The metagenomics of disease-

suppressive soils experiences from

the METACONTROL project
Jan Dirk van Elsas
1
, Rodrigo Costa
1
, Janet Jansson
2,3
, Sara Sjo ling
4
, Mark Bailey
5
,
Renaud Nalin
6
, Timothy M. Vogel
7
and Leo van Overbeek
8
1
Department of Microbial Ecology, Centre for Ecological and Evolutionary Studies University of Groningen, Kerklaan 30, 9750AA
Haren, The Netherlands
2
Department of Microbiology, Swedish University of Agricultural Sciences, Genetics Center, 750 07 Uppsala, Sweden
3
Ecology Department, Earth Sciences Division, Lawrence Berkeley National Laboratory, MS 70A-3317, One Cyclotron Road,
Berkeley, CA 94720, USA
4
School of Life Sciences, So derto rn University College, 141 89 Huddinge, Sweden
5
Molecular Microbial Ecology Group, Centre for Ecology & Hydrology, Manseld Road, Oxford OX1 3SR, UK
6
LibraGen SA, 3 Rue des Satellites 31400, Toulouse, France
7
Environmental Microbial Genomics, Laboratoire AMPERE, Ecole Centrale de Lyon, Universite de Lyon, 36 Avenue Guy de
Collonge, 69134 Ecully, France
8
Plant Research International, PO Box 16, Wageningen, The Netherlands
Soil teems with microbial genetic information that can
be exploited for biotechnological innovation. Because
only a fraction of the soil microbiota is cultivable, our
ability to unlock this genetic complement has been
hampered. Recently developed molecular tools, which
make it possible to utilize genomic DNA from soil, can
bypass cultivation and provide information on the col-
lective soil metagenome with the aim to explore genes
that encode functions of key interest to biotechnology.
The metagenome of disease-suppressive soils is of
particular interest given the expected prevalence of anti-
biotic biosynthetic clusters. However, owing to the com-
plexity of soil microbial communities, deciphering this
key genetic information is challenging. Here, we
examine crucial issues and challenges that so far have
hindered the metagenomic exploration of soil by draw-
ing on experience from a trans-European project on
disease-suppressive soils denoted METACONTROL.
Introduction
Soil is the habitat on Earth that harbours the largest
microbial diversity (see Glossary) per unit mass or volume
[1,2]. Because microorganisms represent the least explored
and richest source of novel bioactive compounds [3], soil
offers promising perspectives for the search of novel func-
tions of biotechnological interest. Traditional microbiolo-
gical approaches already revealed that soils commonly
harbour a broad array of antibiosis-related functions
[46], some of which have been associated with the sup-
pression of plant pathogens [6,7]. In addition, some soil
Review
Glossary
Bacterial artificial chromosome (BAC): an artificially constructed vector for
medium-sized segments of DNA (up to 300 kb in length), which are then
incorporated into a host cell (usually Escherichia coli). BACs serve as cloning
vectors in metagenomics.
Cloning vector: a small DNA vehicle that can accommodate a foreign (cloned)
DNA fragment. Plasmids, cosmids, fosmids and BACs are examples of cloning
vectors.
Cosmid: hybrid plasmid constructed by the insertion of cos sequences, which
are DNA sequences of the bacteriophage Lambda.
Diversity: statistically, it describes the richness as well as distribution of
relative abundance of a species compared to the different species found in a
sample. Microbial diversity in soil is likely to be the highest found in any
ecosystem on Earth.
Fluorescence-activated cell sorting (FACS): sorting of cells that have been
tagged with a fluorescent dye in a flow cytometer.
Fosmid: a hybrid vector consisting of an f-factor cosmid (circular DNA) that is
capable of containing larger pieces of DNA, i.e. up to 50 kb (average 35 kb)
compared to only 10 kb in a plasmid.
Host: in cloning procedures, hosts are organisms that serve as the recipient of
the cloning vectors that each carry a unique copy of foreign DNA directly
extracted from the environment or from another organism. The most common
metagenomic library host is the bacterium E. coli.
Metagenomics: the study of the collective genomes recovered from environ-
mental samples without prior cultivation. It enables the investigation of genome
information on organisms that are not easily cultured in the laboratory. It is
therefore a means of systematically investigating, classifying and manipulating
the entire genetic material isolated from environmental samples.
Plasmids: independent, circular and self-replicating DNA molecules that carry
only a few genes. Plasmids are autonomous molecules and exist in cells as
extra chromosomal genomes, although some plasmids can be inserted into a
bacterial chromosome.
Pyrosequencing: a novel high-throughput nucleotide sequencing method [23]
that is based on multiple parallel extensions from target DNA molecules
coupled to on-line sensitive reads.
Shotgun sequencing: technique in which DNA is broken up randomly into
small segments, which are then sequenced using the chain termination or
pyrosequencing method to obtain reads. Multiple overlapping reads for the
target DNA are obtained and overlapping ends of different reads are
assembled by software packages into contiguous sequences denoted contigs.
Stable isotope probing (SIP): the use of stable isotopes, such as
13
C, as markers
to identify organisms that are actively involved in transforming the
13
C labelled
material (substrate).
Substrate-induced gene expression screening (SIGEX): screening strategy
developed for metagenomic libraries in which the induction of gene
expression is used as the criterion for rapid identification and isolation of
clones.
Suppressive soil: soil that is able to suppress the development of plant
diseases by pathogens that are present.
Corresponding author: van Elsas, J.D. (j.d.van.elsas@rug.nl).
