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© 2005 Nature Publishing Group
*Institute of Environment &
Resources, Technical
University of Denmark,
Kongens Lyngby, DK 2800,
Denmark.
‡Department of
Biochemistry and
Microbiology, Rutgers
University, New Brunswick,
New Jersey 08901, USA.
Correspondence to B.F.S.
e-mail: bfs@er.dtu.dk
Recent years have witnessed an increased appreciation
for the role of microorganisms and their metabolism
in shaping and sustaining life on Earth. Most of life’s
fundamental processes were ‘invented’ during the first
2 billion years of life on Earth, prior to the appearance
of the first eukaryote. This achievement is especially
striking considering that it was accomplished by organ-
isms lacking sexual reproduction, the long-presumed
major mechanism of genetic innovation. Horizontal
gene transfer (HGT) is a process that can compensate
for the otherwise clonal mode of prokaryotic life,
affecting microbial adaptation, speciation and evolu-
tion. Microorganisms occupy — and adapt to occupy
— a plethora of ecological niches on earth, and their
activities in large part control global homeostasis.
Through its attendant effects on microbial adaptation,
HGT poses both challenges and opportunities in the
control of global human and environmental health.
For any gene to be horizontally transferred from
one genome to another, at least four (sometimes five)
distinct steps need to occur (FIG. 1). First, a nucleic-acid
molecule (DNA or RNA) in the donor organism is pre-
pared for transfer. This might entail the active packaging
of nucleic acids into phage particles, plasmid replication
from an origin that leads to conjugal transfer, integron
assembly or passive release of DNA into the environ-
ment upon cell death. Second, the transfer step, which
might or might not require physical contact between
the donor and recipient organism, takes place. Third,
the nucleic acid enters the recipient organisms through
specific or non-specific means. Fourth, the nucleic-acid
molecule is established in the recipient either as a self-
replicating element or through recombination with, or
transposition into, the recipient’s chromosome. This
existence can be transitory, as is the case with many
plasmids of which maintenance by the recipient genome
depends on selective pressure. Last, in step 5, stable
inheritance in the recipient genome might ensue.
Several scientific disciplines are addressing HGT,
each providing their unique perspective and each using
different approaches and methodologies. In general,
evolutionary biology considers HGT events that have
gone to completion (that is, through step 5). Molecular
ecology, on the other hand, tends to focus on HGT
events at the level of step 4. Finally, molecular biology
is most interested in the mechanisms controlling steps
1 through 4. With such distinct perspectives, conflicts
are bound to arise, but opportunities for synthesis are
certain to emerge. The goal of this Focus issue of Nature
Reviews Microbiology is to present HGT from the pers-
pective of these different disciplines and to provide a
path towards the construction of a holistic picture of
HGT and its effects on extant microbial communi-
ties. We argue that efforts towards such synthesis will
accelerate our understanding of the mechanisms and
factors that control HGT, the impacts of HGT on the
evolutionary history of prokaryotes, the effect of HGT
on microbial interactions with each other and their
environment, and the means by which HGT can be
controlled to affect human and environmental health.
HORIZONTAL GENE TRANSFER:
PERSPECTIVES AT A CROSSROADS
OF SCIENTIFIC DISCIPLINES
Barth F. Smets* and Tamar Barkay‡
Horizontal gene transfer (HGT) has a crucial role in microbial evolution, in shaping the structure
and function of microbial communities and in controlling a myriad of environmental and public-
health problems. Here, Barth F. Smets and Tamar Barkay assess the importance of HGT and
place the selection of articles in this Focus issue in context.
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Bacteria Archaea Eukarya
Plastids
Mitochondria
Common ancestral community of primitive cells
12
3,4
5
Molecular evolution
Molecular evolution employs a retrospective approach
to infer HGT by examining the signatures left by HGT
in microbial genomes. This approach has benefited
enormously from the availability of complete microbial
genome sequences. As is described in this Focus issue
by Peter Gogarten and Jeffrey Townsend, HGT can be
inferred from phylogenetic dependent or independent
inspections of genes. In the first approach, the atypi-
cal distribution of genes, inferred from incongruence
between various gene phylogenies, is taken as evidence
of HGT. In the latter approach, genes that seem unu-
sual in their genomic context are considered to have
arrived in their current genome relatively recently
through horizontal transfer. In addition, experimental
approaches that specifically aim at the isolation and
identification of heterologous ‘gene islands’ in closely
related strains1 are also employed.
