treat- strengthens this view. ANAMMOX is a
ment plants. For the continued cycling of relatively newly revealed process (Jetten
nitrogen, organic nitrogen compounds must et al., 1997; Strous et al., 1997) in which
be broken down to release ammonia. Putre- nitrite and ammonia combine to produce
factive metabolism yields considerable quan- dinitrogen gas.
tities of ammonia from biopolymers that
contain nitrogen. Bacteria may also produce
urease, an enzyme that breaks down urea to
liberate carbon dioxide, water and ammo- Approaches to the Study of Microbial
nia. Nitrifying bacteria are responsible for Processes in Biogeochemical Cycles
the biological oxidation of ammonia to
nitrate. Non-genomic methodologies
The carbon and nitrogen cycles are two
of the most important cycles responsible for Microbial processes contributing to bio-
nutrient processing in soil. In addition, soil geochemical cycles are regulated at various
is the site for numerous other activities which steps as outlined in Fig. 1.1. Studies analys-
also finally contribute to nutrient turnover. ing the role of microbes in biogeochemical
Biodegradation of xenobiotics within the cycles can be performed at various levels.
complex soil ecosystem is mostly the result Some parameters that have been used to
of proto-cooperation, i.e. the combined, determine microbiological activity have
mutually beneficial activities of various been heat output, nitrogen mineralization,
types of organisms having different abilities thymidine incorporation, nitrification rate,
at the same time and site. leucine incorporation and potential denitri-
Additionally, microorganisms have a fication activity (Alef and Nannipieri,
role in the biochemical transformation of 1995). The determination of microbial car-
metal ions. Bacteria such as Thiobacillus bon, nitrogen, phosphorus and sulphur con-
ferrooxidans and iron bacteria of the genus tents by fumigation techniques has allowed
Gallionella are capable of oxidizing ferrous a better quantification of nutrient dynamics
(Fe2+) iron into ferric (Fe3+) iron. Many bac- in soil. Conventional methods, such as the
teria can reduce ferric iron to its ferrous most probable number (MPN) and plate-
state by using it as an electron acceptor counting techniques (using specially
instead of O2. Bacteria are also important in formulated media), were designed to deter-
the transformation of manganese ions, where mine the total numbers and/or species of
reactions similar to those seen with iron are microorganisms in a particular soil. How-
observed (Heritage et al., 1999). Microbial ever, they provide only limited information
cycling of heavy metals is especially impor- regarding the functional diversity of the
tant in contaminated sites. One of the microbial community. Apart from the dis-
resistance or detoxification mechanisms of advantage that such methods are laborious,
mercury is enzymatic reduction of Hg2+ to we are now aware that only 1–10% of micro-
Hg0. This occurs in both Gram-negative organisms are culturable.
and Gram-positive aerobic bacteria from a Other approaches include generation of
variety of natural and clinical environments patterns of organic substrate utilization by
across the globe, and as such has become the soil microbial communities, the two most
the best studied mechanisms of mercury commonly used methods being the BIOLOG®
resistance. plate method (Garland and Mills, 1991; Zak
These are a few examples from the known et al., 1994) and the in situ substrate-
wide array of processes contributing to bio- induced respiration (SIR) technique (Degens
geochemical cycles. However, it needs to and Harris, 1997). The latter has the advant-
be mentioned that there might be various age of assessing catabolic diversity without
cycle components of which we are still extracting and cultivating organisms from
unaware. The discovery of anaerobic the soil. However, these methods too suf-
ammonia oxidation (ANAMMOX) further fer from the following limitations: (i) the

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Genomic Analyses of Microbial Processes in Biogeochemical Cycles 3

Microbial structure/
gene pool

Regulation by environmental factors,

repression, co-repression and induction

Transcript pool

Biogeochemical
cycles Regulation by environmental factors, feedback
inhibition, RNA interference and riboswitches

