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com Gut Microbes 415
Gut Microbes 1:6, 415-431; November/December 2010; © 2010 Landes Bioscience
Addendum to: Jones BV, Sun F, Marchesi JR.
Comparative metagenomic analysis of plasmid
encoded functions in the human gut microbi-
ome. BMC Genomics 2010; 11:46; PMID:20085629;
DOI: 10.1186/1471-2164-11-46.
Key words: mobile genetic elements,
mobile metagenome, gut microbiota,
horizontal gene transfer, toxin-antitoxin
addiction module, metagenomics,
hologenome, holobiont
Submitted: 07/22/10
Revised: 10/21/10
Accepted: 11/02/10
Previously published online:
DOI: 10.4161/gmic.1.6.14087
Correspondence to:
Brian V. Jones; Email:
sing the culture independent
TRACA system in conjunction with
a comparative metagenomic approach,
we have recently explored the pool of
plasmids associated with the human gut
mobile metagenome. This revealed that
some plasmids or plasmid families are
present in the gut microbiomes of geo-
graphically isolated human hosts with
a broad global distribution (America,
Japan and Europe), and are potentially
unique to the human gut microbiome.
Functions encoded by the most widely
distributed plasmid (pTRACA22) were
found to be enriched in the human gut
microbiome when compared to micro-
bial communities from other environ-
ments, and of particular interest was the
increased prevalence of a putative RelBE
toxin-antitoxin (TA) addiction module.
Subsequent analysis revealed that this
was most closely related to putative TA
modules from gut associated bacteria
belonging to the Firmicutes, but homo-
logues of the RelE toxin were associated
with all major bacterial divisions com-
prising the human gut microbiota. In this
addendum, functions of the gut mobile
metagenome are considered from the per-
spective of the human host, and within
the context of the hologenome theory of
human evolution. In doing so, our origi-
nal analysis is also extended to include
the gut metagenomes of a further 124
individuals comprising the METAHIT
dataset. Differences in the incidence and
relative abundance of pTRACA22 and
associated TA modules between healthy
individuals and those with inflammatory
bowel diseases are explored, and poten-
tial functions of pTRACA22 type RelBE
The human gut mobile metagenome
A metazoan perspective
Brian V. Jones
Centre for Biomedical and Health Science Research; School of Pharmacy and Biomolecular Sciences; University of Brighton; Brighton UK
modules in the human gut microbiome
are discussed.
Humans share the planet with an esti-
mated 10
prokaryotic cells and interac-
tion with these microbes has shaped the
course of our development.
humans have co-evolved with microbial
communities that have colonized various
habitats offered by the human body, and
now maintain commensal or symbiotic
relationships with their metazoan hosts.
Of the numerous microbial communities
harbored by the human body, the gastro-
intestinal tract (GIT) is home to the larg-
est. The microbiota resident in the distal
colon of an individual adult consists of
an estimated 10
individual pro-
karyotic cells belonging to an estimated
150–800 species, which are derived from
~1,380–4,018 distinct operational taxo-
nomic units observed in collective gut
microbiomes analyzed.
The numerous
beneficial functions undertaken by this
microbial community, and its capacity
to direct development of host physiology,
epitomises the co-evolution of the gut
microbiota with its human host.
influence may even extend to aspects of
human behavior, and the benefits gained
by acquisition of beneficial microbes may
have partly contributed to the evolution of
social behavior and group living, which
facilitates the transmission of microbial
symbionts to new hosts.
Investigation of host-microbe interac-
tion in the GIT has begun to reveal the
mechanisms underlying these beneficial
functions, and emphasised the intricate
416 Gut Microbes Volume 1 Issue 6
For example, the recent genome sequence
of the human gut commensal E. coli strain
SE11 included six plasmids, which carried
genes involved in adherence to epithelial
cells, tetracycline resistance and bacterio-
cine production.
Conversely, plasmid encoded functions
may also contribute to virulence in patho-
genic bacteria, and gut associated patho-
gens harbor some of the best characterized
These virulence plasmids
have been found to carry genes required
for the production of specific virulence
factors such as toxins, or factors that
facilitate colonization and survival in this
In the latter case, there
is potential for pathogens to acquire these
functions from commensal or symbiotic
members of this community through
HGT, while conjugative plasmids in gen-
eral may contribute to colonization of the
human GI tract by both pathogens and
commensals, since conjugation machinery
has been found to facilitate adherence of
bacteria to various surfaces including epi-
thelial cells.
On the whole, genes and functions
encoded by MGE that are long term
members of the gut mobile metagenome
will also reflect the co-evolution of resi-
dent microbes and their human host.

Recent studies indicate that plasmids har-
bored by gut bacteria also display the sig-
natures of long-term association with the
human host evident in the core genomes
of their bacterial hosts, with the observed
patterns of long-term gene convergence
and conservation evident in chromosomes
of gut bacteria, even more pronounced
for plasmids.
These observations sup-
port the hypothesis for a role of the mobile
metagenome in broader aspects of com-
munity development and are likely to be
reflected in the gut mobile metagenome in
The functions encoded by this flex-
ible gene pool, along with the capacity for
MGE to mediate gene flow between dis-
tinct bacterial species, undoubtedly gen-
erates an additional sphere of complexity
and variability in terms of how this com-
munity develops, functions, evolves and
interacts with the human host. Despite
this, the majority of the human gut mobile
metagenome remains unexplored and
undefined, both in terms of its significance
DNA flanking these genes,
and evidence
for an evolutionarily recent transfer of
tetQ from animal to human gut bacterial
species has been presented.
Overall, the gut mobile metagenome
is considered a reservoir for antibiotic
resistance genes.
This view has been
strengthened by recent function-driven
metagenomic studies highlighting the
frequent association of resistance deter-
minants with genes and sequences related
to MGE in the mammalian gut micro-
and the observation that MGE
encoding resistance to antibiotics such as
tetracycline were prevalent in gut isolates
of Bacteriodes sp. collected prior to the
widespread clinical use of these drugs.

However, there is growing interest in other
functions that may be encoded by MGE
resident in the gut microbiota, and the role
of the mobile metagenome in the develop-
ment and functioning of this community.
Characterization of plasmids from
cultivatable commensal and pathogenic
species found in the human GI tract have
illuminated some of the functions encoded
by this sphere of the gut microbiome.

These include functions relevant to sur-
vival in the GIT and interaction with the
human host, such as utilization of car-
bohydrates and other nutrients, bacterio-
cine production, adhesion to host cells,
resistance to bile acids and virulence fac-
A particularly interesting exam-
ple of MGE encoding such functions are
the large megaplasmids that appear to be
widespread in Lactobacillus salivarius, and
in other Lactobacilli sp. resident in the
human GIT.
In the case of the probiotic L. salivarius
UCC118, this organism harbors three
plasmids, including a megaplasmid encod-
ing genes related to bile tolerance, redox
balance, cell wall biosynthesis and bacte-
riocine production.
Bacteriocine produc-
tion has also been identified as a plasmid
encoded function in other gut associated
while genes providing the ability
to resist the antimicrobial effects of bile in
the small intestine appear to be encoded
by plasmids and MGE associated with a
diverse range of gut bacterial species.

Functions involved in gut colonization
and survival have also been characterized
on plasmids infecting the Enterobacterial
population of the human gut microbiota.
and intimate relationship between eukary-
otic host and prokaryotic symbiont.

More recently, metagenomic approaches
which permit access to the greater uncul-
tivated fraction of the gut microbiota have
revealed functions enriched in the gut
microbiome, and highlighted gene fami-
lies shared among the disparate micro-
bial lineages in this ecosystem.
parallel to these studies, there is currently
much effort focused on defining the bac-
terial and archaeal species comprising the
common phylogenetic core of the human
gut microbiome.
Together, such studies
will ultimately lead to the resolution of the
fundamental structure and metabolic out-
puts of this community, which will allow
us to properly define its impact on human
health and development.
However, just as the human gut har-
bors a complex microbial ecosystem, bac-
teria comprising the gut microbiota in
turn play host to their own “hitchhikers”
in the form of mobile genetic elements
(MGE). These include plasmids, transpo-
sons and integrons and collectively the dif-
ferent elements associated with the human
gut microbiota may be referred to as its
mobile metagenome.
also share many of the properties associ-
ated with these elements, and although are
not MGE in the same sense as plasmids,
transposons or integrons, they are capa-
ble of mediating horizontal gene transfer
(HGT) between bacteria, and for the pur-
poses of this article they are considered to
be part of the gut mobile metagenome.
Investigation of MGE infecting key
cultivatable species comprising the human
gut microbiota have highlighted the role
of elements such as plasmids and conju-
gative transposons (CTn) in the mainte-
nance and spread of antibiotic resistance
genes within this community.

Dissemination of erythromycin resis-
tance between Bacteriodes species in the
human gut has been largely attributed to
horizontal transfer of the relevant genes
by conjugative transposons,
the role of MGE in the acquisition and
spread of tetracycline resistance in the
human gut microbiota is also well docu-
Significant similarity has
been identified between tetracycline resis-
tance genes present in human and animal
gut commensals, including regions of Gut Microbes 417
gut microbiota to date, which identified
around 1,000–1,150 prevalent bacterial
species present in the combined gut com-
munities of the 124 individuals investi-
However, only 18 of these species
were common to all individuals, and it
was estimated that only around 38% of
the bacterial genes present in an individu-
al’s gut metagenome are shared with other
This is in stark contrast to
our primary eukaryotic genome, which
in comparison varies little between indi-
viduals (~0.1% by single nucleotide poly-
morphisms, 0.3% by inversions, 1.2% by
copy number variations/insertions or dele-
or even between Homo sapiens
and its closest living relative, Pan troglo-
dytes (~4%).
In addition, our primary eukaryotic
genomes are essentially fixed in terms of
content throughout the life time of the
host, and lack the plasticity inherent in
our secondary prokaryotic metagenomes.
Prokaryotes frequently exchange genetic
information, and the mobile metage-
nome associated with the human micro-
biota may represent an important fraction
of the human hologenome. This highly
flexible tertiary gene pool may benefit
the human host indirectly via effects on
constituent species, or encode functions
that impact directly on fitness of the holo-
Studies of MGE associated with
microbial communities from plants and
invertebrates demonstrate the potential
for the gut mobile metagenome to encode
genes involved in symbiosis of human and
microbe, as well as specific functions of
direct benefit to the human host.
Therefore, while the primary “human”
segment of our genome evolves relatively
slowly due to a long generation time and
its relatively static nature, our secondary
prokaryotic segment has the capacity to
rapidly adapt to new environmental con-
ditions by virtue of short generation times
and recruitment of new genetic material.

A major advantage of this arrangement
for the human host is the retention of a
genetically flexible secondary genome
which may facilitate adaptation to new
environmental conditions such as changes
in available food supply. This model also
affords the possibility that horizontal gene
transfer (HGT) among human-associated
microbes is of direct benefit to the human
prokaryotic and eukaryotic cells, and fre-
quently referred to as superorganisms.

In this context the gut microbiota has
been described as a “virtual organ” that
undertakes a wealth of functions from
which we benefit, but which our eukary-
otic cells have not evolved for themselves.

