REVIEWS

Mechanisms of gene flow in archaea

Alexander Wagner1, Rachel J. Whitaker2,3, David J. Krause4, Jan-Hendrik Heilers1,
Marleen van Wolferen1, Chris van der Does1 and Sonja-Verena Albers1
Abstract | Archaea are diverse, ecologically important, single-celled microorganisms. They have
unique functions and features, such as methanogenesis and the composition of their cell
envelope, although many characteristics are shared with the other domains of life, either through
ancestry or through promiscuous horizontal gene transfer. The exchange of genetic material is a
major driving force for genome evolution across the tree of life and has a role in archaeal
speciation, adaptation and maintenance of diversity. In this Review, we discuss our current
knowledge of archaeal mechanisms of DNA transfer and highlight the role of gene transfer in
archaeal evolution.

TACK superphylum
A recently proposed
superphylum that comprises
the Thaumarchaeota,
Aigarchaeota, Crenarchaeota
and Korarchaeota phyla.
1
Molecular Biology of
Archaea, Institute of Biology II
- Microbiology, Albert Ludwigs
University of Freiburg,
Schaenzlestrasse 1,
79104 Freiburg, Germany.
2
Department of Microbiology,
University of Illinois Urbana-
Champaign, 601 South
Goodwin Avenue, Urbana,
Illinois 61801, USA.
3
Carl R. Woese Institute for
Genomic Biology, University
of Illinois Urbana-Champaign,
1206 South Goodwin Avenue,
Urbana, Illinois 61801, USA.
4
Laboratory of Genetics,
Genome Center of Wisconsin,
Wisconsin Energy Institute,
J. F. Crow Institute for the
Study of Evolution, University
of Wisconsin-Madison,
425-G Henry Mall, Madison,
Wisconsin 53706, USA.
Correspondence to S.V.A. 
sonja.albers@
biologie.uni-freiburg.de
doi:10.1038/nrmicro.2017.41
Published online 15 May 2017
Archaea are single-celled anucleate microorganisms that
are distinct from bacteria and eukaryotes1. Although
they were initially thought to live only in extreme envi-
ronments, archaea have been found in nearly all habi-
tats on Earth; for example, planktonic archaea form a
large proportion of the total microbial biomass in the
oceans2. Moreover, archaea have important roles in soil
ammonia oxidation and in the digestive tract of mam-
mals3,4, and they are model systems for evolutionary
genomics1,5,6. Archaea are highly diverse and encom-
pass different phyla, including the TACK superphylum7,8,
the Euryarchaeota9 and the newly discovered Asgard
­s uperphylum 6. In addition, small, probably parasitic
archaea have been found, which have been grouped into
what is referred to as the DPANN superphylum10.
In recent years, numerous studies have con-
firmed the ecological and evolutionary importance
of archaea1,3,4,11. It became clear that in archaea, which
are not strictly clonal organisms, the process of gene
flow (DNA transfer and the integration of DNA into
the chromosome) is a major driving force for evolution
(BOX 1). Although archaea reproduce ­asexually, DNA
transfer and homologous recombination between
closely related individuals occur frequently in nat-
ural populations and have a role in the formation of
archaeal species12–14. Moreover, horizontal gene trans-
fer (HGT) between more distantly related species can
transfer novel functions and features, without the need
for de novo evolution in the recipient. Remarkable
examples of HGT can be found in the evolutionary
history of life, even if they occur rarely. For example,
a recent analysis of 134 archaeal genomes found that
many contained signatures of horizontally transferred
DNA from bacteria, particularly the methanogens,
and the majority of transferred genes were presumed
to be functional15.
To get a clearer view of the ancient and current evo-
lution of archaea, a better understanding of the different
DNA transfer mechanisms in archaea is required. The
major mechanisms of DNA transfer in bacteria — ­natural
transformation, transduction and conjugation — have
been described in detail (BOX 2). Although mechanistic
insights in archaea are scarce, natural transformation,
transduction and conjugation have been shown to occur
in different archaeal phyla16–18. In addition, new types of
DNA transfer mechanism have been identified. For exam-
ple, halophilic archaea exchange chromosomal and plas-
mid DNA in a cell fusion-based manner 19,20. Moreover,
a recent study identified the first archaea-specific DNA
transport system, which seems to have a pivotal role,
especially under conditions of DNA damage21. In this
Review, we describe examples of signatures of HGT in
archaea and their effect on archaeal evolution. We also
discuss our current knowledge of the mechanisms that
are involved in archaeal DNA transfer and the barriers
that can limit them.
Signatures of HGT in archaea
HGT between divergent evolutionary lineages is thought
to be one of the major drivers for the evolution of single-
celled microorganisms and adaptation to new ecologi-
cal niches. Most of the horizontally transferred genes in
archaea originate from bacteria, the remaining genes are
transferred either from other archaea or, in rare cases, from
eukaryotes22–24. Gene transfer from bacteria to archaea has
been shown to occur five-times more frequently than from
archaea to bacteria15. The reason behind this asymmetry is
unclear, but it has been suggested that the overall domi­
nance of bacteria in most environments might bias stat­
istical analyses of HGT events. It was also speculated that
the incorporation of foreign genes has a lower fitness cost
or higher fitness advantage in archaea than in bacteria23.

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 1

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

Asgard superphylum Many of the horizontally acquired genes in archaea
A recently described are involved in metabolism and cell envelope biogenesis,
superphylum that includes the and probably provide a selective advantage in adaption
proposed Lokiarchaeota, to new ecological niches11,15,25. For example, haloarchaea,
Odinarchaeota, Thorarchaeota
and Heimdallarchaeota phyla.
which are oxygen-respiring heterotrophs, originated from
Asgardarchaeota encode many methanogens, which are strictly anaerobic hydrogen-­
proteins that were previously dependent autotrophs11. A recent study suggested that
suspected to be specific for a single HGT event resulted in the transfer of a large
eukaryotes.
piece of DNA that contained approximately 1,000 bac-
DPANN superphylum terial genes to a methanogen, which became the last
A proposed monophyletic and halo­archaeal common ancestor 11. Many of the acquired
deep-branching group of genes were transferred as functional units, which, for
mainly hyperthermophilic example, are involved in the terminal oxidation of oxy-
archaea that includes the
Diapherotrites, Parvarchaeota,
gen, membrane transport of reduced carbon compounds
Aenigmarchaeota, and the synthesis of menaquinon11. In general, meth-
Nanoarchaeota and anogens acquired many, often metabolic, bacterial genes
Nanohaloarchaeota phyla. that enabled them to adapt to diverse environments15,25,26.
Box 1 | Footprints of recombination in natural populations
Initial studies of archaeal populations that used multilocus sequence typing (MLST)
revealed frequent recombination events in populations of Sulfolobus islandicus13
and Halorubrum spp.14. Recombination between Ferroplasma spp. was identified in
metagenomics data of an acidophilic microbial community140. Population genomics and
whole-genome sequencing enable the identification of gene transfer events with greater
precision and even infer donor–recipient patterns of DNA transfer in isolates. Population
genomics in haloarchaea confirmed the frequent DNA transfer that was suggested by
the MLST data31. Patterns of recombination in population genomic datasets have been
used to identify species barriers and to study genome-wide evolutionary processes in
S. islandicus12,141. In this species, successful recombination occurs in the variable regions
of the chromosome that are distal from the origins of replication7. These regions are also
highly prone to horizontal gene transfer of divergent genes and to the movement of
insertion elements36.
Selection in natural populations leaves different footprints in the genome, depending
on the levels of recombination. Therefore, population genomic analysis can infer the
balance of recombination and selection in archaeal populations and differences in
this balance that occur in different regions of the genome. In regions of the genome
in which rates of recombination are high, positive selection can act on a single locus and
decrease polymorphism at that locus within a population through a selective sweep
and increase divergence between independent populations (see the figure, part a).
In regions in which rates of recombination are low, the same selection can decrease
polymorphism and increase divergence at a locus and all of the linked neutral loci around
it (see the figure, part b). Despite the effect of recombination on the chromosome of
Sulfolobus spp., such events do not occur rapidly enough in this archaeon to enable
genomic loci to evolve independently. This results in large linked ‘continents’ in the
genome and impedes the identification of single loci that are under selection in different
populations36. Recent ongoing work suggests that regions of high recombination are
under strong selection and are associated with selection for combinations of alleles.
a High recombination b Low recombination
Selection Selection
Polymorphism
Polymorphism
Furthermore, phylogenetic analyses revealed high rates
of interdomain HGT in marine Thaumarchaeota and
Euryarchaeota, which acquired 24% and 30% of their
genes from bacteria, respectively 22,23. Similarly to what
was found for methanogens, most of the genes trans-
ferred to these archaea are involved in metabolism and
membrane biogenesis. The acquisition of these genes
probably provided an advantage for life in the cold
oligo­trophic ocean23. Moreover, it was shown that both
lineages recently acquired foreign genes from distant
donors, which suggests that HGT is still ongoing 23.
Haloarchaea, some methanogens, marine
Euryarchaeota and Thaumarchaeota are deeply branch-
ing mesophilic archaea7,23,24. Several studies provide evi-
dence that mesophilic archaea have obtained more
genes from bacteria than hyperthermophilic archaea
(Crenarchaeota, Korarchaeota, Thermococcales,
non-methanogenic Archaeoglobales and Thermo­
plasmatales, and most DPANN species)11,15,23. This is in
agreement with the hypothesis that the adaptation of
archaea to mesophilic environments was the result of the
massive acquisition of bacterial genes24. This assump-
tion is further supported by the finding that halo­
archaea, marine Euryarchaeota and their sister groups
independently acquired a DNA gyrase from bacteria27.
Unlike reverse gyrase, which is found in hyperthermo-
philic archaea and introduces positive supercoils into
DNA, DNA gyrase introduces negative supercoils to
relax supercoils that are produced through transcrip-
tion and replication28. The acquisition of DNA gyrase
might therefore have contributed to the adaptation of
archaea to lower temperatures, at which it is not hot
enough for spontaneous DNA melting and relaxation29.
Moreover, mesophilic archaea contain some bacterial
chaperones that assist in correct protein folding and that
seem to be absent in hyperthermophilic archaea11,23,30.
With the increasing availability of archaeal genomes, it
needs to be shown whether archaea indeed adapted to
the mesophilic lifestyle by obtaining bacterial genes or
whether there are archaea that have adapted to lower
­temperatures without acquiring bacterial genes.
Metagenomic analyses reported rapid HGT between
distinct genera of archaea in Deep Lake, Antarctica31,32.
Interestingly, although large genomic regions of up to
35 kb can be exchanged between phylogenetically distinct
haloarchaea, different genera, such as Halobacterium or
Halorubrum, were maintained in the Deep Lake commu-
nity 32. Owing to low microbial diversity and the slow gen-
eration times of these microorganisms, Deep Lake might
represent a special habitat in which inter-genera HGT
occurs and different genera are maintained, presumably
as a result of niche adaptation32. Conversely, Deep Lake
might also be an example of the evolution of new species