0167-7799/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2008.07.004 Available online 4 September 2008 591
Figure 1. Metagenomic exploration of Wildekamp (NL) soil (W; [6,26]). In total, about 16 000 clones were functionally screened for growth inhibition of Rhizoctonia solani
AG3 and Bacillus subtilis. The clones were also genetically screened for homology with genes involved in polyketide biosynthesis (PKS1 type). (a) Outline of metagenomics
approach [26]. (b) Functional and molecular screening of (W) soil metagenomic library. Functional screening was performed using arrayed Escherichia coli clones on plates
that were overgrown by the target organism R. solani AG3. Inhibition of fungal growth in the vicinity of the E. coli colonies, e.g. slipstream inhibition as indicated in the
figure, was recorded. Genetic screening was performed using an array of E. coli clones, in which duplicated clones overlapped to enable a rapid identification of
hybridization-positive clones. Shown here is hybridization with the PKS1 probe [26].
Review
Trends in Biotechnology Vol.26 No.11
592
microorganisms are naturally resistant to a broad range of
antibiotics [8] and the collective antibiotic resistance genes
present in soil have been referred to as the antibiotic
resistome [9]. The soil microbiota also contains a large
pool of genes that encode enzymes involved in either
biosynthetic or biodegradation processes, including the
degradation of xenobiotics [10,11].
Only a minor fraction of the soil microbiota can be
cultivated using existing cultivation approaches, although
this number is constantly increasing owing to ongoing
development of techniques to support the growth of recal-
citrant species [12]. Nevertheless, soil microbiologists have
increasingly relied on molecular approaches that derive
their strength from the direct exploration of soil-extracted
DNA [1,13,14] using a soil metagenomics approach
(Figure 1a).
Soil metagenomics and its challenges
Soil metagenomics is dened as the study and exploration
of the collective genomes present in a particular soil
sample [15]. To enable such exploration, construction of
soil DNA-based metagenomic libraries in a cloning vector
(see Glossary) is often used. When applied to bulk soil, the
approach mainly addresses genes of the microbial com-
munity, i.e. the soil microbiome. Although metagenomics
already has been successful in unlocking several novel
genes and functions from the soil microbiome, the under-
lying work can be tedious and seemingly inefcient [16
22]. This is a result of technical constraints in: (i) producing
adequate amounts of high-quality soil DNA, which is de-
pendent on soil characteristics; (ii) generating metage-
nomic libraries of sufcient size in suitable metagenomic
hosts; and (iii) screening procedures, which are not always
efcient.
Two different metagenomic approaches to soil are
possible, i.e. either unselective or targeted metagenomics.
Unselective soil metagenomics constitutes a gene shing
expedition, because no a priori selection (e.g. via prior
growth-based selection of the communities under study,
or following PCR amplication of a given gene or DNA
region of interest) takes place before the metagenome is
obtained and analysed. Given the typical distributions of
microbial species in most soils (Box 1), hit rates of target
genes are affected and can be low if targets occur in non-
dominant species. In this context, direct shotgun sequen-
cing of the soil DNA pool is becoming increasingly popular
[23]. This approach bypasses the cloning step and instead
relies on random high-throughput sequencing of soil DNA,
or a target fraction thereof (Box 2). By contrast, in a
targeted soil metagenomic approach, the isolated pool of
DNA is deliberately biased to enhance hit rates, for
instance owing to (i) pretreatment of the microbial com-
munity, (ii) prefractionation of the DNA on the basis of
G+C% [24], or (iii) the amplication of target regions by
Box 1. Soil microbial diversity and library coverage
As most soils support an extensive microbial diversity with
potentially thousands of bacterial and archaeal species [1,52],
sampling the entire diversity in a soil sample would require an
enormous effort, which currently seems impracticable. Two key
parameters are crucial for the distribution of microbial species, the
richness (i.e. the number of species) and their evenness (their
relative abundance). Often, the microbial species distributions in
soil are characterized by the strong dominance of relatively few
species (tens to hundreds) next to the presence of large numbers of
rare species, which form a characteristic tail in rank-abundance
curves [2]. This typical species distribution in soil makes the full
recovery of the extant soil microbiota difficult, although it is
theoretically possible. Therefore, most soil metagenomic libraries
will mainly reflect the genetic make-up of the most abundant
species in the soil. However, in the light of practical limits to library
sizes, these libraries might still contain only fragmented genetic
information for even this pool of species. Any successful soil
metagenomics undertaking will thus have to be guided by prior
knowledge of the prevalence and distribution of target genes within
the microbiota in the soil(s) to be examined. Ideally, gathering such
information should precede construction and screening of the
library. Alternatively, prior information might point the way towards
the introduction of a deliberate bias in the experimental approach,
such as favouring target organisms by substrate inclusions. In the
METACONTROL project, we assumed a high occurrence of polyke-
tide biosynthetic operons in disease-suppressive soils based on
assumptions about the role of antagonism in suppression, which is
supported by the known high prevalence of the pyrrolnitrin
biosynthetic operon [6]. The achievable coverage of metagenomic
libraries, and the success in discovering novel biological functions,
will further depend on the choice of the vector and host strain in
which the libraries are maintained.
Box 2. Environmental shotgun sequencing harnessing a
novel powerful tool
Environmental shotgun sequencing (ESS), the high-throughput
shotgun sequencing of environmental DNA, is a promising
approach to characterize microbial genomes present in an environ-
mental sample. Despite its disadvantages of being highly resource
demanding, it enables acquisition of a wealth of information on
sequences and genomes in natural ecosystems [54] that has no
precedence. Recently developed high-throughput sequencing tech-
nologies, such as the (second generation) genome sequencer GS-
FLX, which uses pyrosequencing (454 Life Sciences, Roche), as
well as the Solexa sequencing platform (Solexa Ltd, Cambridge,
UK), now allow for a rapid generation of large amounts of sequence
information. For instance, at greatly improved accuracy and
throughput, up to 100 Mb of sequence information can be generated
in less than 8 h running time (GS-FLX system). The procedure is
highly unselective and the high amount of sequences of common
housekeeping genes that are generated could overload databases.
