Available online at www.sciencedirect.
com
ScienceDirect
Membrane vesicles and horizontal gene transfer in
prokaryotes
Sara Domingues1,2 and Kaare M Nielsen3,4
Membrane vesicles (MVs) are released from all living cells. MVs
are lumen-containing spheres of lipid-bilayers derived from the
cell surface. MVs are biologically active and contain various
components, including genetic material. Both chromosomal
and plasmid DNA, as well as different types of RNA have been
detected in MVs. Vesicle-mediated transfer of genes coding for
antibiotic resistance, virulence and metabolic traits has been
reported in Gram-negative and Gram-positive bacteria and in
Archaea. MVs can persist over time in natural environments.
Here we review the characteristics of and the role of MVs in
horizontal gene transfer (HGT) processes in prokaryotes.
Addresses
1
Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra,
Portugal
2
Center for Neuroscience and Cell Biology, 3004-517 Coimbra, Portugal
3
Department of Life Sciences and Health, Oslo and Akershus University
College, 0130 Oslo, Norway
4
Genok-Center for Biosafety, SIVA Innovation Center, 9294 Tromsø,
Norway
Corresponding authors: Domingues, Sara (saradomingues@ff.uc.pt),
Nielsen, Kaare M (kaare.nielsen@hioa.no)
Current Opinion in Microbiology 2017, 38:16–21
This review comes from a themed issue on Mobile genetic elements
and HGT in prokaryotes
Edited by Andrew Lang, J Thomas Beatty and Phoebe Rice
For a complete overview see the Issue and the Editorial
Available online 22nd April 2017
http://dx.doi.org/10.1016/j.mib.2017.03.012
1369-5274/ã 2017 Elsevier Ltd. All rights reserved.
Introduction
Living cells produce vesicles that are named differently
accordingly to the taxonomic group that release them.
Generally, they are called membrane vesicles (MVs) or
extracellular vesicles (EVs); membrane vesicles in
Archaea and Mycobacteria; membrane blebs, outer mem-
brane blebs or outer membrane vesicles (OMVs) in Gram-
negative bacteria; membrane vesicles in Gram-positive
bacteria; exosomes, microvesicles or tolerosomes, among
many other designations, in Eukarya (EVpedia; http://
EVpedia.info).
Current Opinion in Microbiology 2017, 38:16–21
Although originally considered a random outcome of cell
lysis, it is now well established that MVs can be produced
by secretion from living cells [1,2]. Several models can
explain the biogenesis (also called vesiculation) of MVs in
processes that are not yet fully understood [2,3]. MVs
are biologically active and a multitude of functions have
been associated with these vesicles [1,2,3]. Here we
review the characteristics of and the role of MVs in
horizontal gene transfer (HGT) processes in prokaryotes.
Formation of membrane vesicles (MVs)
MVs are lumen-containing spheres of lipid-bilayers
derived from the cell surface; occasionally they can be
elongated or elliptical, especially during vesiculation
[4,5]. Their diameter varies and ranges from 10 to
500 nm (Table 1). The amount and composition of vesi-
cles vary even within the same species and population
and depends on growth phase and environmental condi-
tions [1,6,7]. For example, Acinetobacter baumannii
ATCC19606 populations release small outer MVs in
the early log-phase, large MVs in the early and mid-log
phase, and medium sized vesicles called inner and outer
membrane vesicles (IOMVs) comprising elements from
the inner and outer membranes and a putative peptido-
glycan layer, during the stationary phase [8].
MVs are secreted in a variety of environments, including
planktonic and biofilm stages, within eukaryotic host
cells, and when present in different growth media in
the laboratory [6,7]. A conserved general mechanism
for the biogenesis of MVs has not been identified so
far, but several models have been proposed.
The most common MVs from Gram-negative bacteria are
the OMVs, formed from the outer membrane (OM) of the
cells. A multitude of mechanisms have been proposed to
be involved in vesiculation [9–13]. The release of OMVs
appear to occur after the OM budges out in areas where
the OM is detached from the peptidoglycan layer; the
amount of lipids, responsible for the fluidity and curva-
ture of the OM, also influence the formation of the OMVs
[2].
The main question about the biogenesis of thick cell
walled prokaryotes is how the MVs formed from the cell
membrane escape the cell wall and are release in the
environment. Until now there are three, non-mutually
exclusive, proposed models. Briefly, turgor pressure from
the cytoplasm may force the MVs through the pores of the
cell wall; proteases might act to increase the size of cell
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 Membrane vesicles and HGT in prokaryotes Domingues and Nielsen 17