from a single ancestral species. More phylogenetic ana­

ly­ses and experimental evidence are required to under-
stand the role of HGT in haloarchaeal speciation and
species maintenance33.
In the crenarchaeon Sulfolobus islandicus, the fre-
quency of novel genes that are incorporated through
homologous recombination differs within and between
Genome position Genome position species12,13. Homologous recombination in Sulfolobus spp.

Nature Reviews | Microbiology

2 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro
©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

Box 2 | Bacterial DNA transfer mechanisms
Research into the mechanisms of DNA transfer in asexual single-celled organisms has
largely focused on bacteria. This work has uncovered three primary mechanisms, which
are described below.
Natural transformation
Natural transformation is the uptake of DNA from the environment. Exogenous DNA is
bound to a type IV pilus that, following retraction, transfers the DNA to the cytoplasmic
membrane. One DNA strand is degraded and the other is imported through the ComEC
competence transport complex into the cytoplasm. Subsequently, the DNA is integrated
into the host chromosome through homologous recombination43,142–145.
Transduction
Transduction is the contact-independent transfer of DNA between two bacterial cells
by a phage. During phage replication, parts of the chromosomal DNA of the host are
occasionally packaged into phage particles. In specialized and generalized transduction,
specific and nonspecific parts of the chromosomal DNA of the host chromosomal DNA
are packed into phage particles, respectively. In the recipient, the DNA can be integrated
into the genome through homologous recombination44.
Conjugation
Conjugation is the contact-dependent transfer of DNA from a donor to a recipient cell
through a type IV secretion system (T4SS). Cell-to-cell contact is established by a
conjugative pilus, which extends from the membrane-spanning secretion complex.
Before transfer, a relaxase, together with accessory proteins, nicks double-stranded DNA
at the origin of transfer (oriT) and unwinds it to form a single-stranded DNA–relaxase
transfer intermediate. The transfer intermediate is targeted to the secretion complex
by the coupling protein VirD4, which is a membrane-bound hexameric ATPase. The DNA
is transported through the secretion complex, across the cell envelope and into the
recipient cell. After transport, the complementary DNA strand is synthesized again.
The signature protein of the T4SS is the highly conserved ATPase VirB4 (REFS 45,94–98).
populations leads to diverse CRISPR immune profiles34
and shapes the genome architecture35. Moreover, the
integration of genetic elements, such as viruses and plas-
mids, occurs rapidly enough to distinguish highly similar
Sulfolobus spp. in isolated populations36.
Experimental observations of gene flow
Gene transfer and recombination have been inten-
sively studied in members of the Euryarchaeota and
Crenarchaeota 11,19,20,32,33. These two phyla contain
Heterotrophs
most of the cultured and best-studied archaea; for
Organisms that produce example, the Euryarchaeota includes methanogenic
complex organic compounds archaea and the salt-tolerant haloarchaea9, whereas the
from organic carbon. Crenarchaeota includes Sulfolobus spp., which grow in
sulfuric hot springs37. Mating experiments that used
Autotrophs
Organisms that produce genetically tractable model organisms in these two
complex organic compounds phyla have demonstrated DNA transfer, homologous
from inorganic material using ­recombination and HGT under laboratory conditions.
light energy (photosynthesis) A study that used markers to select for recombinants
or chemical energy
(chemosynthesis).
showed that large chromosomal regions of up to 500 kb
in length can be transferred between Haloferax volcanii
Mesophilic archaea and Haloferax mediterranei 19. In addition, in H. volca­
Archaea that grow at nii, the movement of parasitic genetic elements called
temperatures ranging from
inteins has been shown experimentally to change the rate
20–45 °C.
of homologous recombination between strains38.
Type IV secretion system Members of the order Sulfolobales were found to
(T4SS). A secretion system exchange chromosomal auxotrophic markers derived
found in many bacteria and from several regions of the genome39. In Sulfolobus acido­
some archaea that is involved
in the secretion of proteins,
caldarius, DNA transfer and homologous recombination
DNA and protein–DNA frequencies between 10−4 to 10−6 per cell were observed for
complexes into target cells. the auxotrophic marker pyrEF40. Furthermore, treatment
with UV light or the induction of DNA double-strand
breaks (DSB) by bleomycin led to the formation of cel-
lular aggregates and the transfer of chromosomal DNA
fragments of up to 90 kb in length21,41,42. Aggregation and
the transfer of chromosomal DNA were species spe-
cific42, which suggests that DNA transfer among cells of
the same species has an important role in DNA repair by
providing an intact template of homologous DNA.
Mechanisms of archaeal DNA transfer
Three main mechanisms for the transfer of DNA in
asexual microorganisms have been described: natural
transformation, transduction and conjugation (BOX 2).
Natural transformation enables the uptake of environ-
mental DNA through a dedicated competence system43.
Transduction describes the transfer of DNA by viruses
that accidentally package host DNA together with their
own genetic material44. Conjugation is the contact-­
dependent transport of plasmid DNA from one cell into
another cell45. Among bacteria, these mechanisms are
well described at the molecular level, whereas, among
archaea, mechanistic insights were gained only recently.
In addition to the classic DNA transfer systems, other
mechanisms for the transfer of DNA have been described
in archaea. First, members of the Thermococcales are
thought to exchange plasmid DNA through vesicles46.
Second, haloarchaea exchange chromosomal DNA
through cell fusion events19,20. Last, members of the
Sulfolobales exchange chromosomal DNA in species-­
specific aggregates through the recently described
­crenarchaeal exchange of DNA (Ced) system21.
Natural competence. Among archaea, only a few eury­
archaeal species were shown to be naturally compe-
tent 16,47–50 (FIG. 1a). Early studies revealed the uptake
of DNA at very low frequencies in the methanogens
Methanococcus voltae PS and Methanothermobacter
thermautotrophicus Marburg 16,47,51 . Among the
Thermococcales, Thermococcus kodakarensis could be
naturally transformed with both linear and circular
DNA, although also with transformation frequencies less
than 10–7 per cell, and the DNA was incorporated into
the genome48,52. Pyrococcus furiosus can also take up lin-
ear and circular DNA and, with transformation frequen-
cies of up to 10–3, this is the only known archaeon that
takes up DNA with an efficiency that is similar to highly
competent bacteria53. Remarkably, no homologues of
bacterial competence systems have been found in these
species54,55, and the mechanisms through which DNA is
taken up from the environment are currently unknown.
Membrane vesicles. The formation of membrane vesicles
is a universal and ancient process that is hypothesized to
form the foundation of cellular compartmentalization
and eukaryogenesis56. Several thermophilic archaea
have been found to release membrane vesicles through
budding (FIG. 1b). Among the Euryarchaeota, species in
the order Thermococcales frequently release vesicles
that contain both chromosomal and plasmid DNA.
As this DNA was found to be extremely resistant to
high temperatures and unaffected by treatment with