The outstanding challenges associated with most ESS projects is
not the high-throughput sequencing per se, but instead the
assembly and sorting of the sequences and the annotation of
eventual contigs. For this purpose, new bioinformatics tools for the
analysis of ESS databases are constantly being developed and
refined [5560]. As a result of its untargeted nature, ESS enables a
comprehensive analysis of the genomic composition of an environ-
mental sample, in that the genes of the most dominant organisms
are represented to a greater extent than those of rare organisms.
Moreover, ESS is suitable in the aiding of genome reconstruction.
The latter potential of ESS has been demonstrated in the assembly
of one nearly complete and three partially assembled microbial
genomes from a simple community found in an acid mine drainage
[61]. However, genomic reconstructions from most soil samples will
require enormous sequencing power as well as increased bioinfor-
matic input [5560], which raises serious issues of time/money
investment [52]. The collection of large amounts of sequence data of
soils might nevertheless be useful if comparative studies on the
prevalence of proteins are performed. An alternative approach, ESS
of the soil metamobilome, the collective genetic information
encoded in plasmid or phage genomes, might help in facilitating
biotechnological applications of soil metagenomics, because the
metamobilome is expected to contain a large number of relevant
traits [62]. In addition, the microbial consortia associated with
microfauna could also be analysed and explored with ESS [63].
Review Trends in Biotechnology Vol.26 No.11
593
PCR before metagenomic analysis. Pretreatment can con-
sist of, for example, a pre-enrichment of target microbial
groups by adding particular growth substrates, such as
chitin [25], or a selection of particular cells by cell-sorting
procedures [26].
Metagenomic libraries
The metagenomic libraries will thus be derived either from
unbiased DNA in the unselective approach or from a
specic fraction of the microbiota or the DNA pool in the
targeted method. Irrespective of the approach, the DNA
library should ideally encompass all genes of interest that
are targeted in the soil microbial community. However,
such coverage is seldom achieved owing to the difculty of
achieving a representative sample of the soil microbiota,
which would also include rarer microbial species [14] (Box
1). Various approaches, based on functional or sequence
analysis, are commonly used to analyse the obtained DNA
pool and their characteristics are discussed below.
Library screening
A great challenge in soil metagenomics is the screening of
libraries to locate genes of interest. Two approaches are
possible: functional and molecular screenings [14]
(Figure 1b). Functional screening depends on the success-
ful expression of target gene(s) in the metagenomic host,
whereas molecular screening is based on the detection of
regions in the DNA that are recognizable via hybridization
or PCR approaches [14]. An overview of studies that suc-
cessfully screened DNA libraries [2733] can be found in
Table 1. As, depending on the abundance and distribution
of target genes in the soil microbiota, functional screens of
soil-generated libraries can be associated with low hit
rates, molecular screens, in particular those that can be
performed in a high-throughput setup, have become more
widely employed [26,34]. Recently, DNA microarray-based
screening (denominated metagenomic proling) has devel-
oped into a highly promising novel approach because it
enables the efcient screening of a library using (library-
derived) microarrays based on functional or other genes
[35]. Another advanced screening method, substrate-
induced gene expression screening (SIGEX), exploits the
induction of gene expression [36]. In this method, a repor-
ter gene (e.g. the gene encoding green uorescent protein
GFP) enables the sorting of clones, in which the desired
gene has been induced, via uorescence activated cell
sorting (FACS) of GFP-positive cells. In this way, the
identication of clones that carry a desired metabolic
activity, for example a particular step in a biodegradative
pathway, can be facilitated [36]. In one study, multiple
clones could be isolated from a groundwater metagenomic
library consisting of over 150 000 clones that had been
induced by benzoate and naphthalene [36]. Finally, strat-
egies that are based on growth selection can offer excellent
opportunities to enhance screening efciencies.
In the following sections, we examine the power and
pitfalls of the metagenomics-based exploration of agricul-
tural soils suppressive to phytopathogens using our experi-
ence from the European-Union-sponsored project Soil
metagenomics to identify novel mechanisms of antagonism
and antifungal activity for the improved control of phyto-
pathogens (METACONTROL QLK3-CT-200202068)
[5,14,25,26,34,3740]. In particular, we address the tech-
nical and intellectual challenges that need to be overcome
to optimize the potential prot from this novel approach.
The METACONTROL project aims, results and
technological challenges
Aims
It has long been known that particular agricultural soils
suppressive soils can restrict the activity of plant-patho-
genic microorganisms. The suppression is often biotic and
key mechanisms include the production of antibiotics. A
great part of this antagonistic activity might reside in the
as-yet-uncultured fraction of the soil microbiota [7,26] and
hence unlocking this antagonistic potential via a metage-
nomics approach is important. The METACONTROL pro-
ject, which ran as from 2002, is unprecedented in its kind.