Enzyme pool

Regulation by feedback inhibition,

modification and effector molecules

Microbial function/
turnover processes

Regulation by environmental factors

Fig. 1.1. Relationship between microbial community structure and function contributing to
biogeochemical cycles.

inoculum density and incubation time are in the breakdown of organic matter. Urease,
arbitrary; and (ii) the substrate utilization phosphatase and arylsulphatase play import-
profiles obtained do not reflect the nature or ant roles in the mineralization of nitrogen,
structure of the original microbial commu- phosphorus and sulphur compounds. They
nities from which the samples were taken may give indications of the soil’s potential
due to the regulation at every step as out- to perform specific biochemical reactions,
lined in Fig. 1.1. and are important contributors to soil ferti-
Soil enzyme activities have been sug- lity. Enzyme assays, however, do not allow
gested as suitable indicators of soil quality any identification of the microbial species
as they are a measure of microbial activity directly involved in the measured pro-
and they are therefore strictly related to nutri- cesses. Moreover, stable extracellular enzyme
ent cycles and transformations. Moreover, activities are associated with soil colloids
as claimed by several authors (van Beelen and persist even in harsh environments that
and Doelman, 1997; Trasar-Cepeda et al., would limit intracellular activity. Thus,
2000), changes in soil enzyme activities may only strictly intracellular enzyme activities
be regarded as early and sensitive indicators can truly reflect microbial activity (Fig. 1.1).
of the impact of natural and anthropogenic Unfortunately, the presently available assays
factors on ecosystems. Enzyme activities do not distinguish the contribution of intra-
related to the cycling of the biologically cellular from extracellular and stabilized
important nutrients carbon, nitrogen, phos- enzyme activities, and thus they do not
phorus and sulphur have been investigated give valid information on the distribution
in soils. Soil dehydrogenase reflects the soil and comparative importance of reactions
oxidative power. Being present in all micro- mediated by microbes (Nannipieri et al.,
organisms, it may give a measure of the 2002). Active autotrophic bacteria, such as
total viable microbial cells. Invertase and those involved in sulphur and iron cycling,
β-glucosidase catalyse hydrolytic processes can be directly characterized in microbial

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4 S. Sharma et al.

communities by labelling their fatty acid potential sources of biological resources for
methyl esters with [13C]bicarbonate (FAME exploration and application. However, the
analysis; see Lipski, Chapter 8 this volume). determination of the composition of micro-
bial communities in soil is not in itself
sufficient for understanding nutrient trans-
Genomic technologies formations. A holistic approach, based on
the division of the systems into pools and
The latest and most powerful technologies the measurement of fluxes linking these
revolutionizing microbiology are based on pools, is comparatively more efficient. Some
genomics – the mapping and sequencing of of the genes most commonly targeted to
genomes and analysis of gene and genome study processes in biogeochemical cycles
function. These provide a connecting link have been listed in Table 1.1.
between biochemical measurements and
enzyme analysis. Moreover, the entire func-
tional potential of a system can be over-
viewed by analysing its genomics. Methods for Detection and
In general, genomic techniques have Quantification of Gene Expression in
some significant advantages over traditional Biogeochemical Cycles
methods: (i) only very small samples (in the
range of millilitres up to a litre) are required Nucleic acids can be used as markers in a
for most analyses; (ii) the sensitivity of number of different approaches. Selection
many methods is very high, thus enabling of the most appropriate one depends on the
the researcher to detect even single specific desired level of resolution of results. So a
cells among thousands of others; (iii) dead researcher needs to evaluate their benefits
or non-culturable cells can be analysed; and and limitations critically before deciding on
(iv) species-specific data (such as sequences) any one method. In many cases, a combina-
can be obtained without the need to culture tion of several techniques can be well suited
or even isolate a species. to answer the desired questions. Various
However, we emphasize that all these genomic approaches employed in studying
advantages are relative and depend on the microbial processes in biogeochemical cycles
researcher’s requirements, e.g. free DNA from have been summarized in Table 1.2. In the
dead cells could overinterpret the commu- following sections, specific examples are
nity structure. Genomic techniques based cited in some detail to inform the selection
on the polymerase chain reaction (PCR) of appropriate techniques and to evaluate
have several drawbacks. Amplification by the results generated by them.
PCR can be inhibited if contaminants are
not removed by the purification process,
and preferential or selective amplification
in the presence of DNA from mixed commu- PCR and reverse transcription–
nities can occur. Another bias of these PCR (RT–PCR)
techniques is the production of chimeric
or heteroduplex DNA molecules. Despite Early molecular studies used gene probe
these biases, genomic analyses still prove to technology to screen for the presence or
be the fastest and most reliable in extracting absence of structural genes in a soil popula-
information from complex environments. tion (Felske et al., 1996; Griffiths et al.,
More than 100 microbial genome 2000). The detection of DNA sequences
sequences have been completed, providing directly from the soil community has proven
information on > 300,000 predicted genes, to be a useful tool to improve our under-
with approximately half of these being of standing of soil processes. Another approach
unknown function and potentially novel. for studying specific microbial activities is
These novel genes represent exciting to investigate the expression of genes by
new opportunities for future research and analysing mRNA transcripts via their reverse