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 3

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

a Transformation in Euryarchaeota e Cell fusion in haloarchaea
b Vesicle transport in Thermococcales
c Transduction in archaea
Virus
f Chromosomal DNA exchange
in Crenarchaeota
d Conjugation in Sulfolobaceae
Conjugative plasmid
Figure 1 | DNA transfer mechanisms in archaea. Different DNA Reviews
Nature transfer mechanisms
| Microbiology
have been described in archaea. These include the uptake of DNA through
transformation in members of the Euryarchaeota (part a), the transport of membrane
vesicles that contain plasmid or viral DNA in members of the Thermococcales (part b),
the transfer and incorporation of DNA through transduction (part c), the transfer of
conjugative plasmids among members of the Sulfolobaceae (part d), the bidirectional
transfer and incorporation of genomic and plasmid DNA through cell fusion between
haloarchaea (part e), and the bidirectional exchange and incorporation of chromosomal
DNA using the crenarchaeal exchange of DNA (Ced) system in Crenarchaeota (part f).
DNase, the membrane vesicles are thought to protect
the DNA57. Thermococcus onnurineus NA1T produces
chains of vesicles that contain linear fragments of chro-
mosomal DNA58. Vesicles that are produced by the
­ yperthermophile Thermococcus nautilus were shown
h ing them (FIG. 1c). The budding of SSV1 is very similar to
to contain the plasmids pTN1 and pTN3 (REFS 59,60).
When a derivative of the pTN1 plasmid (pLC70) was
Hyperthermophile
An organism that can thrive at
transformed into T. kodakarensis, these cells began to
temperatures around 80 °C or release vesicles that contained pLC70 (REF. 61) and these
higher. vesicles were able to transfer the plasmid to plasmid-free
T. kodakarensis cells46. In addition, vesicles from T. nauti­
Nanopods
lus contained plasmid DNA that included the genome of
Surface structures that project
membrane vesicles from the a defective virus (pTN3), but the vesicles did not contain
cell. Nanopods have been the major viral capsid proteins that are encoded by pTN3
found in bacteria and members (REF. 60). It was therefore suggested that membrane ves-
of the Euryarchaeota. icles have a protective function and enable intercellular
Proviruses
DNA transfer and recombination between viral DNA,
Viral DNAs that are integrated plasmid DNA and cellular chromosomes, without a
into the genome of its host. viral infection60.
Besides budding, members of the Thermococcales
might also produce and project membrane vesicles
through an organelle that resembles bacterial nanopods62.
Both Thermococcus gammatolerans and T. kodakarensis
form tubular structures with a row of internal vesicles63.
Projecting vesicles through nanopods might substantially
increase the distance and efficiency of vesicle, and there-
fore DNA, transfer between cells63. Moreover, nanotubes
that have a diameter of 60–80 nm have been observed
between cells from Thermococcus sp. 5–4 (REF. 63). It is
highly likely that these structures are related to nanopods
and function in the transfer of DNA between cells through
direct cellular contact63. However, for all of the described
euryarchaeal species, the mechanisms of the formation
of vesicles, nanopods or nanotubes are unknown. In cre-
narchaeal species, endosomal sorting complex required
for transport III (ESCRT-III) homologues mediate the
release of membrane vesicles64. ESCRT-III proteins facili-
tate membrane scission in eukaryotes and members of the
TACK phylum7. However, thus far, no crenarchaeal vesi-
cles that contain DNA have been observed. Instead, cer-
tain Sulfolobus strains produce vesicles that contain toxins
(sulfolobicins) that can kill other Sulfolobus strains64,65.
Whether certain crenarchaeal species use ­vesicles to
­transfer DNA remains to be shown.
As genes that are responsible for membrane remodel­
ling and vesicular trafficking were probably present in
pre-eukaryal archaea1, membrane vesicles were probably
involved in the earliest forms of archaeal intercellular
transport. For many organisms from all domains of life,
the formation of membrane vesicles is still essential for the
transport of DNA and other compounds66–69.
Transduction and chromosomally encoded mobile
genetic elements. Genome sequencing has revealed a
large diversity of proviruses in archaeal genomes17. Site-
specific integrases most often insert these viruses into
tRNA genes as target sites70–72. Viruses that are integrated
into archaeal genomes can become degraded after long
periods of time36,73; however, very few have been shown
to have a cost to their hosts. The best-studied provirus
that infects members of the Crenarchaeota is Sulfolobus
spindle-­shaped virus 1 (SSV1)74. This virus and its rela­
tives persist in the cell as episomes or as integrated c­ opies.
First thought to be a plasmid, SSV1 was shown to pro-
duce virus particles that bud from host cells without kill-
the budding process of eukaryotic enveloped viruses75.
The assembly and exit of SSV1 occur simultaneously at
the cytoplasmic membrane of the host cell. The nucleo­
protein complexes are released to the outside of the cell
and the virion matures and forms its spindle shape.
During budding, constricted ring-like structures are
formed that resemble those observed in budding necks
during the exit of some eukaryotic enveloped viruses75.
SSV1 and its relative SSV2 were shown to encapsulate the
DNA of cryptic non-conjugative plasmids (pSSVx and
pSSVi)76,77. These cryptic plasmids are hybrids of viral
and plasmid DNA. Both pSSVx and pSSVi are able to
spread in populations of Sulfolobus solfataricus, but only
in the presence of SSV1 or SSV2 (REFS 76,77).

4 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

Episomes
Fragments of DNA that
exist independently of the
chromosome. Examples of
episomes include insertion
elements, transposons,
plasmids and viruses.
Temperate viruses
Viruses that can integrate into
the chromosome or lyse the
cell following infection or
induction.
Gene transfer agents
A bacteriophage-like element
in bacteria that mediates
horizontal gene transfer by
transducing random host DNA
into a recipient cell.
CRISPR–Cas
An adaptive immune system
in bacteria and archaea that
enables the acquisition of
resistance to foreign genetic
elements, such as plasmids
and viruses.
Partitioning system
A system that ensures the
correct segregation of
chromosomal DNA or plasmids
into the daughter cells of a
dividing bacterial cell.
Hetero-diploid
Cells that have two different
homologous chromosomes.
Members of the Euryarchaeota also maintain
t­ emperate viruses that integrate into their chromo-
somes78–81. Comparative genomics of viruses in archaeal
genomes has been carried out extensively; however,
there are no examples of proviruses that contain genes
of host origin82–84.
Interestingly, M. voltae PS spontaneously produces
virus-derived gene transfer agents that randomly pack-
age chromosomal DNA and transfer auxotrophic mark-
ers85,86. In nature, these agents can probably transfer
random fragments of chromosomal DNA85–87. Besides
proviruses and gene transfer agents, other mobile
genetic elements have been found in different archaeal
phyla, including transposons, insertion sequences and
introns32,38,88,89. It has been shown that archaeal mobile
genetic elements are more likely to be entrapped in
the genome than their bacterial counterparts90. Owing
to their high abundance in archaeal genomes, mobile
genetic elements have been suggested to influence
genome stability and plasticity 88,91.
The single intron of Desulfurococcus mobilis can
be exchanged between S. acidocaldarius cells, which
do not naturally have introns92. A recent phylogenetic
study showed that most archaeal viruses are evolution-
arily linked to other mobile genetic elements84. Thus, it
is tempting to speculate that mobile genetic elements are
transferred from one cell to another by using viruses as
transfer vehicles.
Conjugative plasmids. The process of conjugation
was first described more than 60 years ago for the
F plasmid of Escherichia coli 93. Over the past few dec-
ades, ground-breaking research has revealed detailed
mechanistic and structural insights into the conjuga-
tive systems of bacteria94–98. Although well-studied,
the transfer of the Ti plasmid of Agrobacterium spp.
to plants is currently the only example of inter-king-
dom conjugative transfer in nature99. No conjugative
systems have been found in eukaryotes. In archaea,
little information about conjugation is available. In
1995, the first archaeal conjugative plasmid (pNOB8)
was isolated from the hyperthermophilic Crenarchaeon
Sulfolobus sp. NOB8H2 (REF. 18). In the following years,
several conjugative plasmids were isolated exclusively
in members of the family Sulfolobaceae, which sug-
gests that conjugative plasmids might be limited to
this family 18,100–104 (FIG. 1d). Sequence comparison of six
conjugative plasmids revealed three distinct regions of
genes102. The first region contains a putative origin
of replication. The second region consists of genes
that encode proteins that are probably involved in
plasmid replication and a gene that encodes an inte-
grase102. The integrase enables site-specific integration
of the plasmid into the host chromosome105. The third
region comprises several ORFs that are suggested to
be involved in conjugation102. Highly conserved in the
­latter cluster are two ORFs encoding AAA+ ATPases
that exhibit homology to the ubiquitous ATPase VirB4
and the coupling protein VirD4 of bacterial type IV
secretion systems (T4SSs)102–104,106. Another conserved
ORF encodes a protein with 10–12 transmembrane
helices that possibly forms the translocation pore for
DNA transfer 102. This protein has no homology to bac-
terial T4SS proteins. Although archaeal conjugative
plasmids are self-transmissible, no clear homologues
of known conjugative relaxases or other DNA transfer
and replication proteins, except VirB4 and VirD4, were
identi­fied102. The absence of a known relaxase homo-
logue suggests a novel mechanism of DNA transfer.
Most archaeal conjugative plasmids are stably main-
tained in their natural host, but become unstable after
transfer to a foreign host 100,101,103,106,107. Reasons for this
instability might be recombination at specific sequences
that are spread over the plasmids101,102,106 or the activity
of CRISPR–Cas systems108–110. For example, after conju-
gation of pNOB8 from its original host into S. solfatari­
cus, the plasmid loses a stretch of 8 kb that encodes a
plasmid partitioning system, which results in the plasmid
variant pNOB8‑33 (REFS 18,106,111). pNOB8‑33 initially
replicates to a high copy number, but over prolonged
periods of growth the plasmid is lost 106. The partition-
ing system of pNOB8 belongs to a novel partitioning
system that comprises the centromere-binding pro-
tein AspA, the adaptor protein ParB, which contains a
eukaryotic histone-­like fold, and the bacteria-like par-
titioning ATPase ParA. The tripartite cluster of genes
that encodes the system is widespread in crenarchaeal
genera111. Although conjugative plasmids are present
and functional in members of the family Sulfolobaceae,
neither the mechanism of conjugation nor the selective
advantage of these plasmids is known.
Cell fusion hybrids in haloarchaea. Almost three dec-
ades ago, DNA transfer between H. volcanii cells was
shown to be bi‑directional and dependent on cell–cell
contact 112. Many haloarchaeal species form biofilms,
and frequent DNA exchange occurs in H. volcanii bio-
films113,114. Therefore, it was suggested that biofilms
represent a natural environment in which haloarchaea
live in close contact and exchange DNA and other mol-
ecules114. In 1989, cytoplasmic bridges were discovered
that maintain the cytoplasmic continuity of two con-
nected cells. These bridges were up to 2 μm in length
and 0.1 μm in diameter, and enabled the transfer of DNA
between cells115. As multiple cytoplasmic bridges were
shown to connect two cells at the same time, classical
bacterial conjugation was ruled out as a transfer mecha-
nism. This was later supported by the finding that both
chromosomal and plasmid DNA were exchanged at
similar frequencies, which contrasts with bacterial con-
jugation, in which mostly plasmid DNA is exchanged19.
A cell fusion-based DNA transfer mechanism in
which DNA can be transferred in a bidirectional man-
ner was therefore proposed for the haloarchaea19,20,115
(FIG. 1e). Although the mechanism of cell fusion is still
­unexplored, hetero-diploid cells are formed temporar-
ily during this process19. These cells contain the chro-
mosomal and plasmid DNA of both parental strains.
Recombination between the chromosomes and subse-
quent segregation will occasionally lead to a hybrid of
the parental strains. In addition, plasmids can be divided
unequally, which results in cells with a novel genotype20.