It united seven European laboratories [5,14,25,26,34,37
40], mainly from academia but also including the small
enterprise LibraGen (Toulouse, France), with the main
aim of exploring the biotechnological potential of phyto-
pathogen-suppressive soils. The underlying assumption
was that the microbiota of these suppressive soils would
serve as rich reservoirs of anti-phytopathogen loci, such as
those involved in the production of antibiotics of the poly-
ketide class and chitinase biosynthesis. The metagenomic
exploitation of the aforementioned traits was thus the
principal focus of the METACONTROL endeavour. During
the course of the project, four suppressive soils and one
control soil were identied (indicated by capital letter with
the suppressed species) (Table 2): one in the Netherlands
(W, Rhizoctonia solani AG3), one in Sweden (U, Plasmo-
diophora brassicae), one in France (C, Fusarium) and one
in the UK (Wy, Fusarium). The control soil (France) was
denoted M. Metagenomic libraries were constructed for
these soils and screened for the occurrence of antibiotic
functions [5,14,25,26,34,3739]. In addition, the project
partners developed a range of methodologies that facili-
tated the exploration of the suppressive soil libraries
[21,25,34,37]. In the following, we will discuss the technol-
ogy and the relevant crucial choices that had to be made
before each analytical step (Figure 1) in respect of: (i) the
optimal soil DNA extraction methodology, (ii) the possib-
ility to bias the soil community or DNA, (iii) the suitability
of the metagenomic vector/host system for the target objec-
tives, (iv) the optimal procedure for screening, and (v) the
nal analysis.
Results
Soil DNA extraction and preparation For a robust library
preparation, soil DNA that accurately represent the
diversity of the soil microbiota is required in a quantity
that enables efcient cloning [26,37]. In addition, the DNA
itself needs to be of sufcient quality with regard to
chemical purity, lack of degradation and length of DNA
fragments to be suitable for cloning into a vector [37]. A
required minimal size of roughly 40100 kb will increase
the chance that the entire target pathways, e.g. those
involved in the biosynthesis of polyketide antibiotics,
remain intact [34,40]. The METACONTROL consortium
collectively tested and applied advanced methodology that
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Trends in Biotechnology Vol.26 No.11
594
Table 1. Selected soil metagenomics studies and their relevance for biotechnology and ecology
Soil type/
condition/
location
Host/
vector system
Screening
type/target
Finding(s) Features Relevance Refs
Agricultural
soil, Madison,
US
Escherichia
coli/BAC
Genetic/16S rRNA
genes;
functional/various
traits
Retrieval of clones displaying
heterologous antibacterial,
lipase, amylase, nuclease and
hemolytic activities
Analysis of 16S rRNA gene
diversity revealed
representatives of low GC
Gram-positive bacteria,
Acidobacterium,
Cytophagales, and
Proteobacteria in one of
the BAC libraries
One of the inaugural
studies on soil
metagenomics,
it highlighted the
potential of the
approach in shedding
light on the functional
capabilities of the
soil biota
[27]
Not indicated E. coli/cosmid Functional/
antibacterial
activity
A clone (CSL12) was found that
produces a series of long-chain
N-acyl-L-tyrosine antibiotics
About 700 000 cosmid clones
were screened, 65 of which
were antagonistic to Bacillus
subtilis, the bacterial strain
used in the assays
First report of a
long-chain N-acyl
amino acid
biosynthesis gene
[28]
Uncultivated
soil, New
England, US
E. coli/BAC Functional/
antibacterial
activity
The 27 kb clone mg 1.1 was
found to express many small
molecules related to and
including indirubin
Retrieval of one anti-bacterial
clone per 60 Mb of soil-derived
DNA. Library composed by
DNA fragments of 5120 kb
Indirubin is an
antileukaemic drug
[29]
Soil type not
indicated,
Ithaca, US
E. coli/cosmid Functional/natural
products
Discovery of a 6.7 kb violacein
gene cluster, which is different
from previously reported ones,
present in a blue-coloured clone
Identication of coloured
clones in the cosmid library
was used as a screen criterion
to search for natural product-
producing clones
Violacein is active
against Gram-positive
bacteria and induces
apoptosis in broblast
cells
[30]
Agricultural
soil, Madison,
US
E. coli/BAC Functional/colour
production
Isolation and characterization
of the triaryl cation antibiotics
turbomycin A and B
An example of heterologous
gene expression of previously
uncharacterized organic
molecules
Both turbomycin A
and B display broad-
spectrum activity
against Gram-negative
and positive bacteria
[31]
Arable soil, La
Cote Saint
Andre, France
E. coli
Streptomyces
lividans/
cosmid shuttle
vector
Genetic/type I
polyketide
synthase
(PKS I) and
16S rRNA
genes
Discovery of new PKS genes in
at least eight clones
An analysis of 16S rRNA gene-
based bacterial diversity
present in the E. coli cosmid
library was carried out
16S rRNA gene
sequences analysed
derived primarily
from organisms not
described at that
time. A range of
naturally active
compounds was
shown to be
heterologously expressed
[39]
Uncultivated
soil, New
England, US
E. coli S.
lividans
Pseudomonas
putida/BAC
shuttle vectors
Functional/
antibacterial and
antifungal activity
The three hosts displayed
different heterologous gene
expression capabilities for
known antibiotic clusters
A method is described that
enables the transfer of a
metagenomic library from
E. coli to S. lividans and
P. putida
Highlights the
importance of
increasing the host
range to enhance
the success of
metagenomics
endeavours
[53]
Uncultivated
soil, Madison,
US
E. coli/BAC Functional/
antibiotic
resistance
Nine clones expressing
resistance to aminoglycoside
antibiotics and one expressing
tetracycline resistance were
found
Heterologous gene expression
is required for the detection
of aminoglycoside resistant
clones
Suggests that the
diversity of antibiotic
resistance genes
within soil bacteria is
greater than
previously estimated
[32]
Forest
rhizosphere
soils
E. coli/fosmid Functional/
fungal
antagonism
Identication of eight open
reading frames (ORFs)
conferring antifungal activity
to the E. coli host. These ORFs
were found to be similar to
genes encoding type II family
polyketide synthases
One positive antifungal hit in
a total of 113 700 tested
fosmid clones. Use of the
yeast Saccharomyces
cerevisiae as model fungus
to test for antagonism
Perhaps the rst study
to target antifungal
activity in the
metagenome of
rhizosphere soils
(pine tree rhizospheres).