These include a barrier function against
colonization by intestinal pathogens, sal-
vage of energy from dietary components
exigent to host digestive mechanisms,
development of the immune system, as
well as aspects of intestinal physiology and
control of epithelial cell proliferation in
the GIT.
If we accept the paradigm of humans
as gestalt entities consisting of both pro-
karyotic and eukaryotic cells, with the
gut microbiota as a “virtual organ”, then
we must also acknowledge the human
genome to be composed of both eukary-
otic and prokaryotic components. This
has resulted in the development of eco-
logical and evolutionary models which
integrate both components of the human
genome to explain the co-evolution of
host and microbe.
Recently, Zilber-
Rosenberg & Rosenberg (2008) described
humans and other complex metazoans as
holobionts and presented the hologenome
theory of evolution, in which the overall
unit of selection in evolution is composed
of the total genetic content of the eukary-
otic host organism, plus that of its sym-
biotic microbial partners.
In this model,
our eukaryotic cells carry the primary core
of indispensible, human defining genetic
information, while our prokaryotic sym-
bionts collectively house a secondary, vari-
able portion of the human genome, which
directs beneficial accessory functions not
encoded for by the primary eukaryotic
The content of the principal prokary-
otic segment may be defined as the core
genome content of the collective microbial
species comprising communities such as
the gut microbiota, and is itself a metage-
nome. Consequently this secondary pro-
karyotic metagenome has the potential
to vary in terms of genetic content (and
therefore its functional output and impact
on host fitness) not only between indi-
viduals but also over the lifetime of the
This is highlighted by the
most in depth analysis of the human
to the human host and human evolution,
as well as total genetic content. Overall,
the mobile metagenome is a facet of the
gut microbiota not readily captured by
current efforts to characterize this com-
munity, but one that will be important in
attaining a complete understanding of this
“virtual organ”.
Utilizing a comparative metagenomic
approach we have recently revealed the
existence of plasmids potentially unique to
the human gut microbiome, which appear
to be present in the gut communities of
geographically isolated hosts distributed
across the globe.
Of particular interest
was the unexpected finding that some of
these plasmids encoded genes which are
enriched in the gut microbial community
when compared to other microbial eco-
reinforcing the hypothesis of
the human gut mobile metagenome as a
reservoir of genetic information involved
in key aspects of community function.
This addendum attempts to place the gut
mobile metagenome within the framework
of recent theories of host-microbe co-evo-
lution, considering functions of this mal-
leable genetic resource from the perspective
of the human host. In doing so, our origi-
nal analysis of plasmids in the human gut
mobile metagenome is extended to encom-
pass an additional 124 human gut micro-
and a preliminary exploration of
differences in plasmid encoded functions
in health and disease is presented.
An Integrated View
of the Human Genome: A Place
for the Mobile Metagenome?
The understanding that our prokaryotic
passengers are more than simply oppor-
tunistic hitchhikers, and play an intimate
role in our development and wellbeing,
has resulted in a revision to the view of
humans (and many other complex organ-
isms) as entities composed exclusively of
eukaryotic cells. In light of our increasing
knowledge of host-microbe (prokaryote-
eukaryote) interaction in the gastrointesti-
nal tract and other body sites, the concept
of humans and other animals as organisms
has evolved to encompass our symbiotic
and commensal prokaryotes.
Mammals, including humans, are now
generally accepted to be amalgams of both
418 Gut Microbes Volume 1 Issue 6
environmental stresses arise (exposure to
relevant antibiotics).
Perhaps of greater importance is the
potential for pathogens to acquire new
functions from well adapted gut symbi-
onts, which may include traits that facili-
tate colonization of the gut or inhibit
treatment of infections. In this context the
transfer of antibiotic resistance genes from
members of the normal gut microbiota to
transiently colonizing pathogens is cause
for concern. Species naturally present in
the human gut that survive in the external
environment and cause disease at other
body sites, such as uropathogenic E. coli,
may also serve to disseminate antibiotic
resistance and other genes from the gut
Host fitness and rapid adaptation. On
the scale of the human holobiont, a major
function of the mobile metagenome may
be as a conduit for gene flow between
the secondary prokaryotic portion of
the human genome (in this case the gut
metagenome) and the wider, external pool
of prokaryotic genetic information. The
mobile metagenome facilitates adapta-
tion and evolution of the secondary pro-
karyotic fraction of the human genome by
mediating the introduction of new traits
into this community.
These directly or
indirectly affect the fitness of the human
host, and in so doing, the human holobi-
ont as a whole.
Adding functional capacity to the gut
microbiota through HGT should be a
faster route to acquiring new activities and
adapting to new environmental factors,

while entailing far less risk than recruiting
new member species. Although attaining
new traits by recruitment of new species
is undoubtedly an important mechanism
underpinning the evolution of the gut
microbiome, and the development of this
community in new hosts, in the case of
an established community this entails the
adaptation of an entire organism to the
gut environment and its integration into
a complex and pre-existing metabolic net-
work, without compromising host fitness.
In contrast the acquisition of new genetic
material, and ultimately new functional
capacity through HGT is more likely to
maintain the status quo and essentially
permits the upgrade of an already func-
tioning and well adapted ecosystem.
members of this community may also be
of benefit to the human host.
HGT is a driving force in bacterial evo-
lution and facilitates rapid adaptation to
new environments. This may benefit the
metazoan host either directly by facilitat-
ing development of beneficial functions
of the gut microbiota, or indirectly by
facilitating the adaptation of community
members to the gut environment, and
development of a stable gut microbiota
with associated benefits to the host.

While the levels of HGT extant in the
gut microbiota may be primarily a conse-
quence of the environmental parameters
in the mammalian gut, such as a high
population density in close spatial proxim-
ity, it is possible that this facet of the com-
munity has been of benefit to the human
host and is important in the evolution
of the human holobiont as a whole. This
theory is supported by advantages offered
directly to the human host though HGT
between autochthonous species of the gut
microbiota, as well as between indigenous
and transiently colonizing allochthonous
species: (1) Introduction of new beneficial
traits and rapid adaptation to environmen-
tal change, possibly within the life time
of the host. (2) Generation of functional
redundancy, increasing functional stabil-
ity of the gut microbiota. Both proposed
advantages of HGT to the human holo-
biont are supported by the hologenome
theory of evolution.
In contrast, there is also potential
for such widespread HGT to introduce
functions which may ultimately prove
deleterious to the human host, such as
antibiotic resistance.
However, it
would be expected that the introduction
of overtly harmful traits into the human
hologenome, including those that may
destabilize important functions under-
taken by our prokaryotic components
(such as the gut microbiota), would be
countered by holobiont level selection,
and ultimately reduced in or eliminated
from the gut microbiome. Traits such as
antibiotic resistance may be exceptions,
since carriage of antibiotic resistance
genes does not appear to be directly harm-
ful to the human holobiont, and results in
detrimental effects only when harbored
by community members able to cause
disease, and then only when particular
host, and raises the possibility that HGT
in the gut microbiota (as well as other
human associated microbial communi-
ties) is in itself an important process in
the evolution of the human holobiont as
a whole.
The finding that certain MGE and
the functions they encode are enriched
and generally conserved in the human
gut microbiome,
underscores the
potential contribution of mobile genetic
elements to the development and perhaps
overall functioning of this community,
and therefore also to host-microbe co-
evolution. For example, the genes under-
lying many of the core functions of the
gut microbiota appear broadly distributed
among members of this community, and
HGT seems to have underwritten the
expansion of these beneficial gene sets
ensuring functional stability of the gut
MGE are also capable
of introducing new genetic material into
an existing microbial community, facili-
tating the development of new functional
pathways within an established ecosystem,
which is almost certainly of significance
to community function past, present and
In broader terms, the abil-
ity of MGE to transfer genetic material
between disparate bacterial species may in
itself be an attribute of the mobile metage-
nome as a whole, that has, and continues
to benefit both host and microbe, and
HGT may constitute a key mechanism
underlying the proposed adaptive role of
the prokaryotic fraction of the human
The Gut Mobile Metagenome:
A Role for Microbial HGT
in Human Evolution?
Activities of MGE are generally studied
in terms of their impact on fitness of the
bacterial host. However, activities of bac-
teria belonging to communities associated
with higher host organisms, such as the
gut microbiota, are ultimately selected for
at the level of the metazoan host through
effects on host fitness.
It is therefore
of note that the human gut microbiome
is considered to be a hotspot for genetic
exchange and there is increasing evidence
to support this claim.
If so, then it
follows that a high level of HGT among Gut Microbes 419
Thus, it appears that this activity was
acquired by members of the Japanese
gut microbiota through conjugal trans-
fer of plasmids encoding this ability
from marine bacteria to gut symbionts.
Subsequently its utility resulted in integra-
tion and fixation in the genomes of human
gut symbionts, enhancing functionality of
the Japanese gut microbiome. Notably,
this appears to be an evolutionarily recent
gain in function, and this activity could
not be detected in any of the gut metage-
nomic datasets derived from American
individuals analyzed in this study,
cating that the Japanese gut microbiota
has evolved this trait as a specific adap-
tation to host diet. These findings rein-
force the proposed adaptive role of the
human prokaryotic genome segment in
the human holobiont, and illustrate how
the gut mobile metagenome can facilitate
this process.
Evidence for a role of HGT in the evo-
lution of other key functions of the gut
microbiota relating to dietary energy sal-
vage and maintenance of gut health, have
also recently emerged. Carbohydrates that
escape host digestive processes are fer-
mented by the colonic microbiota result-
ing in the production of short chain fatty
acids (SCFA).
This process not only
liberates additional energy from the diet
that would not normally be accessible to
the host, but certain SCFA are considered
pivotal to gut health.
Butyrate in par-
ticular has been associated with a range of
beneficial effects,
and not only is this
SCFA a primary energy source for colonic
epithelial cells, but has also been impli-
cated in the regulation of epithelial cell
Recent characterization
of butyrate synthesizing bacteria from the
human colon has implicated HGT in the
development of this pathway in intestinal
It seems likely that this origi-
nally arose as a general adaptation to the
gut environment,
and subsequently, this
pathway may have been selected for at
host-level due to its beneficial effects.
Overall, these studies demonstrate the
capacity for the mobile metagenome to
facilitate adaptation of the gut microbiota
to new environmental conditions, such
as composition of the host diet or envi-
ronmental conditions in the GIT itself,
and in doing so facilitate adaptation of
bacteriophage is notable in this context,
and may in part result from selective pres-
sure exerted by the aphid-bacteria-phage
holobiont to provide a functionally stable
gut microbiota, and minimize the disrup-
tion to key prokaryotic components of the
holobiont that could arise from infection
by lytic phage. Overall, this example dem-
onstrates how the mobile metagenome can
add functional capacity to the prokaryotic
fraction of an organisms hologenome, and
in so doing facilitate adaptation and influ-
ence evolution of the holobiont as a whole.
Recent comparisons of functional dif-
ferences between the gut microbiomes of
American and Japanese individuals have
also provided evidence for HGT mediated
adaptation of the human holobiont, and
identified a role of the human gut mobile
metagenome in this process. In this case
a gain in function of direct benefit to
the human host was identified in the
Japanese gut microbiome, and its acquisi-
tion through HGT revealed.
This func-
tional gain relates to a primary activity of
the human gut microbiome: the salvage of
energy from the diet.
It has been esti-
mated that up to 10% of our daily calories
are derived from microbial fermentation
of plant polysaccharides resistant to host
digestive mechanisms,
which recovers
energy from the diet that is otherwise
inaccessible to the human host.
Seaweeds, and in particular Nori, are
major components of the Japanese diet
and marine bacteria colonizing these algae
produce a range of porphyranse and aga-
rase enzymes in order to utilize the poly-
saccharides they generate.
and co workers demonstrated the acquisi-
tion of porphyranse and agarase degrading
ability by the gut microbiota of Japanese
individuals, through horizontal transfer
of these genes from marine bacteria natu-
rally colonizing dietary seaweed (which
are consumed without cooking), to mem-
bers of the Japanese gut microbiota.
this case genes associated with conjugal
transfer machinery were identified in
regions of DNA surrounding the acquired
porphyranase genes in the gut symbi-
ont Bacteriodes plebius.
These showed
homology to plasmid gene sequences
which encode the putative ancestral por-
phyranase genes in the candidate donor
species of marine bacteria.
Examples of holobiont adaptation
mediated by microbial HGT, and the
mobile metagenome of host associ-
ated microbial communities have been
described previously.
instance, the symbiotic relationship
between Rhizobium sp. and the roots of
leguminous plants is heavily influenced by
MGE encoded genes, with the majority of
functions essential for symbiosis carried
by plasmids, and frequently requiring the
acquisition of several distinct MGE for
successful host-microbe interaction.