Table 1 Relocation of genetic material in MVs

Size range of membrane vesicles released by prokaryotes
Prokaryote Diameter (nm)
Gram-negative bacteria 10–500
Gram-positive bacteria 20–400
Mycobacteria 50–300
Archaea 50–230
wall pores; and/or MVs might reach the extracellular
environment after crossing the cell wall inside protein
channels [3].
The cell wall of archaea lacks peptidoglycan and different
species have a high diversity in the composition of the cell
envelope. The formation of MVs nevertheless seems to
resemble that of Gram-positive bacteria. Protein-based
studies suggested that MVs are released from the cell
surface through the action of an endosomal sorting com-
plex required for transport by protrusion of the mem-
brane, followed by disassembly and consequent detach-
ment of vesicles [6,14].
Content of membrane vesicles
In addition to the membrane components needed for the
formation of the membrane sphere itself, MVs contain a
range of other components such as inner-membrane,
periplasmic and cytoplasmic components including pro-
teins, polysaccharides and nucleic acids. The components
may act as toxins, virulence factors, and in antibiotic
degradation. Vesicles can also contain misfolded proteins
or components related to the growth environment of the
cells such as antibiotics and metal ions. MVs are predicted
to have a role in stress response, cell-to-cell communica-
tion, nutrient acquisition, biofilm formation, in defence
and in gene transfer resulting in acquisition of antibiotic
resistance [1,2,3].
Although not yet completely clear, DNA can end up in
MVs by different ways: via a cytoplasmic route, where the
Reference
cytoplasmic content, including DNA, is trapped into
[7,8] IOMVs; via a periplasmic route, where the DNA has to
[3]
[3]
relocate from the cytoplasm to the periplasmic space,
[6,30] followed by trapping in MVs; via an extracellular route,
possibly due to broken MVs that re-annealed after release
from the bacteria; or due to cell death [4,5,15–17]. Phages
may also inject their DNA directly into MVs [18].
The mechanism(s) responsible for the presence of RNA
in MVs is also not fully understood, though different
options have been hypothesized: for instance protein
synthesis close to the membrane site where vesiculation
occurs resulting in trapping of mRNA together with the
ribosomal proteins; and via the routes described for DNA
[19,20].
It remains unclear if DNA/RNA is actively transported
into particular types of MV or if the presence of genetic
material in MV is mainly the outcome of random pro-
cesses. A complication in the study of genetic material
associated with MVs is to distinguish between surface
associated nucleic acids (possible in a nuclease resistant
state) [4,15,21,22] and nucleic acids present in the lumen
[4,15,21–23] as a result of vesicle formation.
Studies of the content of genetic material in membrane
vesicles
Genetic material has been reported to be present in MVs
isolated from a variety of bacterial populations, including
Gram-negative and Gram-positive bacteria, mycoplasma
and archaea (Table 2). Genetic material up to 370 Kbp
has been detected inside MVs, though smaller fragments
are more frequent [24,25,26]. Several studies report
the presence of DNA that can be chromosomal, plasmid