[42]
Pasture soil,
Montrond,
France
E. coli/fosmid Functional/
production and
degradation of
N-acylhomoserin
lactones (NAHL)
Identication of a gene, qlcA,
encoding a NAHL-degrading
lactonase. The fosmid insert
p2H8, containing the glcA gene,
was found to harbour nine
ORFs resembling genes of
Acidobacteria
The qlcA gene was found to
effectively quench quorum-
sensing mediated pathogenic
functions when expressed in
Pectobacterium carotovorum
Knowledge of the
diversity of quorum-
quenching lactonase
extended
[33]
Review Trends in Biotechnology Vol.26 No.11
595
enabled themto obtain pure high molecular weight (HMW)
DNA fromall the soils under study [14,26,34,37]. The most
efcient approach was found to consist of a physical
extraction of microbial cells from soil followed by
gentle DNA extraction and purication using pulsed-
eld gel electrophoresis (PFGE) (Figure 1) [26,37].
Cushion (Percoll and/or Nycodenz) pre-separation of
bacterial cells from soil [26,37] was found to be optimal
for subsequent isolation of the HMW DNA. Moreover, the
microbial growth status in the soil was an important
determinant of soil DNA quality, and the latter could be
boosted by incubation with growth substrates [37].
Typically, this approach produced HMW DNA, often
with size >60100 kb [26,37]. We also found that high
amounts of cells, i.e. minimally ca. 10
11
, were needed to
yield sufcient DNA for efcient library construction [25].
Because soils typically contain in the order of 10
8
to 10
10
cells per g, this nding helped set the rule for the efcient
construction of the soil metagenomic libraries.
Deliberate bias and preselection In the METACONTROL
project, mainly unselective approaches were used to explore
the four selected naturally disease-suppressive soils for
their anti-phytopathogen functions [14,25,26,34,37]. In
doing so, we disregarded the possibility of positive growth
selection because the chosen target genes/operons do not a
priori imply a growth advantage in the metagenomics host,
even in the presence of the target phytopathogens [26,34].
However, some targeted approaches were developed to
assess specic members of the microbial community, for
example the soil was pre-treated with chitin to enhance the
levels of chitinase producers [21,25]. Astrong focus was also
placed on the pre-selection of metabolically active bacterial
cell fractions with the help of FACS analysis. This
introduced bias was hoped to increase the prevalence of
organisms carrying the aforementioned target genes, in
light of their presumed function in the suppressive soil
[25]. Metabolically active cells could indeed be
successfully sorted from the soil [26,41]. However, as a
result of the limited ow rate of the cell sorters used,
throughput was limited (10
6
cells/hour), which resulted in
insufcient biomass for library construction [26]. To meet
the high sorting demands to establish a library, more
sophisticated cell sorters with higher owrates are needed.
Metagenomic library production The clone libraries
obtained from METACONTROL for the four disease-
suppressive soils and the control soil consisted of 6000
to 60 000 clones and were constructed in Escherichia coli
(Table 2). Both large insert size vectors, such as bacterial
articial chromosomes (BACs), that enable the cloning of
inserts up to 200 kb, and fosmids that enable insertion of
3545 kb fragments were used. BAC vectors allow for the
coverage of complex large operons and also facilitate the
analysis of a gene/operon within its original genomic
context. By contrast, fosmids are only able to
accommodate smaller inserts and thereby only enable
the cloning of smaller operons. Using a fosmid vector
system, such as the Epicentre ccFos system, allows for
the positive selection of vectors that have acquired inserts
[21,26,34,37,38]. Three of the nal libraries (Table 2) were
based on fosmid vectors, the reason being the ease of
obtaining appropriately sized libraries within reasonable
time. One library, for the M soil, was successfully
Table 2. Soil metagenomic libraries constructed under the METACONTROL project [26,34,39] and their characteristics
a
Soil description Library
vector/clone
number
Type of screening Number of
positive clones
Remarks and Refs
W Wildekamp
grassland, suppressive
to Rhizoctonia solani
AG3
Fosmid/16 000 Functional: antagonism
against R. solani AG3
and Bacillus subtilis
7 Combined functional and genetic (PKS1) screening
resulted in seven positive clones. Five of those were
conrmed as polyketide synthase PKS1-positive clones.
Three clones were completely sequenced, and one
insert showed high similarity with sequences from
Acidobacterium sp. [26]
Genetic screening with
soil-generated PKS1
probe
Wy Wytham
grassland and
Fusarium-suppressive
agricultural soil
Fosmid/100 000 Functional: antagonism
against Fusarium sp.
indicator strains agar
plate based dual-culture
assay
13 (grassland) Grassland most effective source of functional clones and
greatest diversity. Agricultural bulk soil revealed low
diversity and limited functional traits. End-sequencing
and subcloning of cosmids resulted in mostly
unidentied ORFs. Efcacy of clones lower than control
strains isolated from same source
Average insert
size 35.6 kb
2 (agricultural
soil). Each clone
genetically
distinct
C Chateaurenard
Fusarium-suppressive
soil
Fosmid/51 000 Genetic screening 22 Combination of functional and genetic screening.