Molecular evolution examines HGT from a post-
step-5 position (FIG. 1), and whereas evidence of past
HGT in current genomes is pervasive, the ramification
of these observations to our understanding of life’s his-
tory is hotly contested. Certainly, HGT has challenged
our view of the evolutionary history of organisms and
genes from a tree-like paradigm2 to a network-like
paradigm3, and therefore its influence on microbial
speciation and diversification. This area of ongoing
controversy — the concept of the prokaryotic species
— is discussed in detail by Dirk Gevers and colleagues,
who also propose approaches to find a taxonomic
framework that can accommodate the vast differences
in biology presented by prokaryotes.
Understanding the way HGT has contributed
to microbial evolution can help identify intra- and
extracellular processes that affect the stable inherit-
ance of transferred genes in a new genome (step 5 in
FIG. 1). Crucial among them, according to Gogarten
and Townsend, is the question of selective pressure
for the inheritance of transferred genes in their
new host in light of their observation of selective
neutrality of transferred genes. There remain,
therefore, fundamental questions on the actual
(if any) ecological role of such transferred genes
and the mechanism that controls their mainte-
nance in a host genome. If harmful genes (for
example, antibiotic-resistance or virulence genes) are to
be prevented from spreading horizontally, or beneficial
genes (for example, biodegradative genes) are to be
stimulated to do so, information on the processes that
facilitate inheritance after HGT is essential, and bio-
informatic analyses should provide useful clues.
The traditional view that obligate intracellular
parasitic microorganisms have ‘fixed’ minimal
genomes with little influence of intra- and inter-
genomic genetic (ex)change is challenged by Seth
Bordenstein and William Reznikoff. These authors
argue that the extensive presence of mobile genetic
elements (MGEs) such as prophages, plasmids and
transposons in recently sequenced genomes of obli-
gate parasites suggests a more complex picture. As
the association of eukary otic organisms with obligate
intracellular parasites often leads to pathologies, the
issues raised by this review could have far-reaching
practical implications.
Molecular biology
Molecular biology has long been examining the
mechanisms that govern the first steps in the
gene-transfer process (steps 1–4 in FIG. 1), in part
because MGEs are at the core of much molecu-
lar biological experimentation. Diverse elements
and elegant mechanisms have been discovered
and elucidated. The molecular pro cesses that
govern, as well as those that serve as barriers for,
gene transfer have been thoroughly, yet incompletely,
characterized, as reviewed by Christopher Thomas
and Kaare Nielsen. The authors observe that any
identified explicit barrier to HGT (for example,
surface exclusion, restriction and so on) is subject
to genetic and/or physiological modulation, and is
therefore not impermeable. Remarkably little, how-
ever, is known about environmental and molecular
signals that control expression (or overexpression, if
such exists) of the HGT processes. Yet this question
is central to a correct assessment of HGT in microbial
communities. Is HGT an adaptive phenomenon that
is stimulated in challenging environments or is it a
random process of which the outcome is controlled
by natural selection?
Molecular biology has supplied microbial ecolo-
gists with the tools to interrogate the mobile gene pool
in microbial communities from diverse habitats. The
surprise and lesson from such studies is that the diver-
sity of mobile elements is much broader — and prob-
ably underestimated — than what has been gleaned
from the original molecular biological work that
focused primarily on pathogenic microorganisms.
The diversity of MGEs, and especially the challenges
and opportunities in annotation and cataloguing that
arise as their rate of discovery has accelerated with
Figure 1 | The 5 steps of horizontal gene flow. Horizontal gene transfer and how it has
impacted the evolution of life is presented through a web connecting bifurcating branches that
complicate, yet do not erase, the tree of life. The inset illustrates the continuum of 5 steps that
leads to the stable inheritance of a transferred gene in a new host.
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the sequencing of microbial genomes, is addressed by
Anne Summers and colleagues. Together, these ele-
ments are in the process of being transferred (step 2)
and could be considered to be ‘genes in transit’.
Comparison of this gene pool — with the exclusion
of genes involved in the transfer processes themselves,
such as viral genes or plasmid maintenance genes
— with those that are identified in complete genome
sequences by bioinformatic approaches as laterally
transferred genes should generate new insights and
testable hypotheses on the processes that favour stable
inheritance of transferred genes in a new genomic
context (the transition from step 4 to 5 in FIG. 1).