Table 2

Examples of genetic material present in membrane vesicles

Type of genetic material Species References

DNA
Chromosomal Clostridium perfringens, Escherichia coli, Neisseria gonorrhoeae, Porphyromonas gingivalis, [5,18,21,23,24,26,28]
Prochlorococcus sp., Ruminococcus spp., Shewanella vesiculosa
Plasmid Acinetobacter baumannii, A. baylyi, E. coli, Neisseria gonorrhoeae, Pseudomonas aeruginosa, [4,15,16,22,23,26,29]
Thermococcales kodakaraensis, Thermococcus nautili a
Viral E. coli, T. nautili [22,23]
Not specified Acholeplasma laidlawii [32]
RNA
mRNA E. coli, P. gingivalis, Prochlorococcus [18,19,21]
rRNA E. coli, P. gingivalis [19,21]
sRNA E. coli, Vibrio cholerae [19,20]
tRNA E. coli [19]
Not specified N. gonorrhoeae [26]
a
Formerly Thermococcus nautilus.

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18 Mobile genetic elements and HGT in prokaryotes

Figure 1

Phages

DNA
fragments
Recombination
Genome / replication

Free DNA
Donor cell Recipient cell

Membrane vesicles
Current Opinion in Microbiology

Schematic presentation of membrane-vesicle (MV) mediated horizontal gene transfer from a donor to a recipient cell. Membrane vesicles (10–
500 nm in diameter) may contain nucleic acids in the lumen or bound to the surface. Upon lysis DNA in the lumen will be sensitive to nucleases.
Bacteriophages may also bind to MVs in the environment leading to transduction-like HGT processes. The process is multi-directional.

and/or of phage origin [7]. Both natural and vector plas-
mids have been found inside MVs [4,16].
Different types of RNA, including mRNA, rRNA, sRNA
and tRNA, have also been found in MVs (Table 2). In
some cases, the RNA present covered the majority of the
genes of the genome of the MV producing bacterial strain
[19].
Some MVs, such as the ones detected in Neisseria gonor-
rhoeae, Prochlorococcus sp. and Porphyromonas gingivalis,
carried both DNA and RNA [18,21,26].
HGT by membrane vesicles
MVs carrying genetic material can upon exposure to new
host cells result in gene transfer events (Figure 1;
Table 3). The genetic material can be linked with the
outer surface of MVs [4,15,21,22] or be present in their
lumen [4,15,21–23]. MVs allow transformation in bacte-
rial species that are not considered to efficiently express
competence for natural uptake of DNA, such as Escher-
ichia coli and Salmonella enterica. However, in species that
are efficient at expressing competence for natural DNA
uptake, OMVs-mediated transfer seems to depend also
on the competence machinery in the recipient cell, at
least in Acinetobacter baylyi [4] and in Thermus thermophilus
[27].
MVs confer protection of the DNA in the lumen against
nucleases, as seen by the unaffectedness of the DNA
content in OMVs [18,26,28] and OMV-mediated trans-
fer after addition of DNase [4,16,29]. MVs have also
been reported to protect DNA from thermodegradation
[30].

Table 3

Experimental studies of membrane vesicle mediated HGT in prokaryotes

Genetic material Donor species Recipient species Reference a

Gram-negative bacteria Plasmid DNA Neisseria gonorrhoeae N. gonorrhoeae [26]
Chromosomal DNA Escherichia coli E. coli [36]
Plasmid and phage DNA E. coli E. coli [23]
Phage DNA E. coli Salmonella enterica [23]
Chromosomal DNA Natural seawater E. coli [25]
Plasmid DNA Acinetobacter baumannii A. baumannii [16]
Plasmid DNA Acinetobacter baylyi A. baylyi and E. coli [4]
Chromosomal DNA Porphyromonas gingivalis P. gingivalis [21]
Chromosomal DNA Thermus thermophilus T. thermophilus [27]
Plasmid DNA T. thermophiles and Thermus scotoductus T. thermophilus [27]
Gram-positive bacteria Chromosomal DNA Ruminococcus sp. and Ruminococcus albus Ruminococcus sp. [24]
Archaea Plasmid DNA Thermococcus kodakaraensis T. kodakaraensis [29]
a
Listed by publication date within each taxonomic grouping.