Functional screening used several targets: Fusarium
spore generation and hyphal production, Aspergillus
nidulans growth, Hebeloma cylindrosporum hyphal
generation. Genetic screening included sequencing
of PKS positive clones [34,39]
BAC/60 000 Functional screening
U Uppsala
Plasmodiophora
brassicae-suppressive
soil
Fosmid/8000 Functional: antagonism
against Pythium ultimum
4 Selection of Streptomyces mutomycini, Kitasatospora,
Lentzea, Oerskovia revealed by ngerprinting.
S. mutomycini and Streptomyces clavifer prevalent in
the library. Chitinase genes found in soil, in the library
and in isolates; a cluster that prevailed in soil not
found in the library, whereas a library cluster was not
found in the soil
Genetic: chitinase genes
and 16S rRNA gene
M Montrond (control)
soil
Fosmid/60 000 Genetic and functional
screening
39 Thirty-nine novel PKS1 positive clones, most with
supernatants showing antimicrobial activity, were found
a
PKS1: polyketide synthesis operon for type-I polyketides.
Review
Trends in Biotechnology Vol.26 No.11
596
constructedina BACvector [34,39]. The appliedBACvector
also contained a replicon compatible with a Streptomyces
host, enabling shuttling between E. coli and Streptomyces
metagenomic hosts and consequently enhancing the
probability of heterologous gene expression within the
clones obtained in this library [34,39].
Metagenomic library screening Soil metagenomic
libraries might contain only few clones that actually carry
a gene/operon of interest (Box 1) in addition to any potential
constraints of gene expression in the host strain. In the
METACONTROL project, both functional and molecular
screens were used to uncover potential antagonistic
functions with varying success rates [21,26,34,37,38], but,
overall, low numbers of phytopathogen-suppressive clones
were found in the four libraries [26,34,37] (Table 2).
Functional screening Functional screenings of the libraries
were performed in so-called dual-culture assays, which
enable the target phytopathogenic organisms to grow out
over the metagenomic library clones arrayed on Petri
dishes and scoring for irregularities/inhibitions in growth
of the target organism [26,34,39]. This experimental setup
led to the detection of few positive clones (range 148),
amounting to, on average, <0.05% of positives for all
libraries (Table 2). Such low numbers can be attributed
either to a rare occurrence of target genes/operons in the
vectors used, or to the responsible cellular machineries
being signicantly larger than the vector inserts such as
found for polyketide production loci. Other factors that
might impede the detection of function of the target
genes could be a low expression rate from the DNA
inserts or unsuitable expression conditions on the plates
used in these assays. For instance, the accurate quorum
sensing system, a cellular communication mechanism
commonly found in bacteria, might have been lacking in
the E. coli host used. Indeed, shuttling from E. coli to
Streptomyces as the metagenomic host facilitated the
expression of an antibiotic (amphotericin) production
locus [34,39] as indicated by a high activity against
target fungi.
Considering the potential expression bottlenecks, the
generally low number of functionally positive clones did
not come as a surprise. For instance, a screening of forest
soil libraries for antifungal traits also yielded only one
positive signal among the 113 700 fosmid clones [42]
(Table 1). Hence, substantial methodological improve-
ments are required to boost the percentages of positive
hits and we will present possible strategies below.
Molecular screening The libraries obtained in the
METACONTROL project were also screened using
molecular tools, such as hybridization and PCR
amplication methods (Table 2). It was hypothesized
that the success in detecting novel operons, such as
those involved in polyketide biosynthesis, would depend
on the application of deliberate degeneracy in the probes
and primers used [34,39]. The rationale behind this
contention is that, this way, the molecular screening is
not restricted to previously known exact sequence
information and will enable the broadest possible range
of meaningful positive hits within the metagenomic
library. The approach facilitated the identication of
target genes that are sufciently similar to query
sequences, and thus constituted a rapid crude search
step before a more resource-demanding and thorough
investigation. Using the total soil community DNA as
the target, we PCR-amplied the genes of the polyketide
biosynthesis operon (PKS1) with degenerate primers. The
resulting amplicons were used for the generation of probes
that would thus hybridize with PKS sequences that
abounded in the soil [26,34,38] (Table 2). This
hybridization approach yielded seven positive clones in
one (W) soil library that contained genes that were
probably involved in PKS biosynthesis, which was
subsequently conrmed by end-sequencing [26]. In the
case of the M soil, the roughly 60 000 clones from the
soil metagenomic library were divided into pools, which
were used as templates for PKS-based PCR screenings.
This enabled the detection of >100 positive pools (0.22%hit
rate [34]). The amplicons produced were then sequenced to
check for redundancies within the library and with known
PKS sequences. The redundancy level was low (2%) and
39 unique PKS sequences were found from the pools, all
representing promising novel PKS biosynthesis operons
(Figure 2a). Then, the positive clones in the pools were
identied using colony hybridization with relevant probes,
after which these were tested, following shuttling into
Streptomyces, in assays for antagonistic activity against
the indicator organisms Bacillus subtilis 1A72,
Staphylococcus aureus 21, Enterococcus faecalis 40,
Escherichia coli 9, Pseudomonas aeruginosa 39,
Fusarium oxysporum LNPV, Aspergillus fumigatus Gasp
4707 and Neurospora crassa HK. The positive clones
showed 56% antimicrobial activity against at least B.
subtilis, 13% against S. aureus, 4% against E. faecalis
and <1% partial inhibition of growth of N. crassa
mycelium (provided by LibraGen; Figure 2b). These
molecular screening procedures thus enabled rapid
access to novel PKS sequences (Figure 2a) typical for the
soil community that can be used for production [26].