Here, Rhizobial plasmids have been found
to encode essential functions which facili-
tate adaptation not only to plant-microbe
symbiosis, but also to a free living lifestyle,
such as tolerance of low pH, heat, drought
or starvation.
This strategy pro-
vides Rhizobium with a highly mobile
gene pool that facilitates rapid adaptation
to diverse and often transient ecological
niches, and subsequently allows modula-
tion of core community functions under-
taken by the symbiotic microbiota to be
adapted to the host legume.
As such
the Rhizobial mobile metagenome likely
facilitates rapid adaptation of the plant-
microbe holobiont as a whole.
A further example of how the mobile
metagenome may encompass functions
of direct benefit to the metazoan host
comes from recent studies of the aphid
symbiont Hamiltonia defensa.
H. defensa
protects its aphid host against attack from
parasitoid wasps by killing wasp larvae
before they can fully develop and bring
about the demise of the aphid.
the production of toxins necessary to kill
wasp larvae is not directed by the prin-
cipal genome of H. defensa, but acquired
through infection of the microbial symbi-
ont with a lysogenic bacteriophage carry-
ing the necessary genes.
In this instance,
the phage encodes functions which essen-
tially ensure that the habitat of its bacterial
host (the aphid) is protected and therefore
that suitable host bacteria will be avail-
able for replication. However, since aphids
with a microbiota lacking this ability will
be vulnerable to attack from parasitoid
wasps, the benefit to the metazoan host
will also result in selection for this attri-
bute from the level of the aphid host, and
therefore for the phage providing these
functions. The lysogenic life cycle of this
420 Gut Microbes Volume 1 Issue 6
in the gut environment must be overcome
without adverse effects on host fitness,
and the resulting host-level selection could
drive the dissemination of relevant genes
to many diverse members of this commu-
nity, generating functional redundancy in
the process.
The salvage of energy from the host diet
is a prime example of a core function of the
gut microbiota that exhibits a high level of
redundancy and stability in this commu-
The ability of gut microbes
to utilize carbohydrates and release energy
to the host in the form of SCFA, appears
to be distributed among a diverse array
of species spanning the main bacterial
divisions of the human gut microbiota
(Bacteroidetes, Firmicutes, Actinobacteria
and Proteobacteria).
While it is
likely that some of this redundancy is the
result of early events in the development of
the gut microbiota, reflecting the recruit-
ment of species capable of colonizing
and utilizing available nutrients without
harming the host, recent studies have also
highlighted the pivotal role of the mobile
metagenome in the convergence and
expansion of the required gene sets.
Comparative genomic and metage-
nomic analyses of Bacteroides species from
the normal human gut microbiota with
non-gut Bacteroides sp. have highlighted
the importance of HGT in the adaptation
of gut-associated species to the human
GI tract.
This included the acquisi-
tion of genes related to the interaction of
these organisms with the host immune
system, as well as utilization of carbohy-
drates indigestible to the human host.

This feature of the analyzed genomes
also demonstrates that in these species
MGE mediate access to a diverse genetic
resource, through which adaption to new
ecological niches and environmental con-
ditions may be rapidly achieved. In a more
global analysis, Lozupone et al. (2008)
investigated the distribution of glycoside
hydrolase and glycoside transferase encod-
ing genes among 36 genomes representing
the dominant bacterial divisions in the gut
microbiota, as well as archaeal species.

This illuminated the convergence of key
carbohydrate active gene families among
diverse members of the gut microbiota,
and again indicated HGT as a major pro-
cess facilitating the dissemination and
the gut microbiota to fluctuate through-
out the lifetime of the host, there is scope
for considerable variation in the prokary-
otic section of the human genome, and
therefore the functional output of this
2,3,8,63, 64
Despite this, in the
adult human holobiont, the human gut
microbiota is considered to exhibit a high
level of stability in terms of the major
core functions of this community, such as
energy salvage.
How this functional output is stabilized
and maintained in spite of the variable
nature of the prokaryotic metagenome has
not yet been fully elucidated, but the gen-
eration of functional redundancy within
this community is considered an impor-
tant mechanism.
Redundancy may
be achieved through the wide spread dis-
tribution of genes underlying community
level functions to diverse and disparate
members of the gut community.

This should guard against the loss of key
activities and pathways from the commu-
nity and allow the gut microbiota to main-
tain its overall functional output despite
shifts in community composition.
Although redundancy of core func-
tions may be achieved via the recruit-
ment of diverse species with overlapping
metabolic capabilities, evidence for a key
role of HGT in generating functional
redundancy in this community has also
The movement of MGE
between disparate species belonging to
the mammalian gut microbiota has been
demonstrated both in vitro, in vivo, and
in silico,
and HGT has already
been linked with development of several
primary activities of the gut microbiota
which exhibit a high level of redundancy
in this community.
the mobile metagenome has likely facili-
tated the dissemination of key traits
involved in core community functions to a
wide range of community members, play-
ing a major role in developing a function-
ally stable ecosystem.
In particular, it would be expected
that challenges faced by most or all of the
microbes colonizing a particular environ-
ment, would promote the acquisition of
genes providing solutions from other spe-
cies colonizing the same habitat.
bacteria comprising the human gut micro-
biota, the barriers to survival encountered
the human holobiont as a whole. In par-
ticular, the capacity to rapidly adapt to
new dietary components would serve the
human holobiont by allowing new food
sources to be exploited as they become
available or when access to the normal food
supply is restricted. It is easy to see how
such dietary flexibility, and the ability to
obtain the maximum possible energy yield
from a wide range of food sources would
benefit the ancestors of modern humans,
for whom the source of each meal would
almost certainly vary significantly.
This may be reflected in the division
of the genetic information encoding these
important pathways of dietary energy sal-
vage, which despite the obvious impact on
host fitness, are not present in the primary
eukaryotic portion of our genomes, but
remain in the secondary, flexible prokary-
otic segment. The reason for this, at least
in part, may be the advantage of rapid
adaptation offered by the prokaryotic
metagenome, which through the mobile
metagenome has access to a vast genetic
resource. In the examples discussed here,
HGT rather than changes in species com-
position, has facilitated the adaptation of
the holobiont,
supporting the
hypothesis that HGT in the gut microbi-
ota is of direct benefit to the human host.
This also affords the intriguing pos-
sibility that the observed degree of gene
exchange extant in the gut microbial com-
munity may not only be due to the physi-
cal characteristics of the gut environment
and intrinsic properties of species com-
prising this ecosystem, but also the result
of host-level selection for activities of the
gut microbiota which impact favorably on
host fitness. However, more in depth stud-
ies of the relative levels of HGT between
various host associated microbial com-
munities and those not subject to host
level selection are required to validate this
The mobile metagenome and func-
tional stability of the gut microbiota.
Core functions of the gut microbiota
that impact on host health are ultimately
selected for at the level of the human host,
and there is pressure to ensure they are
stable over time and consistently delivered
by this community.
However, in light of
the plasticity of bacterial genomes and the
potential for the species composition of Gut Microbes 421
Therefore, the identification and charac-
terization of MGE enriched in the gut has
the potential to greatly increase our under-
standing of the gut microbiota, and its
interaction with the human host. However,
cataloguing the MGE comprising the
human gut mobile metagenome, and iden-
tification of gut enriched elements is a chal-
lenging task. In light of the enormous and
predominantly uncharacterized diversity
of the microbial world, the inherent pro-
miscuity of MGE, and the methodological
challenges associated with studying these
elements, identification of MGE poten-
tially unique to or enriched in the gut will
not be straightforward.
membership of the gut mobile metage-
nome, in accordance with the nature of
MGE, is likely to be much less exclusive
or committed than for the core commen-
sal or symbiotic bacterial species compris-
ing this community. As such this section
of the human hologenome is likely to be a
much less clearly definable gene-space than
the principal prokaryotic gut metagenome.
Nevertheless, several recent studies includ-
ing our own, have provided evidence sup-
porting the existence of gut specific MGE,
and offered good candidate elements.
In our recent study, plasmids were iso-
lated from the gut microbiome using the
culture-independent TRACA system,
which is capable of capturing plasmids
from a wide range of bacterial species
comprising the gut microbiota, and facili-
tating their maintenance in surrogate
host species.
The complete nucleotide
sequences of six plasmids captured from
the human gut microbiome were used to
search a range of metagenomic data sets
derived from the human gut,
and terrestrial environ-
In this comparative metage-
nomic analysis, sequences homologous
to two of our plasmids (pTRACA10
and pTRACA22) were detected in mul-
tiple human gut metagenomes, indicat-
ing a broad distribution.
In contrast no
sequences with homology to any of the six
plasmids analyzed were identified in any
of the non-human metagenomes studied.
While none of the metagenomic data
sets currently available afford complete
coverage of their representative com-
munities (particularly for environmental
ecosystems), this nonetheless indicates
MGE Enriched
in the Gut Mobile Metagenome
The proposed integration of the prokary-
otic mobile metagenome into the human
hologenome proper, leads to certain pre-
dictions about the composition of the
pool of MGE comprising the gut mobile
metagenome that may be investigated
in more detail. In particular, if the gut
mobile metagenome constitutes a compo-
nent of the human hologenome as a whole,
then the co-evolution of host and microbe
should also be reflected in the MGE asso-
ciated with the gut microbiota, and the
functions encoded by constituent MGE
will also be subject to selective pressure at
the level of the human host.
This hypothesis is supported by recent
studies of gene convergence in gut associ-
ated bacterial species compared to non-gut
species, in which plasmids carried by gut
bacteria were found to reflect the overall
signatures of increased specialization at
short-phylogenetic distances and long-term
gene convergence at greater phylogenetic
distances, that were observed for bacterial
In addition, the long-term
convergence of gene content identified in
gut associated plasmids also suggest the
general functional conservation exhibited
by the human gut microbiome of indi-
vidual human hosts, may also extend to
aspects of the gut mobile metagenome.
As such it would be expected that the
most successful and well adapted MGE
in this community, which satisfy the
demands of both bacterial and human
hosts, will exhibit the greatest prevalence
in the gut mobile metagenome, and be
common in the gut microbiomes of a
board range of individuals. These gut
“specific” elements would be more likely to
encode factors that provide bacterial hosts
with an advantage in this environment,
such as colonization of and survival in the
gut, as well as to carry genes involved in
host-microbe interaction and functions of
the community influencing host health.
MGE mediated host-microbe interactions
have already been described in bacterial
communities associated with plant and
insect hosts, and in particular the example
of bacteriophage encoded protection of
aphid hosts from parasitoid wasps would
appear to support this theory.
expansion of gene sets involved in core
community functions.
Microbes inhabiting the human gut
also face a number of direct barriers to
colonization which must be overcome
without reducing host fitness.
these are the toxic effects of conjugated
bile acids (CBA) encountered in the small
intestine. HGT has also been implicated
in the dissemination of genes which facili-
tate resistance to CBA (bile salt hydrolase:
BSH) among a broad range of microbes
in the gut community.
The real-
ization that bile acids also function as key
signalling molecules regulating important
aspects of host metabolism and immune
function, means that bile acid modifica-
tion by gut microbes also has the potential
to greatly influence host physiology and
Although our understanding of how
this aspect of the gut microbiota relates to
host health is currently limited, the very
broad distribution of BSH activity and the
resulting stability of this function in the
community indicates host-level selection
for this activity, and therefore a benefit to
the human holobiont as a whole.
In keep-
ing with this, the general enrichment and
excellent functional redundancy of BSH
activity means that although overall capac-
ity for this activity may fluctuate over the
lifetime of the host in response to changes
in population structure of the gut micro-
biota, it is highly unlikely that this func-
tion will be lost from an established gut
It seems likely that much of
the functional redundancy for BSH activ-
ity in this community has been achieved
through HGT, perhaps even transcending
barriers between distinct domains of life
(Bacteria and Archaea).
In our recent comparative metagenomic
study of plasmids from the gut mobile
metagenome, we also provided evidence
for a high level of redundancy and gen-
eral enrichment of certain toxin-antitoxin
(TA) modules in this ecosystem.
was indicated by the broad phylogenetic
distribution of the pTRACA22 type RelE
toxin sequences among all major bacterial
divisions in the gut microbiota. However,
the role of these TA modules, if any, in the
overall functioning of the gut microbiota
remains to be established, and is discussed
in subsequent sections of this article.
422 Gut Microbes Volume 1 Issue 6
level of inter-individual variation in car-
riage of phage GB124 remains to be estab-
Currently, our understanding of
how bacteriophage may influence human
health is limited, but phage not only have
the potential to alter microbial community
dynamics and metabolic output by selec-
tive elimination of species within the gut
microbiota, but may themselves interact
directly with the host via the immune
However, coverage of the gut com-
munity offered by the datasets utilized in
our original analysis of plasmids resident
in the human gut mobile metagenome is
generally low, and it was not possible to
retrieve the complete sequence of the most
widely distributed plasmid, pTRACA22,
from either individual or combined data
In total we found that 81.9% of
the pTRACA22 nucleotide sequence was
represented in these combined datasets at
an identity of 90% or greater, and likely
indicates the general enrichment and con-
servation of a closely related family of plas-
mids of which pTRACA22 is a member.
This suggests that metagenomic data
sets with greatly increased coverage of
the gut microbiota will afford a concor-
dant increase in the degree of coverage
mobile metagenome, may be a manifesta-
tion of community adaptation to modern
environmental stresses such as the use of
Metagenomic studies have also revealed
the high abundance of bacteriophage
associated with all microbial communi-
ties including the human gut microbi-
ome, where it is estimated a minimum
of ~1,000 distinct viruses exist (although
this is likely to be a substantial underesti-
Because the host range for bacte-
riophage typically varies from a few closely
related species down to individual strains,
the existence of gut specific bacteriophage
seems a likely prospect, and candidate
phage apparently unique to the human gut
microbiome have already been described in
reference 93. Phage infecting a common gut
commensal, Bacteroides sp. GB124 which
has been tentatively identified as a strain of
Bacteroides ovatus (Ebdon J, personal com-
munication), were isolated from human
However, this phage was found to
be absent from fecal samples derived from a
wide range of common domestic and wild
animals, and was not present in the general
Furthermore, widespread
carriage among the human population was
also indicated by this study, but the actual
the capture of human gut specific MGE.
The presence of sequences homologous
to pTRACA22 in particular, in multiple
human gut metagenomes derived from
individuals with broad geographic origins
(America, Japan and Europe), suggests
a gut specific family of mobile elements
which has a deep co-evolutionary rela-
tionship with this community and global
A similar broad global distribution
and general enrichment for a family of
conjugative transposons in the human
gut microbiome has also been described
in reference 20. Designated CTnRINT
(Conjugative transposons rich in intes-
tine), these elements are related to the
Tn-1549 family CTns found in a range of
gut associated pathogenic microbes includ-
ing Enterococcus faecalis and Clostridium
CTnRINT elements display
similar gene architecture to Tn-1549 ele-
ments from other bacteria, but differ in
an accessory gene region. It is possible
that the genes encoded by this region of
the CTnRINT elements relate to activi-
ties advantageous to life as a human gut
commensal or symbiont.
the current enrichment of CTnRINT,
as well as other MGE in the human gut
Figure 1 (See opposite page). Incidence and relative abundance of plasmids pTRACA10, pTRACA17, pTRACA18, pTRACA20, pTRACA22 and pTRACA30
in the METAHIT gut metagenome data set. The complete nucleotide sequences of each plasmid were used to search the METAHIT
dataset using
Blastn, and calculate incidence and relative abundance according to methods described previously in reference 21 and 22. For this analysis, only
hits matching the following criteria were considered signifcant: An identity of 90% or greater over 100 nucleotides or more, and an e-value of 1 e