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 Membrane vesicles and HGT in prokaryotes Domingues and Nielsen 19
The specifics of the mechanism of gene delivery at the
host cell surface often remain unclear. Different pathways
seem to occur depending on the recipient species. For
instance, OMVs might attach to the OM surface, followed
by lysis; or attach to the OM and then become internal-
ized by the recipient Gram-negative cell [4].
Successful HGT events require inheritance of the
acquired genetic material, measured as transmission to
subsequent generations. Expression of genes transferred
by MVs has been observed, as well as the heritable nature
of the HGT events in both Gram-positive [24] and
Gram-negative [16,23] bacteria. MVs-mediated gene
transfer can contribute to the dissemination of microbial
fitness determinants such as antimicrobial resistance [16],
metabolic properties [24] and virulence genes [23].
Examples of MV-mediated gene transfer in Gram-
negative bacteria
Fulsundar et al. tracked the route of plasmid DNA from
the bacterial cytoplasm to the lumen of OMVs; the DNA
path was traced by immunogold labelling followed by
visualization by transmission electron microscopy [4]. In
their laboratory study, OMVs-mediated transfer of plas-
mid DNA between populations of A. baylyi strains and
also to E. coli DH5a recipient cells occurred at frequen-
cies between 10 8 to 10 6 gene transfer event per recipi-
ent cell; immunogold labelling of the OMVs with FITC
allowed the observation of attachment and internalization
of the vesicles by the recipient cells. A. baylyi were
transformed at a higher frequency with the OMVs than
with naked DNA in the natural transformation assay used.
Within the same genus, two clinical strains of A. bauman-
nii were also shown to release, during the exponential
growth phase, OMVs carrying plasmid DNA containing a
carbapenemase gene, blaOXA-24. Horizontal transfer of the
plasmid to A. baumannii ATCC 17978 recipient cells was
seen after OMV exposure and resulted in an increase in
the minimal inhibitory concentrations to several b-lactam
antibiotics in the recipient strain [16].
Horizontal transfer of plasmid DNA in MVs between N.
gonorrhoeae strains was demonstrated in early studies [26
]. Chromosomal DNA present in OMVs of P. gingivalis
ATCC 49417 was found to transfer to P. gingivalis ATCC
33277 at a requency of 1.9  10 7. The DNA was inte-
grated into the recipient genome by homologous recom-
bination and subsequently expressed [21].
Although not originally called MVs, but virus-like parti-
cles, MVs-containing DNA present in natural seawater
were able to transform E. coli with a transformation
frequency of 1.0  10 2 [25,31]. Yaron et al. showed vesi-
cles-mediated transfer of plasmid and phage DNA from
E. coli O157:H7 to E. coli JM109 and phage DNA to S.
enterica serovar Enteritidis ATCC 13076 [23].
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However, not all studies of MV transfer have proven
successful. Exposure studies with DNA contained inside
OMVs have also failed. For instance, plasmid-containing
OMVs released by Pseudomonas aeruginosa PAO1 failed to
transform wild-type P. aeruginosa PAO1 and E. coli DH5a
recipient cells under different growth conditions; failure
was possibly associated with the inability of the plasmid
DNA to pass the plasma membrane [15].
MV-mediated HGT in other prokaryotes
Fewer studies on the release of MVs are available in
prokaryotes other than Gram-negative bacteria and only
very few studies have reported the occurrence of MV-
mediated HGT. It is unclear if this is the result of lack of
studies or publication bias.
MVs-containing chromosomal DNA released from the
Gram-positive Ruminococcus sp. and Ruminococcus albus
were shown to transfer DNA to Ruminococcus sp. recipient
cells, but not to Butyrivibrio fibrisolvens [24].
MVs carrying a shuttle plasmid secreted by the hyper-
thermophilic archaea Thermococcus kodakarensis were able
to transform T. kodakaraensis recipient cells, indicating
that MV-mediated gene transfer also occur in this domain
of life [29].
We are not aware of studies that have explored the
possibility of horizontal transfer of RNA mediated by
MVs. Horizontal transfer of RNA molecules are rarely