Technological challenges
What are the remaining challenges in exploring suppres-
sive soils that have become apparent fromthe METACON-
TROL experience? Without any doubt, there is a strong
need to improve the efciencies at each step in soil meta-
genomics, from the application of a deliberate bias to
favour target organisms and genes/operons in the starting
material, to the improvement of cloning and screening
procedures aimed at increasing the throughput and ef-
ciency of metagenomics.
Deliberate bias in sampled communities We learned in
METACONTROL that deliberate manipulation of the
sample communities from soil offers unique possibilities
to enhance metagenomics hit rates. For instance, growth
selection can be applied as outlined before. Here, an
intelligent selection of growth conditions will guide the
bias. Also, FACS can be applied not only to sort the
metabolically active cell fractions, but also to obtain
particular fractions of the community for instance the
Review Trends in Biotechnology Vol.26 No.11
597
high-G+C% Gram-positive bacteria in which antibiotic
production loci are abundantly present following
staining with specic uorescent probes. Another
promising strategy is offered by the use of stable isotopes.
Stable isotope probing (SIP) introduces
13
C-labelled
substrates, for example methane, into soil communities.
Methanotrophs then take up the
13
C and incorporate it in
their cellular DNA. The resulting heavy DNA can be
separated from
12
C-DNA by ultracentrifugation and
sequenced, thus identifying the organisms that captured
the substrate [43,44]. This approach was coupled to soil
metagenomics studies [45] and resulted inthe identication
of a complete methane monooxygenase operon, enabling
insight into the enzyme and its function in soil. Also, a
dominant phenol-degrading bacterium, identied as a
Thauera sp., was found in a wastewater bioreactor [46].
Figure 2. Polyketide synthesis (PKS type 1) positive clones found in the C soil library. (a) Phylogenetic analysis of the sequenced (KS ketosynthase) domains (one
particular gene function involved in the biochemical pathway towards polyketides). The tree reconstruction was computed (OD/Neighbour Joining method) for 43
metagenomic sequences, also including 38 sequences from public databases, and corresponding to 204 informative sites. KS domains from public databases: black;
metagenomic KS domains: red; PKS/non-ribosomal peptide synthesis (NRPS) hybrids: blue. Scale bars (0.1) indicate genetic distance (10%) (also in (b)). Numbers at
junctions indicate bootstrap values (scale 1100). Higher values, generally above 50, indicate robust clustering. (b) Unrooted tree of KS domains and biological activity.
Active clones against only Bacillus subtilis are in red, against Bacillus subtilis and Staphylococcus aureus in blue, against Bacillus subtilis, Staphylococcus aureus and
Enterococcus faecalis in green and against Bacillus subtilis, Staphylococcus aureus and Neurospora crassa in grey. Non-marked clones did not display activity against any
tested organism. Abbreviation: KSLib, ketosynthase sequences of the LibraGen metagenomic library.
Review
Trends in Biotechnology Vol.26 No.11
598
Moreover, by tracking the fate of
13
C-labelled CO
2
xed by
higher plants in soil, major data on plant-responsive
microorganisms that often produce antimicrobials as
secondary metabolites can be achieved [47]. The wide
application of SIP using other organic substrates bears
great potential in future explorative metagenomic studies
in which organisms with particular ecological roles are the
targets.
Searching for better metagenomic library hosts Working
with E. coli as the metagenomics host has clear advantages
in respect of the relative ease of the laboratory work and
the great experience gained with it over many years.
However, it can be limited with regard to the screening
of phenotypes from the soil metagenome as E. coli is not a
typical soil organism. The main restriction arises from the
fact that some intrinsic promoters and associated factors
required for the expression of the inserts might be poorly
recognized in this host. Moreover, essential post-
translational processing and/or transport functions of
the target genes might be missing. Rondon et al. [48]
showed that only 30% of Bacillus traits could be
expressed in E. coli, which indicates that E. coli is at
best a suboptimal host for the heterologous expression of
genes from many non-enteric soil bacteria. Furthermore,
the host can also be sensitive to toxic compounds that are
produced by inserts after their transcription and
translation and these host clones might consequently
disappear from the library. Obviously, these constraints
are true for any host strain selected for metagenomics and
Box 3 details ongoing efforts in developing alternative
hosts. Our work also supports the contention that
working with several hosts instead of just E. coli will
often enable the hit rates to be boosted.
The metagenomic library vector The METACONTROL
experience conrmed that crucial evaluation of the vector
system to be used in soil metagenomics is required [21].
Three types of vectors, i.e. small-, medium- and large-insert
size vectors, are available. The small-insert size vectors that
basically only permit screening for single gene-encoded
functions can be used in shotgun sequencing approaches
and enable construction of libraries from mechanically
sheared DNA. Such an approach was successfully
employed for the detection of small open reading frames
(ORFs) derived fromuncultured prokaryotes fromsediment
[49]. By contrast, fosmid and BAC vectors enable
incorporation of large fragments and even intact operons
within their genomic context, which might provide a better
handle at gene expression. However, the fact that pure
HMW soil DNA is required in sufcient amounts for
efcient cloning into BAC vectors often makes this class
of vectors unsuitable for routine cloning efforts, such as
those required for high-throughput setups. The selection
of vector clones that receivedinserts is alsoacrucial stepand
this could be further improved with the development of
vector systems that facilitate the detection of any inserts,
such as in a conditional host-killing system.