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Genomic Analyses of Microbial Processes in Biogeochemical Cycles 5

Table 1.1. Functional targets of microbial processes in nutrient cycling.

Target genes Process/nutrient cycle Reference

alkB Alkane hydroxylation Tesar et al., 2002; Whyte et al., 2002

amoA Ammonification Wu et al., 2001; Avrahami et al., 2002;
Radajewski et al., 2002
cbbL Autotrophic CO2 fixation Selesi et al., 2005
cbh1 Cellobiohydrolase Lamar et al., 1995; Vallim et al., 1998
chi Chitinase gene Williamson et al., 2000; Metcalfe et al., 2002
dsrAB Dissimilatory (bi) sulfite reduction Loy et al., 2002
lipA, lipC Lignin peroxidation Lamar et al., 1995; Stuardo et al., 2004
mcrA Methyl coenzyme M reduction Ritchie et al., 1997; Castro et al., 2004
mer Mercury resistance Bruce, 1997
mmoX Methane monooxygenase Ritchie et al., 1997; Morris et al., 2002
mnp Manganese peroxidase Bogan et al., 1996
mxaF Methanol dehydrogenation McDonald and Murrell, 1997; Morris et al., 2002;
Radajewski et al., 2002
narG Nitrate reduction Philippot et al., 2002; Deiglmayr et al., 2004
nifH Nitrogen fixation Widmer et al., 1999; Poly et al., 2001
nirK Nitrite reduction Avrahami et al., 2002; Prieme et al., 2002;
Throbäck et al., 2004; Sharma et al., 2005
nirS Nitrite reduction Wu et al., 2001; Prieme et al., 2002;
Rösch et al., 2002; Throbäck et al., 2004
nosZ Nitrous oxide reduction Rösch et al., 2002; Throbäck et al., 2004
pmoA Methane monooxygenase Kolb et al., 2003; Horz et al., 2005

transcription to complementary DNA (cDNA) studies and identification and characteriza-