or lower. In addition to the complete METAHIT data set, incidence and relative abundance of plasmids in sub sets of individuals represented in the
METAHIT dataset was also explored based on nationality (Danish or Spanish), gender, and disease status (healthy, Crohn disease or ulcerative colitis).
For the purpose of this analysis individuals were designated as healthy unless indicated as being diagnosed with ulcerative colitis (UC) or Crohn
disease (CD), regardless of age or body mass index. The statistical signifcance of observed incidence and relative abundance data was explored using
the χ
Where signifcant diferences in relative abundance of plasmids between metagenomic datasets were identifed, the statistical
power aforded by the available sample sizes in each group was calculated using the Piface program.
This indicates the probability that the observed
diferences are truly refective of the wider population, with a probability of 0.8 considered to indicate adequate power. Symbols above bars indicate
the level of signifcance in χ
analysis, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Statistical power is shown in adjacent parentheses, and in all cases has been
computed to p = 0.05 using variation (standard deviation) observed within the total METAHIT dataset. Danish HEALTHY, Spanish HEALTHY = Combined
gut metagenomes from individuals of each nationality in the METAHIT data set, excluding those diagnosed with IBD; Male HEALTHY, Female HEALTHY
= Combined gut metagenomes from individuals of each gender in the METAHIT data set, excluding those diagnosed with IBD; Healthy TOTAL = Com-
bined gut metagenomes from all healthy individuals in the METAHIT dataset; CD, UC = Combined gut metagenomes from individuals diagnosed with
respective IBDs; TOTAL METAHIT = Combined gut metagenomes of all 124 individuals regardless of nationality, gender or disease status. (A) Shows the
incidence of each plasmid (and closely related elements) among the gut metagenomes comprising the METAHIT data set, when grouped by national-
ity, gender and disease statues, as well in the METAHIT cohort as a whole. Incidence is expressed as the percentage of metagenomes in which at least
one signifcant hit to each plasmid was detected, (according to criteria outlined above). In this analysis, only diferences found to be supported by ad-
equate statistical power (0.8 at p = 0.05) were considered signifcant overall. (B) Shows the relative abundance of each plasmid, expressed as hits/Mb,
among the gut metagenomes comprising the METAHIT data set, as grouped in (A).
For this analysis relative abundance was calculated based on the
total number of signifcant hits (according to criteria outlined above) in each group (gender, nationality, disease status), and the average relative abun-
dance in each cohort displayed. INSET: Shows the overall inter-individual variation for each plasmid among all 124 individuals in the METHIT dataset,
with yellow bars representing lowest observed abundance, red bars highest observed abundance, and green bars showing the average abundance
over all 124 individuals. (C) Shows the percentage of each plasmid nucleotide sequence that could be recovered from the total METHIT dataset. Colors
of segments represent % identity of recovered METAHIT sequences corresponding to respective regions of each plasmid, and numbers below pie
charts provide the coordinates in the complete plasmid sequence for each segment. Yellow segments = 92–94% identity; orange segments = 94–96%
identity; red segments = 96–98% identity; black segments = 98–100% identity. Gut Microbes 423
homology could be retrieved (pTRACA17,
pTRACA20, pTRACA30; Fig. 1).

Notably pTRACA22 still exhibited signif-
icantly higher relative abundance (p = 2

or lower) in these human gut microbiomes
compared to the other plasmids, and the
greatest incidence of the six plasmids with
sequences homologous to pTRACA22
detected in 73.3% of the 124 individual
metagenomes comprising the METAHIT
data set (Fig. 1). However, it is not clear
if this is solely due to the greater cover-
age afforded by the METAHIT data set,
or is a result of its European origin and
therefore a reflection of variation in the
gut mobile metagenomes of different eth-
nic groups, but is likely a combination of
both factors.
124 European individuals.
As predicted,
using this data set permitted a much greater
proportion of the pTRACA22 nucleotide
sequence to be retrieved, and in general
homologous sequences with much higher
identity were recovered for all plasmids
analyzed in our original study (Fig. 1C).
In total we were able to recover 99.4% of
the pTRACA22 nucleotide sequence at an
average identity of 97.7% or greater using
the METAHIT data set (Fig. 1C).
The METAHIT data set also permit-
ted the retrieval of sequences homologous
to several other plasmids characterized in
our recent study, which were either poorly
represented in the American and Japanese
data sets originally utilized (pTRACA18),
or for which no sequences with significant
of the plasmids analyzed in our study.
Furthermore, since the general conserva-
tion of plasmid pTRACA22 in the gut
mobile metagenomes of geographically
isolated individuals suggests their asso-
ciation with the human gut microbiota
is ancient, some divergence in plasmids
resident in the gut microbiomes of vari-
ous ethnic groups is to be expected. As
such, use of larger metagenomic data sets
that are also derived from the same broad
ethnic group (European) as the original
pTRACA22 plasmid should also increase
coverage of these plasmids. Therefore,
the recently released METAHIT data set,
which affords the greatest coverage of the
gut metagenome to date, was utilized to
extend our original analysis to a further
Figure 1. For fgure legend, see page 423.
424 Gut Microbes Volume 1 Issue 6
bacterial or human host, or is the result
of the addictive nature of these modules
Perhaps the simplest explanation
for the enrichment of the pTRACA22 type
RelBE modules identified in our study,
stems from the latter possibility, and relates
to the potential for these gene systems to
exist purely as selfish entities.
It is conceiv-
able that these pTRACA22 type TA mod-
ules present no appreciable impact on host
fitness, and in terms of the gut microbiota
and the human holobiont as a whole, are
evolutionarily neutral.
This explanation does not appear to
account for the differential enrichment
of the pTRACA22 RelBE family of TA
modules over other TA systems, and per-
haps even over other RelBE variants.

However, the observed trend in relative
abundance of the TA systems we exam-
ined fits comfortably with this “selfish
DNA” hypothesis, since there is no evi-
dence at present which excludes the pos-
sibility of selection against the other TA
systems, rather than explicit selection for
the pTRACA22 sub-family. Therefore,
the prevalence of the pTRACA22 type
RelBE modules we identified may simply
result from their addictive nature, coupled
with a neutral or insignificant impact on
fitness of the bacterial and ultimately the
human host.
If this is so, then TA modules expanded
in microbial communities such as the
human gut may be under pressure to
minimize any adverse effects on bacte-
rial or human hosts. In this context it
is notable that among the pTRACA22
RelE homologues we retrieved from the
human gut metagenomes analyzed, cer-
tain amino acid residues were found to be
completely or predominantly conserved
specifically among gut associated RelE.

This observation is more significant in
light of the broad phylogenetic distribu-
tion identified for the pTRACA22 type
RelE toxin sequences, and while the
majority of these were affiliated with
members of the Firmicutes division, rep-
resentatives also grouped with members
of the Bacteroidetes, Actinobacteria and
Together, a broad phylo-
genetic distribution, and a conserved gut
associated sequence motif, indicates the
development and expansion of a gut spe-
cific RelE sub type. However, the “selfish
in the dissemination and ultimate enrich-
ment of genes involved in core commu-
nity level outputs, the overall abundance
of genes and functions encoded by gut
specific MGE is of considerable interest.
It would be expected that the genes and
functions encoded by MGE enriched in
this community exhibit an increased prev-
alence in the gut microbiome as a whole.
This is indeed the case for genes encoded
by the CTnRINT elements described
by Kurowkawa et al. (2006),
as well as
certain genes encoded by the pTRACA10
and pTRACA22 plasmids analyzed in our
For CTnRINT, genes encoded by
this gut associated family of MGE were
found to account for around 0.8% of the
5,325 predicted ORFs identified in the
combined metagenomic sequence data
from 15 Japanese gut microbiomes.
genes were also found to be enriched in gut
metagenomes of American individuals,
highlighting CTnRINT as a long standing
member of the gut mobile metagenome.
Utilizing these same metagenomic
data sets, we also observed the enrich-
ment of several ORFs encoded by plas-
mids pTRACA10 and pTRACA22 in the
human gut metagenome.
These enriched
ORFs cover a wide range of functions and
include genes for phosphoesterases or
phophohydrolases, replication proteins, a
TA addiction module, as well as genes of
unknown function.
Of particular inter-
est was the increased prevalence of a puta-
tive RelBE TA addiction module encoded
by pTRACA22, which was unexpected
in this community.
While TA modules
are generally highly abundant in free liv-
ing bacteria and archaea (i.e., those not
forming obligate intracellular relation-
ships with eukaryotic host cells),
analysis revealed that this enrichment in
the gut was confined to homologues of the
pTRACA22 type RelBE module, and was
not observed for MazEF, ParDE or HigBA
This naturally leads to the fol-
lowing questions: why is this particular
family of TA modules enriched in the
human gut microbiota? What role, if any,
do these genes play in the overall opera-
tion of the gut microbial community?
In addressing the first question, it is
important to consider whether the observed
enrichment results from selection for these
modules due to benefits conferred on the
Furthermore, the plasmids isolated in
our original analysis range in size from 3.7
to 10.8 kb. It is possible that this size range
reflects a restriction of the TRACA system
used to isolate them, the dominance of
smaller plasmids in the gut microbiome,
or a combination of these factors.