Outlook the way ahead
Much like in other soil metagenomics projects, several
interesting novel biological functions have been uncovered
in the course of the METACONTROL project. These in-
clude the (partial) biosynthetic machinery for the pro-
duction of particular polyketide antibiotics, e.g. a
leinamycin-like antibiotic, as well as for several other novel
polyketides [26,34,3739] (Table 2). However, at the same
time several intrinsic difculties were found to be associ-
ated with the approach [14,50,51] and this resulted in low
hit rates even for the suppressive soils that were presum-
ably enriched in the target functions. The project actually
conrmed the currently accepted rule-of-thumb in soil
metagenomics that the search for non-housekeeping func-
tions can be compared to looking for a needle in a haystack,
unless preselection is used introducing a bias to
increase the numbers of positive hits. The conditions
imposed by the sample habitat (e.g. the typical rank-
abundance distribution of microbial communities in soil
Box 1) dictate this outcome and require the development
of creative tricks and tools to overcome these limitations.
What are these tricks and tools?
Improvement of the efciency of each step in soil meta-
genomics protocols from DNA preparation to screening/
selection of positive clones remains key to progress in the
mining of the soil microbiota. This will include further
netuning of DNA extraction, that is, extraction of DNA
from community members or metagenome fractions that
most probably contain the target genetic information. For
instance, total plasmid DNA (i.e. the metamobilome) can
be targeted when novel biodegradation, metal or antibiotic
resistance genes that are frequently found on plasmids
are targeted. Also, extraction can be tuned to
13
C-DNA
Box 3. The quest for the most suitable metagenomics host/
vector system
To overcome the limitations associated with the use of Escherichia
coli as a host for soil metagenomics, non-E. coli hosts have been, or
are in the process of being, developed at the premise that such hosts
will enable a greater number of directly obtained genes or operons
to be expressed. Improvements here will enhance the success of
functional screens. Non-E. coli hosts include soil bacteria belonging
to the genera Sphingomonas, Burkholderia, Bacillus, Acidobacter-
ium and/or Verrucomicrobium [64]. Other alternative hosts, such as
Streptomyces, Rhizobium and Pseudomonas spp. [38,53,65] and
mutants of Lysobacter enzymogenes and Pseudomonas fluorescens
mutated in their antagonistic activities [26], have also been used.
Shuttle vectors that allow efficient traffic between these hosts and E.
coli have also become available. For example, a Rhizobium
leguminosarum host was used to test expression of metagenomic
clones and alcohol dehydrogenase as well as tryptophan biosyn-
thetic genes were found in this host, but not in E. coli [65,66]. R.
leguminosarum might enable the expression of a broader range of
genes from typical soil bacteria than E. coli owing to its larger
complement of relevant sigma factors. Pseudomonas spp., which
possess signalling circuits for secondary metabolite production,
might also serve as adequate library hosts for antagonistic functions
and/or attributes promoting plant growth. For example, hetero-
logous expression of genes involved in 2,4-diacetylphloroglucinol
biosynthesis was found in Pseudomonas putida, but not in E. coli
[53]. Finally, vectors that can shuttle between E. coli and eukaryotic
hosts [67] will facilitate the study of eukaryotic genes in a relevant
genomic background. The selection of a specific library host will
depend on the aim of the particular metagenomic study. However, it
has become clear that the preparation of libraries in shuttle vectors,
which allow for gene expression in different or multiple hosts, will
further extend the potential of metagenomics.
Review Trends in Biotechnology Vol.26 No.11
599
incorporated by microorganisms that utilized particular
13
C-labelled substrates or to specic fractions of the chro-
mosomal DNA pool, e.g. high GC content DNA. Other
improvements rely on the introduction of novel positive
selection for desired traits, either based on growth or on
overcoming resistance, as well as improvements in sub-
sequent screens, such as high-throughput formats of
increased accuracy. Moreover, the use of alternative hosts,
with or without shuttle vectors, is expected to yield import-
ant progress. Further, combination of screening (gene
expression) data with high-throughput sequencing should
be fostered. From the METACONTROL experience [26] it
has become apparent that adequate deciphering of meta-
genomic sequence information is paramount here.
Another tool that will support explorative metage-
nomics is based on the provision of prior knowledge on
the incidence, abundance and expression of target genes in
varying habitats. Global-scale gene mapping (GGM
derived from the concept of environmental gene tagging)
enables the description of habitats in terms of their gene
abundance and/or expression [52]. GGM should compare
microbial gene pools across soils and might thus provide a
global perspective on their prevalence. For instance, PKS1-
type polyketide biosynthetic operons were more prevalent
in a soil metagenome than in whale carcass, acid mine
drainage or (Sargasso) sea metagenomes [52]. GGM thus
enables the prediction of metagenomic hit rates. However,
before we reach this objective, crucial questions need to be
answered (Box 4).
We conclude that major advances in the biotechnologi-
cal exploration of soil are expected in the next decade.
Guidance by GGM will be primordial for progress. The
expanded soil metagenomics approaches will enable scien-
tists to (i) mine soil for genes and pathways of interest to
biotechnological applications (Tables 1, 2); (ii) decipher
identity and function of hitherto uncultured microorgan-
isms; and (iii) provide an overall characterization of soil
with regard to function and diversity. However, soil meta-
genomics will continue to depend on labour-intensive tasks
and will thus remain resource-demanding [20]. On the
positive side, the ever-increasing throughput of sequencing
technologies will aid the quick assessment of the preva-
lence of target genes, shedding more light into the soil
genetic reservoir and potential for biotechnological appli-
cation.
Acknowledgements
This work was supported by funding received under the EU
METACONTROL project (QLK3-CT-200202068). R.C. received support
from the Soil Biotechnology Foundation (Groningen).
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