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 5

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

a b c of Sulfolobus) is conserved in the Sulfolobales and is

100 nm 10 nm
75 J per m2
d ment, and that no recombinants could be obtained by
? sion of components of Ups pili is also induced in bio-
Ups pilus
? that the rigid fibres are arranged as a three-stranded
S-layer
Ced system
(CedA, CedB)
Membrane
? that they have an important role in the species-specific
Figure 2 | DNA transfer in Sulfolobales. a | A light microscopy image of UV‑treated
Nature
Sulfolobus solfataricus shows that the majority of the cells are found Reviews | Microbiology
in cellular
|
aggregates. b  Electron microscopy image that shows Ups pili (UV‑inducible pili of
Sulfolobus) on the surface of S. solfataricus cells after exposure to UV light. c | Single-
particle analysis that shows a maximal diameter of 10 nm (left) and the three-stranded
helical arrangement (right) of Ups pili. The pitch of the structure is displayed with
horizontal lines (right) and accounts 15.5 nm. d | Following DNA damage, Ups pili
mediate cell‑to‑cell contact, which leads to the formation of cellular aggregates. It is
unclear whether the surface layer (S-layer) is rearranged during cell–cell contact or
degraded (indicated by the question mark). Within these cellular aggregates,
chromosomal DNA can be transferred and incorporated into the genome of the recipient
cell through homologous recombination, which facilitates DNA repair. Although it is
unknown how the DNA is processed and exported (single-stranded or double-stranded;
represented in the figure by the question marks), the DNA crosses the recipient
membrane through the crenarchaeal exchange of DNA (Ced) system. The Ced system
consists of a polytopic membrane protein, CedA, which presumably assembles into a
translocation pore, and an ATPase, CedB, the activity of which is essential for the
import of DNA. The images in parts a,b are adapted with permission from REF. 41, Wiley.
The image in part c is reproduced with permission from REF. 41, Wiley.
The observation that cell fusion and interspecies
recombination indeed occur is in agreement with phy-
logenetic data that suggest high levels of interspecies
DNA transfer between haloarchaea11,19,31,32. Haloarchaeal
genomes often contain a high density of insertion ele-
ments and transposons32. Therefore, it would be inter-
esting to study their role in gene shuffling during cell
fusion32.
Type IV pilus
A surface appendage found in
The Ups and Ced system. Following DNA damage,
many bacteria and archaea.
Type IV pili are involved in Sulfolobus spp. cells aggregate (FIGS 1f,2a) and chromo-
motility and attachment to somal DNA, which probably provides an intact DNA
surfaces and other cells. template for homologous recombination, is transferred
between the cells. Some of the genes for which expres-
Polytopic membrane
protein
sion was most highly induced by UV radiation are
A protein that has multiple part of an operon that encodes a type IV pilus assem-
transmembrane domains. bly system 116,117. This ups operon (UV-induced pili
required for the expression of UV-inducible pili and
the formation of cellular aggregates after DNA dam-
age42,118 (FIG. 2b). Sulfolobus spp. cells that are incapable
of producing Ups pili are less viable following treatment
with UV light than wild-type cells, which suggests that
cell‑to‑cell contact through the pili is essential for DNA
transfer 42. This is supported by the finding that chromo-
somal marker exchange is insensitive to DNase treat-
mixing cells with lysate or purified DNA39. The expres-
films, which suggests that Ups-dependent DNA transfer
also occurs under other conditions119. Single-particle
analysis of isolated Ups pili from S. solfataricus showed
helix with a maximal diameter of 10 nm (FIG.  2c) .
Although the diameter of Ups pili from different species
is similar, their length varies from 0.1 μm in S. acido­
caldarius to a maximum of 0.3 μm in S. solfataricus 41.
As Ups pili mediate cellular aggregation, it is possible
recognition.
A recent study identified a group of proteins that
are not related to the biogenesis of Ups pili in S. acido­
caldarius, but that are essential for DNA transfer 21.
Homologues of these proteins were found exclusively
in crenarchaeal phyla and were named the crenarchaeal
system for exchange of DNA (Ced). Similarly to the ups
operon, the expression of the ced genes is stimulated by
UV light 21,116,117. CedA is a polytopic membrane protein
that presumably forms the transport channel, which
might resemble ComEC of bacterial competence sys-
tems or VirB6 of conjugation systems, through which
DNA might pass the membrane43,120. CedB is a mem-
brane-bound homologue of VirB4 and HerA, and
its ATPase activity is essential for DNA transfer 21. In
Sulfolobus spp., DNA damage triggers the expression
of Ups pili and the Ced system (FIG. 2d). In the first step,
Ups pili mediate species-specific cellular aggregation.
Through an unknown mechanism, DNA is exported
from the donor cell after cell‑to‑cell contact is initi-
ated. DNA is then actively imported by the Ced system.
Although it is unknown whether single-stranded DNA
or double-stranded DNA is transported, incoming
DNA can be used as a template for homologous recom-
bination to repair damaged DNA. A contact-dependent
DNA import system has thus far not been observed in
other archaea, or in bacteria or eukaryotes21.
It is currently unknown whether conjugative plas-
mids of members of the Sulfolobaceae might use Ups
pili or the Ced system for transfer. Further studies should
reveal the mechanisms of DNA transport by conjugation
and through the Ups and Ced systems, and whether any
connections exist between these two processes.
Barriers to DNA transfer
Extracellular factors and cellular processes have fun-
damental roles in the establishment and maintenance
of species by preventing DNA transfer and the incor-
poration of foreign DNA (FIG. 3). Physical barriers in