Our expanded view on the diversity and distribu-
tion of MGEs has, to a large extent, been made possible
by the progression in microbial ecology from the
study of pure cultures (and the genetic elements
residing within them) to the direct isolation of nucleic
acids and mobile elements from microbial communi-
ties (for example, exogenous plasmid isolation, direct
sequencing of viral DNA). This newly found diversity
should be matched by an attempt at characterizing
these mobile elements beyond their sequence com-
position and understanding the manner in which
they might enhance microbial genome evolution.
An essential tool for progress towards this goal is
the establishment of curated and carefully annotated
databases and repositories of molecular information
specifically for the mobile gene pool, or ‘mobilome’,
which spans all kingdoms of life. Challenges associ-
ated with this effort are further discussed by Summers
and colleagues.
Microbial ecology
The role of HGT in adaptation of microbial com-
munities to changing environmental conditions has
intrigued microbial ecologists for at least three decades.
Although the advantage of spreading ‘ready made’
genes that enhance fitness under altered conditions
relative to their de novo evolution by the slow process
of mutations acted upon by natural selection is obvi-
ous, obtaining solid evidence of this occurrence in
extant microbial communities has been elusive. Most
evidence to date consists of observations that imply
HGT’s role in response to changing environments.
Chief among them is the frequent association of envi-
ronmentally beneficial genes, such as antibiotic- and
metal-resistance genes and xenobiotic-compound-deg-
radation genes, with MGEs, as described by Summer
and colleagues in this Focus issue. Observations of
such MGEs in man-impacted environments, and in
related but pristine environments, has led to the fas-
cinating hypothesis that the horizontal transfer events
that led to the dissemination of such genes are induced
by the introduction of substances such as antibiotics,
metals or organic contaminants into the environment.
However, the observation of HGT under apparently
selective conditions does not necessarily imply that
the environmental forces caused HGT; it could simply
mean that these elements were enriched to detectable
concentrations.
The documented incidence of HGT, as revealed
from comparative genome-sequence analysis, and
the discovery of an increased diversity of MGEs have
nevertheless given credence to the notion that HGT
could be an important determinant in shaping the
microbial community metagenome. Analytical and
experimental tools developed by molecular evolu-
tion and molecular biology are now routinely used to
examine strains and nucleic-acids pools from different
environments. For example, incongruence between gene
trees has been invoked to suggest horizontal transfer of
metal-homeostasis4 and 2,4-dichloro phenoxyacetic-
acid-degradation genes5 among micro organisms from
aquatic and terrestrial environments.
Perhaps most exciting are new experimental
approaches that facilitate the real-time demonstration
of HGT in undisturbed microbial communities. Søren
Sørensen and colleagues describe the issues, chal-
lenges and achievements in the study of HGT in extant
microbial communities. These methods are largely
driven by technological advances in optical detection
and biomarker construction to permit observations of
single-cell and single-MGE dynamics in undisturbed
microbial communities. Whereas achievements to
date have mostly focused on methods development,
future research employing these methods should place
HGT within the context of contemporary microbial
communities and their activities. The consequences
of such studies to enhance our ability to modulate
interactions with and among the microorganisms
around us might result in a better control of disease
processes and improved environmental management
(see below).
HGT: why care?
While the concept of HGT frequently engenders
joyful intellectual contemplation and lively philo-
sophical exchanges, it carries more than just ivory
tower relevance. The evidence indicates that HGT is a
central process in microbial activities that control our
health and the environment, and that it holds promise
as a tool for their improvement.
The increased global documentation of human
pathogenic bacteria (for example, Streptococcus
pneumoniae, Staphylococcus aureus and Pseudomonas
aeruginosa) that are resistant to multiple classes of
antibiotics — identified as one of the key challenges
to contemporary infectious-disease control — is one
example in which proficient HGT has resulted in
undesirable consequences6. The improper and exces-
sive administration of antibiotics (conferring selective
advantage), combined with the ready bacterial ability
to transfer antibiotic-resistance genes through plasmids
and transposons and the presence of large transfer
communities (for example, the gastrointestinal tract) in
places such as hospitals or animal husbandry facilities,
promotes the widespread dissemination of these genes.