and its amplification by PCR (RT–PCR). The tion of the microbial members.
usefulness of transcription analysis is based
on the short half-life of mRNA, which is
Fingerprinting techniques
sometimes in the order of minutes (Alifano
et al., 1994). Regulation at the transcription Fingerprinting techniques allow a researcher
level almost immediately affects the rate of to screen rapidly for similarities and differ-
protein synthesis, and detection of gene ences between samples. They are useful
expression can be used for investigation of when performing multiple sample analyses
real, defined microbial processes (i.e. phe- of complex communities and are based
nomena occurring under in situ conditions). on differences in the gene sequence from
Thus, detection of mRNA provides a strong different organisms.
indication of gene expression at the time One such method is terminal restriction
of sampling, which can be correlated with fragment length polymorphism (T-RFLP;
the prevailing physicochemical conditions see also Blackwood, Chapter 5 this volume).
(Nogales et al., 2002; Alfreider et al., 2003). The technique itself depends on the ampli-
Primers for genes involved in nutrient fication of DNA with a primer set, one of
cycling can be used to detect and quantify the which is fluorescently end labelled, and
gene/transcript copies in the environment. restriction of the resulting product with fre-
The technique relies on the availability of quently cutting enzymes. Due to sequence
large numbers of sequences in databases for variations, the terminal restriction site for
the design of efficient primers. The amplicon each species in the community should be
achieved can be further analysed for diversity different. The output is digital and provides

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Composite Default screen

6 S. Sharma et al.

Table 1.2. Various genomic approaches employed to study biogeochemical cycles in soil.

Molecular approach Target Reference

PCR/RT–PCR-cloning mnp Bogan et al., 1996

nifH, nirS, nirK, nosZ Rösch et al., 2002
mcrA Castro et al., 2004
cbbL Selesi et al., 2005
PCR/RT–PCR-DGGE-cloning amoA, nirK Avrahami et al., 2002
nirK, nirS, nosZ Throbäck et al., 2004
Sulphate reducers Dar et al., 2005
PCR-RFLP/T-RFLP-cloning narG Philippot et al., 2002
nirK, nirS Wolsing & Prieme, 2004
amoA, nifH Yeager et al., 2005
Real-time PCR pmoA Kolb et al., 2003
nirK Henry et al., 2004
narG López-Gutiérrez et al., 2004
PCR-probe hybridization amoA Bruns et al., 1999
narG, nifH, nirK, nirS, nosZ Mergel et al., 2001
sub, npr Sharma et al., 2004
cPCR cbh1, lipA, lipC Lamar et al., 1995
mnp Bogan et al., 1996
amoA Bjerrum et al., 2002
FISH Ammonia oxidizers Briones et al., 2002
Nitrifiers, ammonia-oxidizers & Meyer et al., 2005
nitrite-oxidizers
Macroarray/Microarray amoA, nirK, nirS, pmoA Wu et al., 2001
dsrAB Loy et al., 2002
amoA, nifH, nirK, nirS Taroncher-Oldenburg et al., 2003
amoA, dsrAB, nifH, nirK, nirS, Tiquia et al., 2004
pmoA
SIP mxaF, mmoX, pmoA Morris et al., 2002
amoA, methylotrophs Radajewski et al., 2002
PAH degraders Singleton et al., 2005

information on the size of the product in nitrite reductase (nirK and nirS) gene frag-
base pairs (i.e. species) and the intensity of ments. The analyses were performed three
fluorescence or relative abundance of the times during the year: in March, July and
various community members. T-RFLP ana- October. Fragments of the nirK and nirS
lysis of nifH (Widmer et al., 1999), nirK genes were amplified using primer sets nirK1F
(Avrahami et al., 2002) and narG (Philippot and nirK5R for nirK, which give a 512–515 bp
et al., 2002) has been used to compare product, and nirS1F and nirS6R for nirS,
denitrifying community patterns in differ- which give an approximately 890 bp prod-
ent environments (see also Philippot and uct (Braker et al., 1998). nirK gene fragments
Hallin, Chapter 9 this volume). could be amplified in all three months,
Wolsing and Prieme (2004) explored whereas nirS gene fragments could be
the temporal and spatial variation of com- amplified only in March. For T-RFLP, the
munities of soil denitrifying bacteria at sites forward primers were fluorescently labelled
receiving mineral fertilizer and cattle manure with FAM (nirS1F) or TET (nirK1F). Each
using T-RFLP analyses of PCR-amplified PCR product was divided in three equal

16
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