6 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

are expressed and the precursor CRISPR RNA is tran-

Chromosomal
DNA
Plasmid
Geographical Virus
Ecological barrier
barrier (isolation) (pH, ion strength,
S-layer temperature, etc)
Surface REase
exclusion A
T T–A modules MTase
Homologous
recombination
cas genes CRISPR
Figure 3 | Barriers to HGT. Barriers to horizontal gene transfer (HGT) have a fundamental
Nature Reviews
role in the establishment and maintenance of species by preventing | Microbiology
DNA transfer and the
incorporation of foreign DNA. Physical barriers prevent random gene flux and lead to
the geographical isolation of microorganisms121. Environmental factors, such as pH, ion
strength or temperature, can destabilize or degrade external DNA, and thereby prevent
DNA transfer122,123. Foreign DNA, including plasmid, virus and chromosomal DNA (red),
has to cross the species-specific surface layer (S‑layer) and the membrane of a recipient
cell. DNA transport proteins, which are encoded on a plasmid and are anchored in the
membrane, reduce the import of other plasmid DNA in a process that is known as surface
exclusion124. In the cytoplasm, foreign DNA can be recognized and degraded by the Cas
proteins of the CRISPR–Cas immune system or through a restriction–modification
system.126–128. In restriction–modification, a restriction endonuclease (REase) only cuts
non-modified DNA, whereas the chromosomal DNA is protected (blue dots) by a
respective modification enzyme (MTase). Furthermore, the spread of viral and plasmid
DNA can be prevented by chromosomally encoded toxin–antitoxin modules (T–A)130.
The homologous recombination machinery will only incorporate foreign DNA that is
homologous to chromosomal DNA135–137. Dashed arrows indicate the genetic source of
the respective system.
the environment prevent gene flow and lead to the
geographical isolation of microorganisms, as has been
shown for hyperthermophilic Sulfolobus spp.121. In the
same habitat, ecological factors, such as pH, temperature
and salt concentrations, can negatively affect the stabil-
ity of DNA and thereby interfere with the exchange of
genetic material122,123.
Foreign DNA, including plasmid, virus or chro-
mosomal DNA, has to cross the surface layer (S‑layer)
and the membrane of a recipient cell. For members of
the Sulfolobales, it is speculated that species-specific
DNA transfer depends on the recognition of the S‑layer
by Ups pili 42. Indeed, with decreasing similarity of
S‑layer subunits, fewer HGT events between different
strains of S. islandicus were observed12. Furthermore,
plasmid-­encoded DNA transport proteins can decrease
the import of other plasmid DNA124. This process is
known as surface exclusion and might also be a rele­
vant barrier for HGT of the conjugative plasmids of
the Sulfolobaceae.
In the cytoplasm, phage and plasmid DNA can be
recognized by the CRISPR–Cas system, an adaptive
immune system in bacteria and archaea125. To achieve
this, short foreign DNA fragments are integrated in the
CRISPR locus. Following infection, the Cas proteins
scribed, and the Cas proteins are involved in the mat-
uration of the precursor CRISPR RNA125. This enables
the recognition of invading DNA and subsequent degra-
dation by a Cas nuclease. CRISPR loci have been identi-
fied in 84% of all archaeal genomes, which suggests that
CRISPR–Cas mediated immunity has an important role
in defence of archaea against viral infection126. Other
intracellular defence mechanisms against foreign DNA
are restriction–modification systems. In these systems,
a modification enzyme (usually a methyltransferase)
modifies self-DNA, which protects the chromosome
from the activity of a restriction endonuclease127,128.
Conversely, incoming foreign non-modified, or differ-
ently modified, DNA will be degraded. A recent genome
analysis showed that 129 of the 144 tested archaeal
species encode at least one restriction–modification
system129. In addition, the spread of phages can be pre-
vented by chromosomally encoded toxin–antitoxin
modules, which are prevalent in archaea130. These mod-
ules are composed of a toxin and a neutralizing anti-
toxin. Some toxins become activated following phage
infection and can lead to the suicide of the cell, which
prevents the spread of the phage in the population131–133.
Archaeal toxin–antitoxin modules are poorly investi-
gated in vivo; therefore, future studies should address
questions about their influence on phage propagation
and HGT through transduction134.
Finally, DNA divergence may present a barrier to
the incorporation of foreign DNA135–137. Interestingly,
no relationship between DNA divergence and homolo­
gous recombination was observed in natural popula-
tions of Sulfolobus spp.12. This may be due to the lack
of MutSL mismatch recognition in Sulfolobus spp.,
which prevents divergent DNA transfer in most other
microorganisms12,138,139.
Conclusions and future perspectives
With the increasing number of available archaeal
genome sequences and the development of meta­
genomic techniques, we are beginning to understand
the importance of DNA transfer in archaeal evolution.
Initial multilocus sequence typing (MLST) studies
(BOX 1) illustrated that in populations of the crenarchae-
ote S. islandicus 13 and the euryarchaeote Halorubrum
spp. 14 recombination occurs frequently. Moreover,
interdomain DNA transfer from bacteria to several
archaeal species (for example methanogens) supports
the strong evolutionary force of HGT in archaeal speci-
ation15. Future genomic and metagenomic studies will
undoubtedly identify novel archaea, which will not only
help to uncover more HGT events among archaea but
also between archaea, bacteria and eukaryotes. This
will enable us to get a deeper understanding of the
requirements for adaptation to new ecological niches,
the speed of evolution in certain habitats and the origin
of eukaryotes.
Classic bacterial HGT mechanisms, such as natural
transformation, conjugation and transduction, have
been identified in a small number of archaeal species
(FIG. 1). However, unlike for bacteria, the proteins that

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 7

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

are involved in the actual transfer of DNA are unknown
for most of these species. This can be explained by the
fact that, except for VirB4 and VirD4 in conjugation
systems, and possibly type IV pili-like structures, such
as Ups pili, no homologues of bacterial transport pro-
teins have been identified in archaeal genomes, which
suggests different mechanisms in archaea. Most of our
knowledge about archaeal DNA transfer mechanisms
is based on work with Sulfolobus spp. and haloarchaea.
Therefore, it needs to be determined whether the obser-
vations that were made in these organisms are specific
for these lineages or whether they are general features
of archaea. With an increasing number of genetically
tractable archaeal species available, we will be able
to elucidate the underlying processes of gene flow in
archaea. Future structural and biochemical work will
uncover new insights into the respective DNA transfer
mechanisms.