While the smaller size of these plasmids
do not alter the observation that some
plasmid families are generally enriched
and broadly distributed in the human gut
similar studies of larger
plasmids would be useful in understand-
ing how plasmid size may relate to cover-
age in metagenomic datasets.
Overall, MGE potentially enriched in
the human gut mobile metagenome such as
pTRACA22 family plasmids, CTnRINT
type transposons and phage GB124, along
with numerous as yet uncharacterized
gut specific or gut enriched MGE, could
conceivably constitute the mobile metage-
nome equivalent of a conserved “phyloge-
netic core”. It also seems most probable
that MGE enriched in the human gut
mobile metagenome will be associated
with numerically dominant members of
this community. Given the potential for a
bacterial host to support a range of MGE,
those elements that are most frequently
associated with abundant, and presum-
ably successful and well adapted species,
in the gut microbiota are likely to confer
selective advantages. While enrichment of
these elements or association with numeri-
cally dominant members of the commu-
nity does not necessarily indicate a role
in community function or survival in the
gut environment, characterization of such
elements is most likely to provide insights
into the role of MGE in the development
and functioning of the gut microbiota,
particularly given that plasmids (and
most likely other MGE comprising the
mobile metagenome) also appear to be
susceptible to long-term habitat associ-
ated convergence of gene content in this
Plasmid Encoded Functions
Enriched in the Human
Gut Microbiome
In light of the prevalence of certain MGE
within the human gut mobile metage-
nome, and the prominent role of HGT Gut Microbes 425
question of what role these gene systems
may play in the human gut microbiota
or for the human holobiont as a whole,
and there are a range of possible answers.
TA modules have been associated with
a wide variety of activities which may
be relevant to microbes inhabiting the
human gut. These include contribution
to fitness of the bacterial host through
modulation of gene expression,

the establishment of new regulatory gene
formation of persister cells
and resistance to environmental stresses
such as nutrient limitation,
as well
as the potential for direct host-microbe
interaction through toxic effects of RelE
on eukaryotic cells.
Known or pro-
posed functions of TA modules that may
be relevant to the human gut microbi-
ome are summarized in Table 1 along
with the potential utility of these attri-
butes to gut microbes and implications
for both the human host and microbiota
as a whole.
Importantly, in the case of mecha-
nisms underlying bacterial colonization
and persistence in the gut, these must not
only permit bacteria to overcome particu-
lar environmental stresses, but must do so
without any negative effect on fitness of
the human host, and TA modules would
appear to fulfil this remit. It is also con-
ceivable that discrete functions mediated
by TA modules are not only differentially
selected for in various host bacteria, (with
different host species benefiting from dis-
tinct functions of the same TA module),
but are also of most benefit at particu-
lar stages in the development of the gut
microbiota or under specific environmen-
tal parameters which are not a constant
feature of the gut environment. If so, the
observed prevalence of these TA modules
may result from the wide range of func-
tions potentially mediated by TA (Table 1),
which make them of benefit to a broad
range of community members under a
wide range of conditions. The addictive
properties of these gene systems would
only serve to reinforce any positive selec-
tion for these modules and stabilize them
within the community.
When considered from the perspective
of the human host and within the con-
text of the hologenome theory of human
evolution, there also exists the possibility
selective effects, rather than a general
cloning bias. This is reinforced by the well
established presence and functionality of
the TA systems we examined in E. coli
(RelBE, MazEF, ParDE, HigBA).
However, to investigate this issue more
definitively, the METAHIT data set was
again utilized,
and the relative abun-
dance of the pTRACA22 RelBE family of
TA modules in the combined gut metage-
nomes of the 124 individuals represented
was assessed (Fig. 2). This illuminated the
large inter-individual variation in the rela-
tive abundance of pTRACA22 like RelBE
modules between the individuals in this
dataset (Fig. 2A), and demonstrated a
pervading trend of a higher abundance
of RelB anti-toxin in the majority of indi-
viduals (Fig. 2B). The incidence of these
pTRACA22 like RelBE TA modules was
high, and we were able to detect homolo-
gous TA modules in 93% of individuals
represented in the METAHIT data set
(Fig. 2B).
Notably the METHIT data set was
generated using distinctly different
methods for DNA extraction, sequenc-
ing, and library construction than the
previously analyzed data sets, including
the use of small and very small insert
libraries unlikely to harbor intact TA
Regardless of these differ-
ences in methodology, no significant
differences were observed in the relative
abundance of RelBE modules between
our previous analysis (based on the
combined American and Japanese data-
and that calculated from the
METAHIT data (Fig. 2C).
The varia-
tion in pTRACA22 type TA module
abundance between the data sets utilized
in our original study and healthy indi-
viduals in the METAHIT dataset was
found to be 0.004 and 0.012 hits/Mb
for RelE and RelB respectively. Thus it
appears highly unlikely that the observed
enrichment of the pTRACA22 family of
RelBE TA modules is an artefact of the
metagenomic approach.
Apart from the “selfish DNA” hypoth-
esis, there also remains the possibility
that the pTRACA22 family RelBE mod-
ules have been selected due to a beneficial
function or attribute they confer upon
members of the gut community or even
the human host. This leads back to the
DNA” hypothesis for the observed enrich-
ment of pTRACA22 type RelBE modules
in the human gut microbiome is neither
supported, nor refuted by our findings,
and further investigation of the role of
these systems in the gut microbiota is
required to test this hypothesis.
Alternatively, the addictive proper-
ties of TA modules could account for
the observed enrichment through more
nefarious mechanisms which lead to
perturbations in metagenomic data sets.
The contribution of TA modules to gen-
erational stability of plasmids is well
established, and plasmid encoded TA
modules ensure that daughter cells retain
a copy of the plasmid through a post seg-
regation killing mechanism (PSK).

Loss of the plasmid effectively results in
loss of the unstable antitoxin, leading to
cell death or growth arrest through the
continued activity of the stable toxin
component which persists in plasmid
free cells.
PSK may account for the
enrichment of certain RelBE TA mod-
ules by stabilizing metagenomic clones
in the surrogate E. coli hosts used to
construct metagenomic libraries. This
could lead to the artificial enrichment of
TA module harboring clones in the final
metagenomic data set. In addition, the
issue of biases generated by use of differ-
ent extraction procedures and sequencing
strategies used to generate metagenomic
data sets, may also be an underlying fac-
tor in the observed trends. Despite this,
there is much evidence indicating that
the prevalence of pTRACA22 type TA
modules in the human gut microbiome
is not the result of bias in these data sets,
and reinforces the utility of available
metagenomic data.
The existence of orphan toxin genes
identified in metagenomic sequences
argues against bias induced by TA medi-
ated stabilization of metagenomic clones,
and no significant differences in terms
of relative gene abundance were identi-
fied between the Japanese and American
data sets utilized in our previous study.

This is in spite of the reported paucity of
sequences from Bacteroides sp. in the lat-
ter data set.
Furthermore, the differential
enrichment of pTRACA22 family RelBE
modules compared to other TA systems in
itself points to enrichment though specific
426 Gut Microbes Volume 1 Issue 6
metagenome may be an auxiliary, flex-
ible gene pool that introduces additional
variation at a level distinct from that of
the principal gut metagenome (which is
based on the core genome content of con-
stituent species). Changes in the structure
and metabolic output of the human gut
microbiota are being increasingly linked
to human disease, and there is potential
for the mobile metagenome to contribute
to such changes.
Bacteriophage in particular may be
involved in the pathogenesis of disorders
such as inflammatory bowel diseases
(IBD). As well as mediating HGT and
MGE, Community Output and
Intestinal Disease: A Preliminary
Our original comparative metagenomic
analysis also hinted at the inter-individual
variation of the gut mobile metagenome,

and this has been further supported by the
analysis of the METAHIT dataset pre-
sented in this article (Fig. 1). Variation in
this sphere of the human hologenome, as
with other sections, has the potential to
contribute to differences in the metabolic
output of gut communities between indi-
In this respect the gut mobile
that the primary reason for the observed
enrichment may be selection for these
pTRACA22 type RelBE TA modules at
the level of the human host. In this case,
carriage of these modules may impose a
fitness cost on the bacterial host which is
ultimately offset by increased fitness of the
holobiont as a whole. However, further
study is required to determine if these
modules simply persist as selfish entities
in the human hologenome, with no tan-
gible impact on the human holobiont or
contribute to functions undertaken by the
gut microbiota and its interaction with the
human host.
Figure 2. For fgure legend, see page 427. Gut Microbes 427
pTRACA10 in healthy Spanish compared
to healthy Danish individuals, although
power calculations indicated this is almost
certainly not a true representation of the
wider population (Fig. 1B). The incidence
of all plasmids was found to vary between
different groups, particularly healthy indi-
viduals and those diagnosed with CD, but
no significant differences were observed
(Fig. 1A). However, significant differences
in relative abundance of pTRACA10,
pTRACA22 and pTRACA30 were identi-
fied in individuals with CD or ulcerative
colitis (UC), compared to healthy indi-
viduals, but in most cases available sample
sizes do not provide adequate statistical
power to infer an association in the wider
population (Fig. 1B).
Interestingly, the relative abundance
of pTRACA22 type TA modules was also
altered in both CD and UC disease states
(Fig. 2C). In the CD cohort the pervad-
ing trend for higher abundance of RelB
antitoxin over the RelE toxin in healthy
individuals was reversed, with RelE toxin
elevated and RelB antitoxin reduced in
comparison to healthy individuals and
those with UC, although this was not sta-
tistically significant (Fig. 2C), despite the
distinct separation of the gut microbiomes
of healthy individuals and those with CD
and UC reported by Qin et al. (2010).
the case of UC, a statistically significant
In the case of the human gut specific
phage infecting Bacteroides GB124, the
observation that the host species for these
phage may be a strain of Bacteroides ova-
tus also has implications for their involve-
ment in CD. The titre of serum antibodies
against Bacteroides ovatus is significantly
elevated in patients with CD, in contrast
to that of other common gut associated
Bacteriodes sp. (B. fragilis and B. vulga-
While it is presently unclear if this
is indicative of a role for B. ovatus in the
pathogenesis of CD, the immunogenicity
of this organism, and the increased levels
of both IgG and IgA anti-ovatus antibod-
ies in CD patients, suggests a possible pro-
tective role for bacteriophage capable of
killing this organism.
The METAHIT data set also allowed
us to explore more fully the incidence
and relative abundance of plasmids cap-
tured in our original study (pTRACA10,
pTRACA22 and pTRACA30) among
individuals of different nationalities,
genders or disease status (Fig. 1). For all
plasmids a large inter-individual varia-
tion in incidence and relative abundance
was observed (Fig. 1B inset), but when
group data was analyzed, no significant
difference between healthy individuals by
nationality or gender was identified, with
the exception of relative abundance of
harboring genes involved in important
activities of the gut microbiota, bacterio-
phage may also be a major force behind
changes in the species composition of the
gut community within an individual.
Selective attack on dominant species in
the gut microbiota opens new niches for
less abundant members, and phage driven
population shifts are well documented in
other bacterial ecosystems.
This aspect
of phage-bacteria interaction is of particu-
lar significance in light of recent obser-
vations by Lepage et al. (2008),
described distinct differences in abun-
dance of bacteriophage between healthy
individuals and those suffering from
Crohn disease (CD). This raises the pos-
sibility that bacteriophage drive the dys-
biosis of the gut microbiota observed in
CD patients, and that phage driven shifts
in community structure may be involved
in disease initiation or progression.