described in bacteria and the biological relevance of such
events remain unclear. This is in contrast to the RNA
content of mammalian exosomes. Based on the observed
effects of transfer of RNA carried by eukaryotic exosomes
in new host cells, Sjöström et al. suggested that similar
mechanisms can be involved in bacteria–bacteria inter-
actions resulting in modulation of expression of target
genes in recipient cells [20].
Environmental influence on vesiculation and HGT
Several external stimuli induce the release of MVs,
including temperature, desiccation, starvation, UV light,
quorum sensing molecules, oxygen stress and exposure to
subinhibitory antibiotics of different classes, such as
ceftazidime, chloramphenicol, ciprofloxacin, gentamicin
and imipenem [4,8,32–34]. It can therefore be hypothe-
sized that HGT rates mediated by OMVs may be
enhanced by stress. Some stress factors may also increase
the overall concentrations of DNA in the cytoplasm.
Hence more DNA may be part of the lumen content
of the MVs [4] under such conditions.
Discussion
Prokaryotes maintain several mechanisms to take up
DNA from the environment and other host cells. MVs
add to the arsenal of gene transfer mechanisms and
provide a shielded environment for various genetic
Current Opinion in Microbiology 2017, 38:16–21
20 Mobile genetic elements and HGT in prokaryotes
materials outside replicating host cells [3,15,18]. More-
over, nucleic acids may be transferred between cells with
associated regulatory proteins. The release of MVs is not
necessarily coupled with DNA uptake in time or space. In
this way, MVs gene transfer resembles transduction. For
some Gram-negative bacteria, a transformation-like
mechanism is suggested. For most bacteria, the mecha-
nistic aspect of MVs formation and interaction with
exposed recipient cells remains to be elucidated.
The experimental studies of MVs-mediated gene transfer
described above were all conducted in vitro and with
purified MVs. Thus, such gene transfer processes are yet
to be described quantitatively under natural conditions. It
has, however, been observed that marine microorganisms
produce and release vesicles in the seawater [18], which
suggest that this mechanism of gene transfer can occur at
large scales in the environment. However, as DNA-car-
rying MVs and viral particles may have similar dimen-
sions, morphology and molecular composition, it can be
challenging to distinguish between these entities in
environmental samples [35]. MVs will protect and pro-
long the half-life of extracellular DNA in the environ-
ment, enhancing the probability of HGT events upon
successful interactions with new host cells. Biller
et al. reported that MVs present in the open ocean
remained stable for two weeks in the laboratory [18]
and Blesa and Berenguer observed that EVs produced
by Thermus spp. and stored at 4 C for 20 months in the
presence of DNase were still active in transformation
experiments [27]. It has also been shown that bacteria
living in extreme environments produce MVs [5,27]. The
genetic content and half-live of the different types of MV
produced by the large variety of prokaryotes present in
different natural environments remains to be described.
The observation that MVs can carry different types of
genetic material highlights their potential in the dissemi-
nation of different genes and metabolic functions across
space, time and environments. Key remaining knowledge
gaps include: the identification of the genes and regula-
tory pathways involved in MVs biogenesis, to what extent
DNA is part of vesicles due to random processes or the
outcome of active transport/packaging, to what extent the
different size ranges and vesicles serve different biologi-
cal functions, the role and extent of MVs formation in
prokaryotes other than Gram-negative bacteria, the iden-
tification of the genes and regulatory pathways, if any,
that control the recipient’s ability to take up DNA from
MVs, and to what extent and how the genetic content of
MV contributes to evolutionary processes within and
between species over time.
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Current Opinion in Microbiology 2017, 38:16–21
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