1. Spang, A. et al. Complex archaea that bridge the gap
between prokaryotes and eukaryotes. Nature 521,
173–179 (2015).
2. Cavicchioli, R. Cold-adapted archaea. Nat. Rev.
Microbiol. 4, 331–343 (2006).
3. Chen, X.‑P., Zhu, Y.‑G., Xia, Y., Shen, J.‑P. & He, J.‑Z.
Ammonia-oxidizing archaea: important players in
paddy rhizosphere soil? Environ. Microbiol. 10,
1978–1987 (2008).
4. Offre, P., Spang, A. & Schleper, C. Archaea in
biogeochemical cycles. Annu. Rev. Microbiol. 67,
437–457 (2013).
5. Makarova, K. S., Yutin, N., Bell, S. D. & Koonin, E. V.
Evolution of diverse cell division and vesicle formation
systems in Archaea. Nat. Rev. Microbiol. 8, 731–741
(2010).
6. Zaremba-Niedzwiedzka, K. et al. Asgard archaea
illuminate the origin of eukaryotic cellular complexity.
Nature 541, 353–358 (2017).
7. Guy, L. & Ettema, T. J. G. The archaeal ‘TACK’
superphylum and the origin of eukaryotes. Trends
Microbiol. 19, 580–587 (2011).
8. Williams, T. A., Foster, P. G., Nye, T. M. W., Cox, C. J. &
Embley, T. M. A congruent phylogenomic signal places
eukaryotes within the Archaea. Proc. Biol. Sci. 279,
4870–4879 (2012).
9. Woese, C. R., Kandler, O. & Wheelis, M. L. Towards a
natural system of organisms: proposal for the domains
Archaea, Bacteria, and Eucarya. Proc. Natl Acad. Sci.
USA 87, 4576–4579 (1990).
10. Rinke, C. et al. Insights into the phylogeny and coding
potential of microbial dark matter. Nature 499,
431–437 (2013).
11. Nelson-Sathi, S. et al. Acquisition of 1,000 eubacterial
genes physiologically transformed a methanogen at
the origin of Haloarchaea. Proc. Natl Acad. Sci. USA
109, 20537–20542 (2012).
This paper suggests that a single gene transfer
event transformed a methanogen in the last
haloarchaeal common ancestor.
12. Cadillo-Quiroz, H. et al. Patterns of gene flow define
species of thermophilic Archaea. PLoS Biol. 10,
e1001265 (2012).
13. Whitaker, R. J., Grogan, D. W. & Taylor, J. W.
Recombination shapes the natural population
structure of the hyperthermophilic archaeon
Sulfolobus islandicus. Mol. Biol. Evol. 22, 2354–2361
(2005).
14. Papke, R. T., Koenig, J. E., Rodríguez-Valera, F. &
Doolittle, W. F. Frequent recombination in a saltern
population of Halorubrum. Science 306, 1928–1929
(2004).
15. Nelson-Sathi, S. et al. Origins of major archaeal clades
correspond to gene acquisitions from bacteria. Nature
517, 77–80 (2015).
This study provides evidence for high bacteria-to-
archaea gene transfer at the origin of deeply
branching archaeal taxa.
16. Worrell, V. E., Nagle, D. P., McCarthy, D. & Eisenbraun, A.
Genetic transformation system in the archaebacterium
Methanobacterium thermoautotrophicum Marburg.
J. Bacteriol. 170, 653–656 (1988).
17. Held, N. L., Herrera, A., Cadillo-Quiroz, H. &
Whitaker, R. J. CRISPR associated diversity within a
population of Sulfolobus islandicus. PLoS ONE 5,
e12988 (2010).
18. Schleper, C., Holz, I., Janekovic, D., Murphy, J. &
Zillig, W. A multicopy plasmid of the extremely
thermophilic archaeon Sulfolobus effects its transfer to
recipients by mating. J. Bacteriol. 177, 4417–4426
(1995).
This study describes the first observation of an
archaeal conjugative plasmid.
19. Naor, A., Lapierre, P., Mevarech, M., Papke, R. T. &
Gophna, U. Low species barriers in halophilic archaea
and the formation of recombinant hybrids. Curr. Biol.
22, 1444–1448 (2012).
20. Naor, A. & Gophna, U. Cell fusion and hybrids in
Archaea: prospects for genome shuffling and
accelerated strain development for biotechnology.
Bioengineered 4, 9–8 (2013).
21. van Wolferen, M., Wagner, A., van der Does, C. &
Albers, S.‑V. The archaeal Ced system imports DNA.
Proc. Natl Acad. Sci. USA 113, 2496–2501 (2016).
This study, for the first time, describes proteins
that are involved in DNA transport machinery in
archaea.
22. Brochier-Armanet, C. et al. Complete-fosmid and
fosmid-end sequences reveal frequent horizontal gene
transfers in marine uncultured planktonic archaea.
ISME J. 5, 1291–1302 (2011).
23. Deschamps, P., Zivanovic, Y., Moreira, D., Rodriguez-
Valera, F. & López-García, P. Pangenome evidence for
extensive interdomain horizontal transfer affecting
lineage core and shell genes in uncultured planktonic
Thaumarchaeota and Euryarchaeota. Genome Biol.
Evol. 6, 1549–1563 (2014).
24. López-García, P., Zivanovic, Y., Deschamps, P. &
Moreira, D. Bacterial gene import and mesophilic
adaptation in archaea. Nat. Rev. Microbiol. 13,
447–456 (2015).
25. Allen, M. A. et al. The genome sequence of the
psychrophilic archaeon, Methanococcoides burtonii:
the role of genome evolution in cold adaptation.
ISME J. 3, 1012–1035 (2009).
26. Deppenmeier, U. et al. The genome of
Methanosarcina mazei: evidence for lateral gene
transfer between bacteria and archaea. J. Mol.
Microbiol. Biotechnol. 4, 453–461 (2002).
27. Raymann, K., Forterre, P., Brochier-Armanet, C. &
Gribaldo, S. Global phylogenomic analysis disentangles
the complex evolutionary history of DNA replication
in archaea. Genome Biol. Evol. 6, 192–212 (2014).
28. Wang, J. C. DNA topoisomerases. Annu. Rev.
Biochem. 65, 635–692 (1996).
29. López-García, P. DNA supercoiling and temperature
adaptation: a clue to early diversification of life?
J. Mol. Evol. 49, 439–452 (1999).
30. Groussin, M. & Gouy, M. Adaptation to environmental
temperature is a major determinant of molecular
evolutionary rates in archaea. Mol. Biol. Evol. 28,
2661–2674 (2011).
31. Williams, D., Gogarten, J. P. & Papke, R. T. Quantifying
homologous replacement of loci between haloarchaeal
species. Genome Biol. Evol. 4, 1223–1244 (2012).
32. DeMaere, M. Z. et al. High level of intergenera gene
exchange shapes the evolution of haloarchaea in an
isolated Antarctic lake. Proc. Natl Acad. Sci. USA 110,
16939–16944 (2013).
33. Papke, R. T. et al. Horizontal gene transfer, dispersal
and haloarchaeal speciation. Life (Basel) 5,
1405–1426 (2015).
34. Held, N. L., Herrera, A. & Whitaker, R. J.
Reassortment of CRISPR repeat-spacer loci in
Sulfolobus islandicus. Environ. Microbiol. 11,
3065–3076 (2013).
35. Krause, D. J. & Whitaker, R. J. Inferring speciation
processes from patterns of natural variation in
microbial genomes. Syst. Biol. 64, 926–935 (2015).
36. Reno, M. L., Held, N. L., Fields, C. J., Burke, P. V.
& Whitaker, R. J. Biogeography of the Sulfolobus
islandicus pan-genome. Proc. Natl Acad. Sci. USA
106, 8605–8610 (2009).
37. Brock, T. D., Brock, K. M., Belly, R. T. & Weiss, R. L.
Sulfolobus: a new genus of sulfur-oxidizing bacteria
living at low pH and high temperature. Arch.
Mikrobiol. 84, 54–68 (1972).
38. Naor, A., Lazary, R., Barzel, A., Papke, R. T. &
Gophna, U. In vivo characterization of the homing
endonuclease within the polB gene in the halophilic
archaeon Haloferax volcanii. PLoS ONE 6, e15833
(2011).
39. Grogan, D. W. Exchange of genetic markers at
extremely high temperatures in the archaeon
Sulfolobus acidocaldarius. J. Bacteriol. 178,
3207–3211 (1996).
40. Ghane, F. & Grogan, D. W. Chromosomal marker
exchange in the thermophilic archaeon Sulfolobus
acidocaldarius: physiological and cellular aspects.
Microbiology 144, 1649–1657 (1998).
41. Fröls, S. et al. UV‑inducible cellular aggregation of the
hyperthermophilic archaeon Sulfolobus solfataricus
is mediated by pili formation. Mol. Microbiol. 70,
938–952 (2008).
42. Ajon, M. et al. UV‑inducible DNA exchange in
hyperthermophilic archaea mediated by type IV pili.
Mol. Microbiol. 82, 807–817 (2011).
43. Chen, I. & Dubnau, D. DNA uptake during bacterial
transformation. Nat. Rev. Microbiol. 2, 241–249
(2004).
44. Salmond, G. P. C. & Fineran, P. C. A century of the
phage: past, present and future. Nat. Rev. Microbiol.
13, 777–786 (2015).
45. Guglielmini, J. et al. Key components of the eight
classes of type IV secretion systems involved in bacterial
conjugation or protein secretion. Nucleic Acids Res. 42,
5715–5727 (2014).
46. Gaudin, M. et al. Hyperthermophilic archaea
produce membrane vesicles that can transfer DNA.
Env. Microbiol. Rep. 5, 109–116 (2013).
This paper describes, for the first time, the transfer
of archaeal plasmids through vesicles.
47. Bertani, G. & Baresi, L. Genetic transformation in the
methanogen Methanococcus voltae PS. J. Bacteriol.
169, 2730–2738 (1987).
48. Sato, T., Fukui, T., Atomi, H. & Imanaka, T. Targeted
gene disruption by homologous recombination in the
hyperthermophilic archaeon Thermococcus
kodakaraensis KOD1. J. Bacteriol. 185, 210–220
(2003).
49. Waege, I., Schmid, G., Thumann, S., Thomm, M. &
Hausner, W. Shuttle vector-based transformation
system for Pyrococcus furiosus. Appl. Env. Microbiol.
76, 3308–3313 (2010).
50. Grogan, D. W. & Stengel, K. R. Recombination of
synthetic oligonucleotides with prokaryotic
chromosomes: substrate requirements of the
Escherichia coli /λRed and Sulfolobus acidocaldarius
recombination systems. Mol. Microbiol. 69,
1255–1265 (2008).
51. Patel, G. B., Nash, J. H., Agnew, B. J. & Sprott, G. D.
Natural and electroporation-mediated transformation
of Methanococcus voltae protoplasts. Appl. Environ.
Microbiol. 60, 903–907 (1994).
52. Sato, T., Fukui, T., Atomi, H. & Imanaka, T. Improved
and versatile transformation system allowing multiple
genetic manipulations of the hyperthermophilic
archaeon Thermococcus kodakaraensis. Appl. Env.
Microbiol. 71, 3889–3899 (2005).
53. Lipscomb, G. L. et al. Natural competence in the
hyperthermophilic archaeon Pyrococcus furiosus
facilitates genetic manipulation: construction of
markerless deletions of genes encoding the two
cytoplasmic hydrogenases. Appl. Env. Microbiol. 77,
2232–2238 (2011).
54. Averhoff, B. Shuffling genes around in hot environments:
the unique DNA transporter of Thermus thermophilus.
FEMS Microbiol. Rev. 33, 611–626 (2009).
55. Claverys, J.‑P., Martin, B. & Polard, P. The genetic
transformation machinery: composition, localization,
and mechanism. FEMS Microbiol. Rev. 33, 643–656
(2009).
56. Gould, S. B. et al. Bacterial vesicle secretion and the
evolutionary origin of the eukaryotic endomembrane
system. Trends Microbiol. 24, 525–534 (2016).