Alternatively, the observed potential for
bacteriophage to translocate from the gut
environment and themselves act as anti-
gens raises the possibility that phage play
a direct role in the pathogenesis of CD,

and also fits with their increased preva-
lence in CD patients.
In both hypoth-
eses, the relapsing and remitting nature
of CD may correspond to predator-prey
population dynamics of relevant phage
and their bacterial hosts.
Figure 2 (See opposite page). Relative abundance of pTRACA22 type RelBE modules in the METAHIT dataset and variation in infammatory bowel
disease. The relative abundance of pTRACA22 type RelBE modules in the gut microbiomes of individuals represented in the METHIT
dataset was cal-
culated exactly as described previously in reference 22. METHIT data was searched with pTRACA22 RelB and RelE amino acid sequences using tBlastn
to identify homologous sequences, and only hits generating e-values of 1 e
or lower with a length of 30 aa or over were considered signifcant, and
used to calculate hits/Mb.
For the purpose of this analysis individuals were designated as healthy unless indicated as being diagnosed with ulcerative
colitis (UC) or Crohn disease (CD), regardless of age or body mass index. Statistically signifcant diferences in relative abundance or incidence of RelBE
modules within and between metagenomic datasets were explored using the χ
Where signifcant diferences in relative abundance of
RelBE homologues were identifed within or between groups comprised of metagenomes from the METAHIT dataset, the statistical power aforded by
the available sample sizes in each group was calculated using the Piface program.
This provides the probability that the observed diferences refect
the wider population, with a probability of 0.8 generally considered to indicate adequate power. Symbols above bars indicate the level of signifcance
in χ
analysis, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Statistical power is shown in adjacent parentheses, and in all cases has been computed to p = 0.05
using variation (standard deviation) observed within the total METAHIT dataset. (A) Shows the relative abundance of pTRACA22 type RelBE homo-
logues in the individual gut microbiomes of all 124 individuals represented in the METAHIT dataset. Each bar represents the relative abundance data
(Hits/Mb) for RelB (blue) overlaid with that of RelE (red). Purple regions show the least abundant ORF in each individual metagenome, while the color
of upper regions indicates the abundance of the most prevalent ORF in each individual metagenome. Individuals in which the RelB anti-toxin is most
abundant are indicated by blue upper regions of bars, while those in which the RelE toxin is most abundant are indicated by red upper regions of bars.
Individuals in which the level of RelB and RelE are equal are indicated by exclusively purple bars. (B) Shows the percentage of individuals represented
in the entire METAHIT dataset with varying ratios of RelB:RelE abundance. Blue = % of individuals in which RelB dominates, red = % of individuals in
which RelE dominates, purple = % of individuals with equal relative abundance of both RelB and RelE; white = % of individuals in which neither RelB
nor RelE could be detected within the criteria employed in this study. (C) Comparison of overall relative abundance in combined human gut metage-
nomes of Japanese and American individuals (utilized in our original analysis), and the METAHIT dataset.
Red bars show relative abundance of
RelE homologues and Blue bars show relative abundance of RelB homologues. Am&Jp = American and Japanese gut metagenomes utilized in our
original study;
METAHIT Healthy = Combined gut metagenomes of individuals from the METAHIT dataset without IBD; METAHIT CD = Combined
gut metagenomes of individuals from the METAHIT dataset diagnosed with Crohn disease; METAHIT UC = Combined gut metagenomes of individuals
from the METAHIT dataset diagnosed with ulcerative colitis; METAHIT TOTAL = Combined gut metagenomes of all 124 individuals represented in the
METAHIT dataset.
428 Gut Microbes Volume 1 Issue 6
and disease risk, not only between indi-
viduals but also within an individual over
The gut mobile metagenome is a partic-
ularly enigmatic and little explored aspect
of the human holobiont. MGE encoding
genes required for bacteria to establish
symbiotic relationships with metazoan
host organisms, as well as MGE conferring
a direct benefit to the metazoan host, have
already been described among the mobile
metagenomes of microbial communi-
ties colonizing plants, invertebrates and
also humans.
Therefore genes
important for life in the gastrointestinal
tract, as well as activities that impact on
host-microbe or microbe-microbe interac-
tions, are likely to be encoded by the gut
mobile metagenome.
Perhaps of more significance is the over-
all capacity of the mobile metagenome to
move information both into and out of the
gut community. This has profound impli-
cations for the human holobiont, in that
the prokaryotic mobile metagenome effec-
tively links the human hologenome with
the wider prokaryotic world. In this view,
the hologenome of an individual human
may be likened to an island in an ocean
of prokaryotic genetic information. The
primary eukaryotic core of the human
hologenome constitutes the rocky heart
of the island which changes only slowly.
Our principal prokaryotic metagenome
sits at the interface between the island and
the ocean forming a beach of relatively
stable but ever shifting sands. The ter-
tiary mobile metagenome forms the shal-
low waters of the surrounding reef which
connects to the wider ocean beyond, and
through which new genetic material enters
the lagoon, washes onto the prokaryotic
beach and is distributed along the shore.
I would like to thank Dr. Caroline Jones
for valuable discussion and constructive
criticism of the manuscript, and Elizabeth
Cheek for expert advice regarding sta-
tistical analyses of the data presented.
Research in the laboratory of Dr. B.V.
Jones is currently supported by funding
from the Medical Research Council, The
Hospital Infection Society, The Society
for Applied Microbiology, The Royal
Society and the Centre for Biomedical
(power = 0.796; Fig. 1C). Although, for
functions analyzed here, even when suf-
ficient samples are available to provide
the required statistical power, it is not yet
clear if a statistically significant difference
between gut metagenomes from distinct
groups equates to any meaningful biologi-
cal effect and vice versa.
Nonetheless, although far from proven
a role for the mobile metagenome in intes-
tinal disease cannot yet be precluded, par-
ticularly as MGE may also be enriched or
reduced by the microbial dysbiosis char-
acteristic of IBD (Fig. 1B). These initial
findings are in agreement with numer-
ous other studies, including the original
METHIT study,
showing differences in
structure and function of the gut micro-
biome in IBD patients and potentially
extends the dysbiosis of the gut microbiota
observed in IBD to the mobile metage-
nome and specifically the plasmid popu-
lation (Fig. 1B). Such disturbances could
lead to alterations in the abundance of
MGE and the genes they encode, includ-
ing pTRACA22 type RelBE modules, in
the gut microbiomes of individuals with
IBD and therefore perturb or amplify
any effects they may have on the human
host. Further research will be required to
substantiate or refute these preliminary
findings, establish their relevance to ini-
tiation, progression or diagnosis of IBD,
and answer the question: Do attributes of,
or functions encoded by, the human gut
mobile metagenome play any role in the
pathogenesis of IBD or other intestinal
If we accept the paradigm of humans as
chimeras of both eukaryotic and prokary-
otic cells, with the gut microbiota consti-
tuting a “virtual organ”, and forming an
integral part of the human (holo)genome,
then we must also accept that our knowl-
edge of how our bodies function, and the
mechanisms of many prevalent diseases
will remain obscure until we have also
gained an in-depth understanding of our
prokaryotic components. The gut micro-
biota especially is far more versatile than
any of our own tissues (both metaboli-
cally and genetically), and undoubtedly
contributes greatly to differences in health
reduction in both RelB and RelE homo-
logues was observed, and analysis of indi-
vidual gut metagenomes showed that in
33.33% of this cohort no pTRACA22
type RelBE modules could be detected,
compared to only 2.02% of individuals in
the healthy group (Fig. 2A).
These observations potentially associ-
ate pTRACA22 type TA modules with
aspects of these diseases (Fig. 2). While
several suggested functions of TA mod-
ules, particularly their potential toxicity
towards eukaryotic cells (Table 1),

appear to fit with an association with IBD,
the actual role of these gene systems in the
human gut remains unknown, and toxic-
ity of RelE has not yet been demonstrated
outside artificial delivery to, and expres-
sion in mammalian cells via specialized
Overall, the most probable
explanation remains that the observed
changes are the result of the well estab-
lished population shifts characteristic of
CD, rather than a direct involvement in
the disease, and it is perhaps not surprising
that the mobile metagenome also appears
disrupted in individuals diagnosed with
these diseases.
While these initial findings are intrigu-
ing and indicate that this aspect of the
gut metagenome should be subjected to
further study, it should also be noted that
only a small number of individuals with
CD or UC (n = 4, and n = 21 respec-
tively) are present in the METAHIT
data set. Although significant differences
were observed between the metagenomes
analyzed here, power calculations clearly
demonstrate that in most cases the small
numbers of gut metagenomes from indi-
viduals with CD or UC do not provide
adequate power (generally accepted to be
0.8 at p = 0.05). Where statistical power
is low, the probability that the observed
differences are truly reflective of the wider
population is correspondingly low, and as
such any apparently significant differences
should be treated with caution.
However, adequate statistical power
appears to be available to demonstrate the
greater number of individuals in which
pTRACA22 type RelBE modules were
undetected among the UC group (Fig.
2A); and possibly the greater relative
abundance of pTRACA10 in individuals
with UC compared to healthy individuals Gut Microbes 429
Table 1. Proposed or confirmed functions of TA addiction modules and relevance to the human gut microbiota
Confirmed or proposed function of TA modules Significance/relevance to human gut microbiome
• Plasmid stabilization
• Stabilize vertical transmission of neighbouring
DNA segments
The stabilization of plasmids though post-segregational killing (PSK) by TA modules should
allow the retention of plasmids within the gut microbiome in the absence of direct selective
pressure for functions they encode.
Chromosomally encoded TA modules may stabilize neigh-
bouring gene regions, and ensure vertical transmission of associated genes is maintained even
in the absence of direct selective pressure for functions they encode.
Together, these stabili-
zation effects may serve to retain important functions within the gut microbiome that are not
under continual selection. These may be functions required only intermittently, or during key
stages of holobiont development, such as transmission of the microbiota to new hosts, or initial
formation of the community.
• Plasmid anti-addiction modules
• Bacteriophage countermeasure
Chromosomally encoded TA modules may function as anti-addiction modules, preventing PSK
by plasmid encoded TA systems and destabilising the plasmid within the host bacterial popula-
Both plasmid and chromosomally encoded TA systems may also function as phage exclu-
sion mechanisms, restricting the capacity of bacteriophage to infect a bacterial population.