An urgent need for a more prudent use of antibiotics,
combined with a better grasp of the ecology of HGT,
is essential to avoid a return to a pre-antibiotic area of
infectious-disease control7.
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Transgenic organisms hold great promise for
improved food production. Concerns about HGT from
these organisms have, however, shrouded and limited
their application. The appropriateness of current risk-
assessment models8 and monitoring protocols9 to depict
the potential for recombinant gene transfer through
HGT to unintended target organisms are issues that
are subject to fierce debate. A fuller understanding of
the mechanisms and constraints for HGT could ensure
development of effective gene-containment strategies
within target species and ultimately allow the full
realization of the promise of biotechnology.
On the other hand, the spread of genes by HGT
to microorganisms in contaminated environments is
a desired outcome of gene-augmentation strategies10.
In these strategies, donor cells carrying an MGE that
encodes essential genes for the biodegradation of a
target contaminant are introduced into the relevant
environment, and dissemination of the genes to indig-
enous bacteria, followed by expression of the degra-
dative genes in their new hosts, leads to accelerated
contaminant degradation. Although some promising
results have been obtained to date, more studies are
required to evaluate whether the concept of HGT-
based environmental management can be sustained.
The ability to control harmful effects and to enhance
desired attributes of HGT depends on the integrated
understanding of HGT as a continuum spanning
steps 1–5 of the gene-transfer paradigm (FIG. 1) and its
integra tion within an ecological framework.
Conclusions
Clearly, HGT has contributed to prokaryotic evolu-
tion and is an ongoing process in extant microbial
communities. The ‘mobilome’ is therefore receiving
unprecedented attention from a range of scientific
disciplines. The purpose of this themed issue is to
synthesize the state of our knowledge from these dif-
ferent perspectives. We believe that such a synthesis
will be mandatory to obtain a more precise appraisal
of HGT as a force in shaping prokaryotic evolution,
diversity and activity and, therefore, in modulating
the history of life on Earth.
1. Nesbo, C. L. & Doolittle, W. F. Targeting clusters of transferred genes
in Thermotoga maritima. Environ. Microbiol. 5, 1144–1154 (2003).
2. Woese, C. R. Interpreting the universal phylogenetic tree.
Proc. Natl Acad. Sci. USA 97, 8392–8396 (2000).
3. Bapteste, E. et al. Do orthologous gene phylogenies really support
tree-thinking? BMC Evol. Biol. 5, 33 (2005).
4. Coombs, J. M. & Barkay, T. Molecular evidence for the evolution of
metal homeostasis genes by lateral gene transfer in bacteria from the
deep terrestrial subsurface. Appl. Environ. Microbiol. 70, 1698–1707
(2004).
5. McGowan, C., Fulthorpe, R., Wright, A. & Tiedje. J. M.
Evidence for interspecies gene transfer in the evolution of
2, 4-dichlorophenoxyacetic acid degraders. Appl. Environ. Microbiol.
64, 4089–4092 (1998).
6. Monroe, S. & Polk, R. Antimicrobial use and bacterial resistance.
Curr. Opin. Microbiol. 3, 496–501 (2000).
7. Levy, S. B. & Marshall, B. Antibacterial resistance worldwide: causes,
challenges and responses. Nature Med. 10, S122–S129 (2004).
8. Heinemann, J. A. & Traavik, T. Problems in monitoring horizontal gene
transfer in field trials of transgenic plants. Nature Biotechnol. 22,
1105–1109 (2004).
9. Nielsen, K. M. & Townsend, J. P. Monitoring and modeling horizontal
gene transfer. Nature Biotechnol. 22, 1110–1114 (2004).
10. Springael, D. & Top, E. M. Horizontal gene transfer and microbial
adaptation to xenobiotics: new types of mobile genetic elements
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(2004).
Acknowledgements
The authors would like to thank the US National Science Foundation (BES
pogramme) and the US Department of Energy (NABIR programme) for sup-
port of research on HGT in their laboratories. This article and special issue
were inspired by a workshop on ‘Horizontal Gene Flow in Microbial
Communities’ that was co-chaired by the authors in Warrenton, Virginia,
USA, in June 2004, and sponsored by the National Science Foundation
(MO/MIP programme) and the Department of Energy (NABIR programme).
These agencies, as well as the US National Aeronautics and Space Agency
(Astrobiology Programme) provided gracious support to the production of
this issue.
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