8 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
 REVIEWS

57. Soler, N., Marguet, E., Verbavatz, J.‑M. & Forterre, P.
Virus-like vesicles and extracellular DNA produced by
hyperthermophilic archaea of the order Thermococcales.
Res. Microbiol. 159, 390–399 (2008).
58. Choi, D. H. et al. Extracellular vesicles of the
hyperthermophilic archaeon Thermococcus
onnurineus NA1 T. Appl. Environ. Microbiol. 81,
4591–4599 (2015).
59. Soler, N., Gaudin, M., Marguet, E. & Forterre, P.
Plasmids, viruses and virus-like membrane vesicles
from Thermococcales. Biochem. Soc. Trans. 39, 36–44
(2011).
60. Gaudin, M. et al. Extracellular membrane vesicles
harbouring viral genomes. Environ. Microbiol. 16,
1167–1175 (2014).
61. Santangelo, T. J., Cubonová, L. & Reeve, J. N. Shuttle
vector expression in Thermococcus kodakaraensis:
contributions of cis elements to protein synthesis in a
hyperthermophilic archaeon. Appl. Env. Microbiol. 74,
3099–3104 (2008).
62. Shetty, A., Chen, S., Tocheva, E. I., Jensen, G. J. &
Hickey, W. J. Nanopods: a new bacterial structure and
mechanism for deployment of outer membrane
vesicles. PLoS ONE 6, e20725 (2011).
63. Marguet, E. et al. Membrane vesicles, nanopods and/or
nanotubes produced by hyperthermophilic archaea of
the genus Thermococcus. Biochem. Soc. Trans. 41,
436–442 (2013).
64. Ellen, A. F. et al. Proteomic analysis of secreted
membrane vesicles of archaeal Sulfolobus species
reveals the presence of endosome sorting complex
components. Extremophiles 13, 67–79 (2009).
65. Prangishvili, D. et al. Sulfolobicins, specific
proteinaceous toxins produced by strains of the
extremely thermophilic archaeal genus Sulfolobus.
J. Bacteriol. 182, 2985–2988 (2000).
66. Mashburn-Warren, L. M. & Whiteley, M. Special
delivery: vesicle trafficking in prokaryotes.
Mol. Microbiol. 61, 839–846 (2006).
67. Kulp, A. & Kuehn, M. J. Biological functions and
biogenesis of secreted bacterial outer membrane
vesicles. Annu. Rev. Microbiol. 64, 163–184 (2010).
68. Deatherage, B. L. & Cookson, B. T. Membrane vesicle
release in bacteria, eukaryotes, and archaea:
a conserved yet underappreciated aspect of microbial
life. Infect. Immun. 80, 1948–1957 (2012).
69. Nieuwland, R. & Sturk, A. Why do cells release
vesicles? Thromb. Res. 125 (Suppl.), S49–S51 (2010).
70. Rice, G. et al. Viruses from extreme thermal
environments. Proc. Natl Acad. Sci. USA 98,
13341–13345 (2001).
71. Witte, A. et al. Characterization of Natronobacterium
magadii phage ΦCh1, a unique archaeal phage
containing DNA and RNA. Mol. Microbiol. 23,
603–616 (1997).
72. Liu, Y. et al. Identification and characterization of
SNJ2, the first temperate pleolipovirus integrating into
the genome of the SNJ1‑lysogenic archaeal strain.
Mol. Microbiol. 98, 1002–1020 (2015).
73. Krupovic, M., Spang, A., Gribaldo, S., Forterre, P.
& Schleper, C. A thaumarchaeal provirus testifies for
an ancient association of tailed viruses with archaea.
Biochem. Soc. Trans. 39, 82–88 (2011).
74. Schleper, C., Kubo, K. & Zillig, W. The particle SSV1
from the extremely thermophilic archaeon Sulfolobus
is a virus: demonstration of infectivity and of
transfection with viral DNA. Proc. Natl Acad. Sci. USA
89, 7645–7649 (1992).
75. Quemin, E. R. J. et al. Eukaryotic-like virus budding in
Archaea. mBio 7, e01439‑16 (2016).
76. Arnold, H. P. et al. The genetic element pSSVx of the
extremely thermophilic crenarchaeon Sulfolobus is a
hybrid between a plasmid and a virus. Mol. Microbiol.
34, 217–226 (1999).
77. Wang, Y. et al. A novel Sulfolobus non-conjugative
extrachromosomal genetic element capable of
integration into the host genome and spreading in the
presence of a fusellovirus. Virology 363, 124–133
(2007).
78. Iro, M. et al. The lysogenic region of virus φCh1:
identification of a repressor-operator system and
determination of its activity in halophilic Archaea.
Extremophiles 11, 383–396 (2007).
79. Mei, Y. et al. Induction and preliminary
characterization of a novel halophage SNJ1 from
lysogenic Natrinema sp. F5. Can. J. Microbiol. 53,
1106–1110 (2007).
80. Schnabel, H. et al. Halobacterium halobium phage øH.
EMBO J. 1, 87–92 (1982).
81. Zhang, Z. et al. Temperate membrane-containing
halophilic archaeal virus SNJ1 has a circular dsDNA
genome identical to that of plasmid pHH205. Virology
434, 233–241 (2012).
82. Prangishvili, D., Garrett, R. A. & Koonin, E. V.
Evolutionary genomics of archaeal viruses: unique viral
genomes in the third domain of life. Virus Res. 117,
52–67 (2006).
83. Pina, M., Bize, A., Forterre, P. & Prangishvili, D. The
archeoviruses. FEMS Microbiol. Rev. 35, 1035–1054
(2011).
84. Iranzo, J., Koonin, E. V., Prangishvili, D. &
Krupovic, M. Bipartite network analysis of the
archaeal virosphere: evolutionary connections
between viruses and capsidless mobile elements.
J. Virol. 90, 11043–11055 (2016).
85. Bertani, G. Transduction-like gene transfer in the
methanogen Methanococcus voltae. J. Bacteriol. 181,
2992–3002 (1999).
86. Eiserling, F., Bertani, G., Pushkin, A. & Gingery, M.
Bacteriophage-like particles associated with the
gene transfer agent of Methanococcus voltae PS.
J. Gen. Virol. 80, 3305–3308 (1999).
87. Lang, A. S., Zhaxybayeva, O. & Beatty, J. T.
Gene transfer agents: phage-like elements of genetic
exchange. Nat. Rev. Microbiol. 10, 472–482 (2012).
88. Spang, A. et al. The genome of the ammonia-oxidizing
Candidatus Nitrososphaera gargensis: insights into
metabolic versatility and environmental adaptations.
Environ. Microbiol. 14, 3122–3145 (2012).
89. Redder, P. & Garrett, R. A. Mutations and
rearrangements in the genome of Sulfolobus
solfataricus P2. J. Bacteriol. 188, 4198–4206
(2006).
90. She, Q., Peng, X., Zillig, W. & Garrett, R. A. Gene
capture in archaeal chromosomes. Nature 409, 478
(2001).
91. Gudbergsdottir, S. et al. Dynamic properties of the
Sulfolobus CRISPR/Cas and CRISPR/Cmr systems when
challenged with vector-borne viral and plasmid genes
and protospacers. Mol. Microbiol. 79, 35–49 (2011).
92. Aagaard, C., Dalgaard, J. Z. & Garrett, R. A.
Intercellular mobility and homing of an archaeal rDNA
intron confers a selective advantage over intron- cells
of Sulfolobus acidocaldarius. Proc. Natl Acad. Sci.
USA 92, 12285–12289 (1995).
93. Cavalli, L. L., Lederberg, J. & Lederberg, E. M.
An infective factor controlling sex compatibility in
Bacterium coli. J. Gen. Microbiol. 8, 89–103 (1953).
94. Alvarez-Martinez, C. E. & Christie, P. J. Biological
diversity of prokaryotic type IV secretion systems.
Microbiol. Mol. Biol. Rev. 73, 775–808 (2009).
95. Cruz, F. De et al. Towards an integrated model of
bacterial conjugation. FEMS Microbiol. Rev. 39,
81–95 (2014).
96. Christie, P. J., Whitaker, N. & González-Rivera, C.
Mechanism and structure of the bacterial type IV
secretion systems. Biochim. Biophys. Acta 1843,
1578–1591 (2014).
97. Chandran Darbari, V. & Waksman, G. Structural
biology of bacterial type IV secretion systems.
Annu. Rev. Biochem. 84, 603–629 (2015).
98. Ilangovan, A., Connery, S. & Waksman, G. Structural
biology of the Gram-negative bacterial conjugation
systems. Trends Microbiol. 23, 301–310 (2015).
99. Lacroix, B. & Citovsky, V. Transfer of DNA from
bacteria to eukaryotes. mBio 7, e00863‑16 (2016).
100. Prangishvili, D. et al. Conjugation in archaea: frequent
occurrence of conjugative plasmids in Sulfolobus.
Plasmid 40, 190–202 (1998).
101. Stedman, K. M. et al. pING family of conjugative
plasmids from the extremely thermophilic archaeon
Sulfolobus islandicus: insights into recombination
and conjugation in Crenarchaeota. J. Bacteriol. 182,
7014–7020 (2000).
102. Greve, B., Jensen, S., Brugger, K., Zillig, W. &
Garrett, R. A. Genomic comparison archaeal
conjugative plasmids from Sulfolobus. Archaea 1,
231–239 (2004).
103. Erauso, G., Stedman, K. M., van den Werken, H. J. G.,
Zillig, W. & van der Oost, J. Two novel conjugative
plasmids from a single strain of Sulfolobus.
Microbiology 152, 1951–1968 (2006).
104. Basta, T., Smyth, J., Forterre, P., Prangishvili, D.
& Peng, X. Novel archaeal plasmid pAH1 and its
interactions with the lipothrixvirus AFV1.
Mol. Microbiol. 71, 23–34 (2009).
105. She, Q., Shen, B. & Chen, L. Archaeal integrases and
mechanisms of gene capture. Biochem. Soc. Trans. 32,
222–226 (2004).
106. She, Q. et al. Genetic profile of pNOB8 from
Sulfolobus: the first conjugative plasmid from an
archaeon. Extremophiles 2, 417–425 (1998).
107. Wang, H., Peng, N., Shah, S. A., Huang, L. & She, Q.
Archaeal extrachromosomal genetic elements.
Microbiol. Mol. Biol. Rev. 79, 117–152 (2015).
108. Erdmann, S. & Garrett, R. A. Selective and
hyperactive uptake of foreign DNA by adaptive
immune systems of an archaeon via two distinct
mechanisms. Mol. Microbiol. 85, 1044–1056
(2012).
109. Garrett, R. et al. CRISPR–Cas adaptive immune
systems of the sulfolobales: unravelling their
complexity and diversity. Life 5, 783–817 (2015).
110. Liu, G., She, Q. & Garrett, R. A. Diverse CRISPR–Cas
responses and dramatic cellular DNA changes and
cell death in pKEF9‑conjugated Sulfolobus species.
Nucleic Acids Res. 44, 4233–4242 (2016).
111. Schumacher, M. A. et al. Structures of archaeal DNA
segregation machinery reveal bacterial and eukaryotic
linkages. Science 349, 1120–1124 (2015).
112. Mevarech, M. & Werczberger, R. Genetic transfer in
Halobacterium volcanii. J. Bacteriol. 162, 461–462
(1985).
This study is the first to show gene transfer in
archaea using auxotrophic mutants of H. volcanii.
113. Fröls, S., Dyall-Smith, M. & Pfeifer, F. Biofilm
formation by haloarchaea. Environ. Microbiol. 14,
3159–3174 (2012).
114. Chimileski, S., Franklin, M. J. & Papke, R. T. Biofilms
formed by the archaeon Haloferax volcanii exhibit
cellular differentiation and social motility, and
facilitate horizontal gene transfer. BMC Biol. 12, 65
(2014).
115. Rosenshine, I., Tchelet, R. & Mevarech, M.
The mechanism of DNA transfer in the mating system
of an archaebacterium. Science 245, 1387–1389
(1989).
116. Fröls, S. et al. Response of the hyperthermophilic
archaeon Sulfolobus solfataricus to UV damage.
J. Bacteriol. 189, 8708–8718 (2007).
117. Götz, D. et al. Responses of hyperthermophilic
crenarchaea to UV irradiation. Genome Biol. 8, R220
(2007).
118. Fröls, S., White, M. F. & Schleper, C. Reactions to
UV damage in the model archaeon Sulfolobus
solfataricus. Biochem. Soc. Trans. 37, 36–41 (2009).
119. Koerdt, A., Gödeke, J., Berger, J., Thormann, K. M.
& Albers, S.‑V. Crenarchaeal biofilm formation under
extreme conditions. PLoS ONE 5, e14104 (2010).
120. Beijersbergen, A., Smith, S. J. & Hooykaas, P. J.
Localization and topology of VirB proteins of
Agrobacterium tumefaciens. Plasmid 32, 212–218
(1994).
121. Whitaker, R. J., Grogan, D. W. & Taylor, J. W.
Geographic barriers isolate endemic populations of
hyperthermophilic archaea. Science 301, 976–978
(2003).
This paper describes the role and importance of
geographical barriers in the evolution of
hyperthermophilic archaea.
122. Bauer, T., Hammes, W. P., Haase, N. U. & Hertel, C.
Effect of food components and processing parameters
on DNA degradation in food. Environ. Biosafety Res.
3, 215–223 (2004).
123. Tan, Z.‑J. & Chen, S.‑J. Nucleic acid helix stability:
effects of salt concentration, cation valence and size,
and chain length. Biophys. J. 90, 1175–1190 (2006).
124. Achtman, M., Kennedy, N. & Skurray, R. Cell–cell
interactions in conjugating Escherichia coli: role of
traT protein in surface exclusion. Proc. Natl Acad. Sci.
USA 74, 5104–5108 (1977).
125. Marraffini, L. A. & Sontheimer, E. J. CRISPR
interference: RNA-directed adaptive immunity in
bacteria and archaea. Nat. Rev. Genet. 11, 181–190
(2010).
126. Rath, D., Amlinger, L., Rath, A. & Lundgren, M. The
CRISPR–Cas immune system: biology, mechanisms
and applications. Biochimie 117, 119–128 (2015).
127. Mruk, I. & Kobayashi, I. To be or not to be: regulation
of restriction–modification systems and other toxin–
antitoxin systems. Nucleic Acids Res. 42, 70–86
(2014).
128. Loenen, W. A. M., Dryden, D. T. F., Raleigh, E. A.,
Wilson, G. G. & Murray, N. E. Highlights of the DNA
cutters: a short history of the restriction enzymes.
Nucleic Acids Res. 42, 3–19 (2014).
129. Oliveira, P. H., Touchon, M. & Rocha, E. P. C. The
interplay of restriction–modification systems with
mobile genetic elements and their prokaryotic hosts.
Nucleic Acids Res. 42, 10618–10631 (2014).
130. Yamaguchi, Y., Park, J.‑H. & Inouye, M. Toxin–
antitoxin systems in bacteria and archaea. Annu. Rev.
Genet. 45, 61–79 (2011).