These attributes could constitute a mechanism which controls the rate of HGT in the gut micro-
biome by restricting the range of MGE that may participate in this process.
TA modules may also limit the impact of bacteriophage on the gut microbiota, stabilising the
community against bacteriophage “attacks”. This attribute fits well with the observed enrich-
ment of pTRACA22 type RelBE modules in the gut microbiome,
and is of particular interest in
light of the potential role of bacteriophage in dysbiosis of the gut microbiota in CD.
• Modulation of host gene expression
• Formation of new regulatory gene networks
Specific mechanisms through which plasmids and other MGE alter global gene expression of
the bacterial host have recently been described.
Modulation of gene expression and the for-
mation of new regulatory gene networks in bacterial cells have also been linked with TA mod-
These potential functions of TA systems could serve to facilitate rapid adaptation of
gut associated bacterial species to the GIT, or changing conditions within this environment. This
may be of significance during colonization of pristine gut environments, as well as survival of
bacterial cells in the external environment during transmission to new hosts. This feature of TA
systems may also allow members of an established gut microbiota to deal with shifts in envi-
ronmental conditions, such as changes in host diet.
• Formation of persister cells and survival of
exposure to environmental stresses
TA modules have been consistently associated with the formation of persister cells, and survival
of environmental stresses.
These include nutrient limitation and exposure to antimicro-
bial agents, both of which are likely to be encountered during colonization of the human gut.
In addition, the ability to survive under nutrient limiting conditions may also be of benefit to
members of an established community. For example the ability to survive under nutrient limit-
ing conditions may be important during changes in host diet which reduce the levels of usable
nutrients to particular members of the gut community, or during periods of host starvation. In
particular, wide variation in diet and periods of starvation were undoubtedly more common in
the ancestors of modern humans, and access to a consistent food supply is still a problem for a
significant fraction of the human population alive today. Alternatively, the association of per-
sister cells with survival of exposure to antimicrobial agents, may indicate a potential role for TA
systems in the ability of the gut microbiota to recover following exposure to antibiotics.
• Biofilm dispersal
The recent finding that TA modules may play a role in regulating aspects of biofilm formation is
also of relevance to the human gut microbiome.
The formation of biofilm communities associ-
ated with the gut mucosa, or the surface of indigestible food particles, is generally considered
to be an important and consistent behaviour in the gut microbial community, which may be
involved in the persistence of microbes in the gut, and also in the pathogenesis of intestinal
• Control of epithelial cell proliferation
Perhaps the most contentious of the proposed functions for RelBE TA modules is the potential
for a direct role of these gene systems in host-microbe interaction.
This theory stems from the
observation that RelE toxins are active in eukaryotic cells via the same mechanism employed to
inhibit growth of or kill bacterial cells.
Importantly, in cultured human cells expression of
bacterial RelE leads to cell death by induction of apoptosis. As such these systems may contrib-
ute to homeostasis of the intestinal epithelium, as has been documented for other activities of
the gut microbiota. However, this activity has to date only been observed using artificial mam-
malian gene expression systems,
and it is not yet known if RelE expressed by microbial cells
in the gut microbiota, or in even in model co-culture systems, will recreate these effects. The
observed enrichment of the pTRACA22 type RelBE in the human gut microbiome argues for
further study of this potential activity of RelBE TA modules.
Previously presented functions or activities of TA modules which may be relevant to the human gut microbiota are described, and the significance
of these functions to the gut microbiota is considered. All functions of TA that are discussed are based on previous studies which have provided evi-
dence for particular roles of TA modules, and relevant literature cited. However, further research is required to ascertain the relevance of these func-
tions, and the enriched pTRACA22 type RelBE modules, if any, to the human gut microbiome.
430 Gut Microbes Volume 1 Issue 6
41. Li Y, Canchaya C, Fang F, Raftis E, Ryan KA, van
Pijkeren JP, et al. Distribution of megaplasmids in
Lactobacillus salivarius and other Lactobacilli. Appl
Environ Microbiol 2007; 189:6128-39.
42. Oshima K, Toh H, Ogura Y, Sasamoto H, Morita
H, Park SH, et al. Complete genome sequence and
comparative analysis of the wild-type commensal
Escherichia coli strain SE11 isolated from a healthy
adult. DNA Res 2008; 15:375-86.
43. Millette M, Dupont C, Sharek F, Ruiz MT,
Archambault D, Lacroix M. Purification and iden-
tification of the pediocine produced by Pediococcus
acidilactici MM33, a new human intestinal strain.
J Appl Microbiol 2007; 104:269-75.
44. Claesson MJ, Li Y, Leahy S, Canchaya C, van Pijkeren
JP, Cerdeño-Tárraga C, et al. Multireplicon genome
architecture of Lactobacillus salivarius. Proc Natl Acad
Sci USA 2006; 103:6718-28.
45. Moon K, Sonnenburg J, Salyers A. Unexpected effect
of a Bacteriodes conjugative transposons, CtnDOT, on
chromosomal gene expression in its bacterial host. Mol
Microbiol 2007; 64:1562-72.
46. Chassey B, Gibson EM, Giuffrida A. Evidence for
plasmid associated lactose metabolism in Lactobacillus
casei ssp. casei. Curr Microbiol 1978; 1:141-4.
47. Muriana PM, Klaenhammer RT. Conjugal transfer of
plasmid-encoded determinants for bacteriocin produc-
tion and immunity in Lactobacillus acidophilus 88. Appl
Environ Microbiol 1987; 53:553-60.
48. Tannock GW, Munro K, Harmsen HJ, Welling GW,
Smart J, Gopal PK. Analysis of the faecal microflora of
human subjects consuming a probiotic product con-
taining Lactobacillus rhamnosus DR20. Appl Environ
Microbiol 2000; 66:2578-88.
49. Zaneveld JR, Lozupone C, Gordon JI, Knight R.
Ribosomal RNA diversity predicts genome diversity in
gut bacteria and their relatives. Nucleic Acids Res 2010;
50. Blum-Oehler G, Oswald S, Eitejörge K, Sonnenborn
U, Schulze J, Kruis W, et al. Development of strain-
specific PCR reactions for the detection of the probiotic
Escherichia coli strain Nissle 1917 in faecal samples. Res
Microbiol 2003; 154:59-66.
51. Shankar N, Baghdayan AS, Gilmore MS. Modulation
of virulence within an pathogenicity island in a van-
comycin resistant Enterococcus faecalis. Nature 2002;
52. Tzipori S, Gibson R, Montanaro J. Nature and distri-
bution of mucosal lesions associated with enteropatho-
genic and enterohemorrhagic Escherichia coli in piglets
and the role of plasmid-mediated factors. Infect Immun
1989; 57:1142-50.
53. Dudley EG, Abe C, Ghigo JM, Latour-Lambert P,
Hormazabal JC, Nataro JP. An IncI1 plasmid contrib-
utes to the adherence of the atypical enteroaggregative
Escherichia coli strain C1096 to cultured cells and abi-
otic surfaces. Infect Immun 2006; 74:2102-14.
54. Rotger R, Casadesús J. The virulence plasmids of
Samonella. Internatl Microbiol 1999; 2:177-84.
55. Boyd EF, Hartl D. Salmonella virulence plasmid:
Modular acquisition of the spv virulence region by an
F-plasmid in Salmonella enterica subspecies I and inser-
tion into the chromosome of subspecies II, IIa, IV and
Vii isolates. Genetics 1998; 149:1183-90.
56. Venkatesan MM, Goldberg MB, Rose DJ, Grotebeck
EJ, Burland V, Blattner FR. Complete DNA sequence
and analysis of the large virulence plasmid of Shigella
flexneri. Infect Immun 2001; 96:3271-85.
57. Xiong Z, Tang X, Yang F, Zhang X, Yang J, Chen L,
et al. Comparison of the virulence plasmid genomes
of two strains of Shigella which lost the ability to bind
Congo red. Sci China C Life Sci 2006; 49:141-8.
58. Zagaglia C, Casalino M, Colonna B, Conti C, Calconi
A, Nicoletti M. Virulence plasmids of enteroinvasive
Escherichia coli and Shigella flexneri integrate into a
specific site on the host chromosome: Integration greatly
reduces expression of a plasmid-carried virulence genes.
Infect Immun 1991; 59:729-99.
21. Jones BV, Begley M, Hill C, Gahan CGM, Marchesi
JR. Functional and comparative metagenomic analysis
of bile salt hydrolase activity in the human gut micro-
biome. Proc Natl Acad Sci USA 2008; 105:13580-5.
22. Jones BV, Sun F, Marchesi JR. Comparative metage-
nomic analysis of plasmid encoded functions in the
human gut microbiome. BMC Genomics 2010; 11:46.
23. Jones BV, Marchesi JR. Transposon-aided capture
(TRACA) of plasmids resident in the human gut
mobile metagenome. Nat Methods 2007; 4:55-61.
24. Jones BV, Marchesi JR. Accessing the mobile metage-
nome of the human gut microbiota. Mol Biosyst 2007;
25. Nikolich MP, Hong G, Shoemaker NB, Salyers AA.
Evidence for natural horizontal transfer of tetQ
between bacteria that normally colonize humans and
bacteria that normally colonize livestock. Appl Environ
Microbiol 1994; 60:3255-60.
26. Salyers AA, Shoemaker NB, Stevens AM, Li LY.
Conjugative transposons: an unusual and diverse set of
integrated gene transfer elements. Microbiol Rev 1995;
27. Kazimierczal KA, Scott KP, Kelly D, Aminov RI.
Tetracycline resistome of the organic pig gut. Appl
Environ Microbiol 2009; 75:171-1722.
28. Shoemaker NB, Vlamakis H, Hayes K, Salyers AA.
Evidence for extensive resistance gene transfer among
Bacteroides spp. and among Bacteroides and other
genera in the human colon. Appl Environ Microbiol
2001; 67:561-8.
29. Gupta A, Vlamakis H, Shoemaker N, Salyers AA. A new
Bacteroides conjugative transposon that carries an ermB
gene. Appl Environ Microbiol 2003; 69:6455-63.
30. Wang YP, Wang GR, Shoemaker NB, Whitehead TR,
Salyers AA. Distribution of the ermG gene among
bacterial isolates from porcine intestinal contents. Appl
Environ Microbiol 2005; 71:4930-4.
31. Salyers AA, Gupta A, Wang YP. Human intestinal bac-
teria as reservoirs for antibiotic resistance genes. Trends
Microbiol 2004; 12:412-6.
32. Kazimierczak KA, Flint HJ, Scott KP. Comparative
analysis of sequences flanking tet(W) resistance genes
in multiple species of gut bacteria. Antimicrob Agents
Chemother 2006; 50:2632-9.
33. Melville CM, Brunel R, Flint HJ, Scott KP. The
Butyrivibrio fibrisolvens tet(W) gene is carried on the
novel conjugative transposon TnB1230, which con-
tains duplicated nitroreductase coding sequences. J
Bacteriol 2004; 186:3656-9.
34. Melville CM, Scott KP, Mercer DK, Flint HJ. Novel
tetracycline resistance gene, tet(32), in the Clostridium-
related human colonic anaerobe K10 and its transmis-
sion in vitro to the rumen anaerobe Butyrivibrio fibrisol-
vens. Antimicrob Agents Chemother 2001; 45:3246-9.
35. Scott KP, Melville CM, Barbosa TM, Flint HJ.
Occurrence of the new tetracycline resistance gene
tet(W) in bacteria from the human gut. Antimicrob
Agents Chemother 2000; 44:775-7.
36. Shears P, Suliman G, Hart CA. Occurrence of multiple
antibiotic resistant and R plasmids in Enterobacteriaceae
isolated from children in the Sudan. Epidem Inf 1988;
37. Trobos M, Lester CH, Olsen JE, Frimondt-MØller N,
Hmmerum AM. Natural transfer of sulphonamide and
amplicllin resistance between Escherichia coli residing in
the human intestine. J Antimicrob Chemother 2009;
38. Sommer MOA, Dantas G, Church GM. Functional
characterisation of the antibiotic resistance reservoir in
the human microflora. Science 2009; 325:1128-31.
39. Goran MG, Carmeli Y, Schwaber MJ, Chmelnitsky I,
Schechner V, Navon-Venezia S. Transfer of carbapen-
em-resistant plasmid from Klebsiella pneumonias ST258
to Escherichia coli in patient. Emerg Infect Dis 2010;
40. Licht TN, Wilcks A. Conjugative gene transfer in the
gastrointestinal environment. Adv Appl Microbiol
2006; 58:77-95.
and Health Science Research at the School
of Pharmacy and Biomolecular Sciences,
University of Brighton.
1. Whitman WB, Coleman DC, Wiebe WJ. Prokaryotes:
the unseen majority. Proc Natl Acad Sci USA 1998;
2. Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA,
Gordon JI. Host-bacterial mutualism in the human
intestine. Science 2005; 307:1915-20.
3. O’Hara AM, Shanahan F. The gut flora as a forgotten
organ. EMBO Rep 2006; 7:688-93.
4. Zilber-Rosenberg I, Rosenberg E. Role of microorgan-