NATURE REVIEWS | MICROBIOLOGY ADVANCE ONLINE PUBLICATION | 9

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.
REVIEWS

131. Hazan, R. & Engelberg-Kulka, H. Escherichia coli
mazEF-mediated cell death as a defense mechanism
that inhibits the spread of phage P1. Mol. Genet.
Genomics 272, 227–234 (2004).
132. Engelberg-Kulka, H., Amitai, S., Kolodkin-Gal, I. &
Hazan, R. Bacterial programmed cell death and
multicellular behavior in bacteria. PLoS Genet. 2,
e135 (2006).
133. León-Sobrino, C., Kot, W. P. & Garrett, R. A.
Transcriptome changes in STSV2‑infected Sulfolobus
islandicus REY15A undergoing continuous CRISPR
spacer acquisition. Mol. Microbiol. 99, 719–728
(2016).
134. Shah, S. A. & Garrett, R. A. in Prokaryotic Toxin–
Antitoxins (ed. Gerders, K.) 225–238 (Springer Berlin
Heidelberg, 2013).
135. Zawadzki, P., Roberts, M. S. & Cohan, F. M. The log-
linear relationship between sexual isolation and
sequence divergence in Bacillus transformation is
robust. Genetics 140, 917–932 (1995).
136. Majewski, J. & Cohan, F. M. Adapt globally, act locally:
the effect of selective sweeps on bacterial sequence
diversity. Genetics 152, 1459–1474 (1999).
137. Wolf, Y. I., Rogozin, I. B., Kondrashov, A. S. &
Koonin, E. V. Genome alignment, evolution of
prokaryotic genome organization, and prediction of
gene function using genomic context. Genome Res.
11, 356–372 (2001).
138. Majewski, J., Zawadzki, P., Pickerill, P., Cohan, F. M. &
Dowson, C. G. Barriers to genetic exchange between
bacterial species: Streptococcus pneumoniae
transformation. J. Bacteriol. 182, 1016–1023
(2000).
139. Datta, A., Adjiri, A., New, L., Crouse, G. F. & Jinks
Robertson, S. Mitotic crossovers between diverged
sequences are regulated by mismatch repair proteins
in Saccharomyces cerevisiae. Mol. Cell. Biol. 16,
1085–1093 (1996).
140. Eppley, J. M., Tyson, G. W., Getz, W. M. &
Banfield, J. F. Genetic exchange across a species
boundary in the archaeal genus Ferroplasma.
Genetics 177, 407–416 (2007).
141. Krause, D. J., Didelot, X., Cadillo-Quiroz, H. &
Whitaker, R. J. Recombination shapes genome
architecture in an organism from the archaeal
domain. Genome Biol. Evol. 6, 170–178 (2014).
142. Ambur, O.‑H., Engelstadter, J., Johnsen, P., Miller, E.
& Rozen, D. Steady at the wheel: conservative sex and
the benefits of bacterial transformation. Phil. Trans.
R. Soc. B. Biol. Sci. 371, 20150528 (2016).
143. Rocha, E. P. C. Using sex to cure the genome.
PLoS Biol. 14, 1–7 (2016).
144. Mell, J. C. & Redfield, R. J. Natural competence and
the evolution of DNA uptake specificity. J. Bacteriol.
196, 1471–1483 (2014).
145. Johnston, C., Martin, B., Fichant, G., Polard, P. &
Claverys, J.‑P. Bacterial transformation: distribution,
shared mechanisms and divergent control. Nat. Rev.
Microbiol. 12, 181–196 (2014).
Acknowledgements
R.J.W. and D.J.K. acknowledge funding support from the US
National Science Foundaion (NSF; grant DEB #1355171) and
NASA Exobiology and Evolutionary Biology (grant
NNX09AM92G). A.W., M.v.W. and J.H. were supported by a
European Research Council (ERC) starting grant (grant
ARCHAELLUM, 311523). J.H. received further support from
the CRC746 (DFG).
Competing interests statement
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

10 | ADVANCE ONLINE PUBLICATION www.nature.com/nrmicro

©
2
0
1
7
M
a
c
m
i
l
l
a
n
P
u
b
l
i
s
h
e
r
s
L
i
m
i
t
e
d
,
p
a
r
t
o
f
S
p
r
i
n
g
e
r
N
a
t
u
r
e
.
A
l
l
r
i
g
h
t
s
r
e
s
e
r
v
e
d
.