isms in the evolution of animals and plants: the hologe-
nome theory of evolution. FEMS Microbiol Rev 2008;
5. Rosenberg E, Sharon G, Zilber-Rosenberg I. The
hologenome theory of evolution contains Lamarckian
aspects within a Darwinian framework. Environ
Microbiol 2009; 11:2959-62.
6. Dethlefsen L, McFall-Ngai M, Relman DA. An ecolog-
ical and evolutionary perspective on human-microbe
mutualism and disease. Nature 2007; 449:811-8.
7. Wilks M. Bacteria and early human development. Early
Hum Dev 2007; 83:165-70.
8. Ley RE, Peterson DA, Gordon JI. Ecological and evolu-
tionary forces shaping microbial diversity in the human
intestine. Cell 2006; 124:837-48.
9. Turnbaugh PJ, Quince C, Faith JJ, McHardy AC,
Yatsunenko T, Niazi F, et al. Organismal, genetic and
transcriptional variation in the deeply sequenced gut
microbiomes of identical twins. Proc Natl Acad Sci
USA 2010; 107:7503-8.
10. Qin J, Li R, Raes J, Arumugaum M, Burgdorf KS,
Manichanh C, et al. A human gut microbial gene cata-
logue established by metagenomic sequencing. Nature
2010; 464:59-65.
11. Tap J, Mondot S, Levenez F, Pelletier E, Caron C, Furet
JP, et al. Towards the human intestinal microbiota phy-
logenetic core. Environ Microbiol 2009; 11:2574-84.
12. Backhed F, Ding H, Wang T, Hooper LV, Koh Y, Nagy
A, et al. The gut microbiota as an environmental fac-
tor that regulates fat storage. Proc Natl Acad Sci USA
2004; 101:15718-23.
13. Rawls JF, Mahowald MA, Ley RE, Gordon JI.
Reciprocal gut microbiota transplants from zebrafish
and mice to germ-free recipients reveal host habitat
selection. Cell 2006; 127:423-33.
14. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V,
Mardis ER, Gordon JI. An obesity-associated gut
microbiome with increased capacity for energy harvest.
Nature 444:1027-31.
15. Hooper LV, Xu J, Falk PG, Midtvedt T, Gordon JI. A
molecular sensor that allows a gut commensal to con-
trol its nutrient foundation in a competitive ecosystem.
Proc Natl Acad Sci USA 1999; 96:9833-8.
16. Stappenback TS, Hooper LV, Gordon JI. Development
regulation of intestinal angiogenesis by indigenous
microbes via Paneth cells. Proc Natl Acad Sci USA
2002; 99:15451-5.
17. Zoetendal EG, Vaughan EE, de Vos WM. A microbial
world within us. Mol Microbiol 2006; 59:1639-50.
18. Lombardo P. Access to mutualistic endosymbiotic
microbes: an underappreciated benefit of group living.
Behav Ecol Sociobiol 2008; 62:479-97.
19. Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ,
Samuel BS, et al. Metagenomic analysis of the human
distal gut microbiome. Science 2006; 312:1355-9.
20. Kurokawa K, Itoh T, Kuwahara T, Oshima K, Toh H,
Toyoda A, et al. Comparative metagenomics revealed
commonly enriched gene sets in human gut microbi-
omes. DNA Res 2007; 14:169-81. Gut Microbes 431
99. Tsilibaris V, Maenhaut-Michel G, Mine N, Van
Melderen L. What is the benefit to Escherichia coli of
having multiple toxin-antitoxin systems in its genome?
J Bacteriol 2007; 189:6101-8.
100. Christensen SK, Pedersen K, Hansen FG, Gerdes
K. Toxin-antitoxin loci as stress-response-elements:
ChpAK/MazF and ChpBK cleave translated RNAs
and are counteracted by tmRNA. J Mol Biol 2003;
101. Christensen SK, Mikkelsen M, Pedersen K, Gerdes K.
ReIE, a global inhibitor of translation, is activated dur-
ing nutritional stress. Proc Natl Acad Sci USA 2001;
102. Anantharaman V, Aravind L. New connections in the
prokaryotic toxin-antitoxin network: Relationship with
the eukaryotic nonsense-mediated RNA decay System.
Genome Biol 2003; 4:81.
103. Vázquez-Laslop N, Lee H, Neyfakh AA. Increased per-
sistence in Escherichia coli caused by controlled expres-
sion of toxins or other unrelated proteins. J Bacteriol
2006; 188:3494-7.
104. Nieto C, Sadowy E, de la Campa AG, Hryniewicz W,
Espinosa M. The relBE2Spn toxin-antitoxin system of
Streptocococcus pneumonia: Role in antibiotic tolerance
and functional conservation in clinical isolates. Plos
One 2010; 5:11289.
105. Yamamoto TA, Gerdes K, Tunnacliffe A. Bacterial toxin
RelE induces apoptosis in human cells. FEBS Lett
2002; 519:191-4.
106. Kristoffersen P, Jensen GB, Gerdes K, Piskur J. Bacterial
toxin-antitoxin gene system as containment control in
yeast cells. Appl Environ Microbiol 2000; 66:5524-6.
107. Andreev D, Hauryliuk V, Terenin I, Dmitriev S,
Ehrenberg M, Shatsky I. The bacterial toxin ReIE
induces specific mRNA cleavage in the A site of the
eukaryote ribosome. RNA 2008; 14:233-9.
108. Rohwer F, Prangishvili D, Lindell D. Roles of viruses in
the environment. Environ Microbiol 2009; 11:2771-4.
109. Lepage P, Colombet J, Marteau P, Sime-Ngando T, Doré
J, Leclerc M. Dysbiosis in inflammatory bowel disease: a
role for bacteriophages? Gut 2008; 57:424-5.
110. Saitoh S, Noda S, Aiba Y, Takagi A, Sakamoto M,
Benno Y, et al. Bacteroides ovatus as the predominant
commensal intestinal microbe causing a systemic anti-
body response in inflammatory bowel disease. Clin
Diagnost Lab Immunol 2002; 9:54-9.
111. Lenth RV. Java applets for power and sample size
[Piface.jar Version 1.70, March 2009], http://www.stat.
112. Saavedra De Bast M, Mine N, Van Melderen L.
Chromosomal toxin-antitoxin systems may act as anti-
addiction modules. J Bacteriol 2008; 190:4603-9.
113. Pecota CD, Wood TK. Exclusion of T4 phage by the
hok/sok killer locus from plasmid R1. J Bacteriol 1996;
114. Doyle M, Fookes M, Ivens A, Mangan MW, Wain
J, Dorman CJ. A H-NS-like stealth protein aids
horizontal gene transmission in bacteria. Science 2007;
115. Kim Y, Wang X, Ma Q, Zhang XS, Wood TK.
Toxin-antitoxin systems in Escherichia coli influence
biofilm formation through YigK (TabA) and fimbriae.
J Bacteriol 2009; 191:1258-67.
116. Bollinger RR, Barbas AS, Bush EL, Lin SS, Parker W.
Biofilms in the normal human large bowel: Fact rather
than fiction. Gut 2007; 56:1481-2.
117. Kleessen B, Blaut M. Modulation of gut mucosal bio-
films. Br J Nutr 2005; 93:35.
79. Barbour WM, Elkan GH. Relationship of the presence
and copy number of plasmids to exopolysaccharide
production and symbiotic effectiveness in a Rhizobium
fredii ASDA 206. Appl Environ Microbiol 1989;
80. McNiel NI. The contribution of the large intestine to
energy supplies in man. Am J Clin Nutr 1984; 39:338-42.
81. Pryde SE, Duncan SH, Hold GL, Stewart CS, Flint HJ.
The microbiology of butyrate formation in the human
colon. FEMS Microbiol Lett 2002; 217:133-9.
82. Cummings JH, Antoine JM, Azpiroz, Bourdet-Sicard
R, Brandtzaeg P, Calder PC, et al. PASSCLAIM
health and immunity. Eur J Nut 2004; 43:118-73.
83. McIntyre A, Gibson PR, Young GP. Butyrate produc-
tion from dietary fiber and protection against large
bowel cancer in a rat model. Gut 1993; 34:86-391.
84. Sponheimer M, Passey BH, de Ruiter DJ, Guatelli-
Steinberg D, Cerling TE, Lee-Thorp JA. Isotopic
evidence for dietary variability in the early hominin
Paranthropus robustus. Science 2006; 314:980-9822.
85. Sponheimer M, Lee-Thorp JA. Isotopic evidence for
dietary variability in the early hominid Australopithecus
africanus. Science 1999; 283:368-70.
86. Igimi S, Ryu CH, Park SH, Sasaki Y, Sasaki T, Kumagai
S. Transfer of conjugative plasmid pAMbeta1 from
Lactococcus lactis to mouse intestinal bacteria. Lett Appl
Microbiol 1996; 23:31-5.
87. Tuohy K, Davies M, Rumsby P, Rumney C, Adams
MR, Rowland IR. Monitoring transfer of recombinant
and non-recombinant plasmids between Lactococcus
lactis strains and members of the human gastrointes-
tinal microbiota in vivo -impact of donor cell number
and diet. J Appl Microbiol 2002; 93:954-64.
88. Fang F, Li Y, Bumann M, Raftis EJ, Casey PG, Cooney
JC, et al. Allelic variation of bile salt hydrolase genes
in Lactobacillus salivarius does not determine bile resis-
tance levels. J Bacteriol 2009; 191:5743-57.
89. Begley M, Hill C, Gahan CG. Bile salt hydrolase
activity in probiotics. Appl Environ Microbiol 2006;
90. Venter JC, Remington K, Heidelberg JF, Halpern
AL, Rusch D, Eisen JA, et al. Environmental genome
shotgun sequencing of the Sargasso Sea. Science 2004;
91. Tringe SG, von Mering C, Kobayashi A, Salamov AA,
Chen K, Chang HW, et al. Comparative metagenomics
of microbial communities. Science 2005; 308:554-7.
92. Breitbart M, Hewson I, Felts B, Mahaffy JM, Nulton
J, Salamon P, et al. Metagenomic analyses of an uncul-
tured viral community from human feces. J Bacteriol
2003; 185:6220-3.
93. Ebdon J, Muniesa M, Taylor H. The application of
a recently isolated strain of Bacteroides (GB-124) to
identify human sources of faecal pollution in a temper-
ate river catchment. Water Res 2007; 41:3683-90.
94. Górski A, Wazna E, Dabrowska BW, Dabrowska
K, Switała-Jelen K, Miedzybrodzki R. Bacteriophage
translocation. FEMS Immunol Med Microbiol 2006;
95. Pandey DP, Gerdes K. Toxin-antitoxin loci are highly
abundant in free-living but lost from host-associated
prokaryotes. Nucleic Acids Res 2005; 33:966-76.
96. Van Melderen L, De Bast MS. Bacterial toxin-antitox-
insystems: More than selfish entities? Plos Genet 2009;
97. Overgaard M, Borch J, Jørgensen MG, Gerdes K.
Messenger RNA interferase RelE controls relBE tran-
scription by conditional cooperativity. Mol Microbiol
2008; 69:841-57.
98. Gerdes K, Møller-Jensen J, Bugge Jensen R. Plasmid
and chromosome partitioning: surprises from phylog-
eny. Mol Microbiol 2000; 37:455-66.
59. McCormick B, Siber AM, Maurelli AT. Requirement
of the Shigella flexneri virulence plasmid in the ability
to induce trafficking of neutrophils across polarized
monolayers of the intestinal epithelium. Infect Immun
1998; 66:4237-43.
60. Gigho JM. Natural conjugative plasmids induce bacte-
rial biofilm development. Nature 2001 412: 4442-5.
61. Flint HJ, Duncan SH, Scott KP, Louis P. Interactions
and competition within the microbial community
of the human colon: links between diet and health.
Environ Microbiol 2007; 9:1101-11.
62. Huycke MM, Gaskins HR. Commensal bacteria, redox
stress and colorectal cancer: mechanisms and models.
Exp Biol Med 2004; 229:586-97.
63. Claesson MJ, Cusak S, O’Sullivan O, Greene-Dinaz
R, de Weerd H, Flannery E, et al. Composition, vari-
ability and temporal stability of the intestinal micro-
biota of the elderly. Proc Natl Acad Sci USA 2010;
64. Biagi E, Nylund L, Candela M, Ostan R, Bucci L, Pini
E, et al. Through ageing, and beyond: Gut microbiota
and inflammatory status in seniors and centinarians.
Plos ONE 2010; 5:10667.
65. Pang AW, MacDonald JR, Pinto D, Wei J, Rafiq MA,
Conrad DF, et al. Towards a comprehensive struc-
tural variation map of an individual human genome.
Genome Biol 2010; 11:52.
66. The Chimpanzee Sequencing and Analysis Consortium.
Initial sequence of the chimpanzee genome and
comparison with the human genome. Nature 2005;
67. Denarie J, Boistard P, Cassedelbart F, Atherly AG, Berry
JO, Russell P. Indigenous plasmids of Rhizobium. Int
Rev Cytol 1981; 13:225-46.
68. Martinez E, Romero D, Palacios R. The Rhizobium
genome. Crit Rev Plant Sci 1990; 9:59-93.
69. Top EM, Moenne-Loccoz Y, Pembroke T, Thomas
CM. Phenotypic traits conferred by plasmids. In:
Thomas CM, Ed. The Horizontal Gene Pool, Bacterial
Plasmids and Gene Spread, Amsterdam: Harwood
Academic Publishers 2000; 249-85.
70. Oliver KM, Degnan PH, Hunter MS, Moran NA.
Bacteriophages encode factors required for protection
in a symbiotic mutualism. Science 2009; 325:992-4.
71. Hehemann JK, Correc G, Barbeyon T, Helbert W,
Czjzek M, Michel G. Transfer of carbohydrate-active
enzymes from marine bacteria to Japanese gut micro-
biota. Nature 2010; 464:908-12.
72. Luzopone CA, Hamady M, Cantral BL, Coutinho PM,
Henrissat B, Gordon JI, et al. The convergence of carbo-
hydrate active gene repertoires in human gut microbes.
Proc Natl Acadn Sci USA 2008; 105:15076-81.
73. Xu J, Mahowals MA, Ley RE, Lozupone CA, Hamady
M, Martens EC, et al. Evolution of symbiotic bacteria
in the distal human intestine. PLOS Biol 2007; 5:156.
74. Louis P, McCrae SI, Charrier C, Flint HJ. Organization
of butyrate synthetic genes in human colonic bacteria:
phylogenetic conservation and horizontal gene transfer.
FEMS Microbiol Lett 2007; 269:240-7.
75. Netherwood T, Bowden R, Harrison P, O’Donnell AG,
Parker DS, Gilbert HJ. Gene transfer in the gastrointes-
tinal tract. Appl Environ Microbiol 1999; 65:5139-41.
76. Bardin S, Dan S, Osteras SM, Finan TM. A phosphate
transport system is required for symbiotic nitrogen
fixation by Rhizobium meliloti. J Bacteriol 1996;
77. Chen HC, Gartner E, Rolfe BG. Involvement of genes
on a megaplasmid on the acid-tolerant phenotype of
Rhizobium leguminsarum. Appl Environ Microbiol
1993; 59:1058-64.
78. Baldani JI, Weaver RW. Survival of clover rhizobia
under heat and drought stresses in soil after curing
plasmids. Soil Biol Biochem 1992; 24:737-42.