REVIEWS

Types and origins of bacterial

membrane vesicles
Masanori Toyofuku1*, Nobuhiko Nomura1 and Leo Eberl 2
*
Abstract | Most bacteria release membrane vesicles (MVs) that contain specific cargo molecules
and have diverse functions, including the transport of virulence factors, DNA transfer,
interception of bacteriophages, antibiotics and eukaryotic host defence factors, cell
detoxification and bacterial communication. MVs not only are abundant in nature but also show
great promise for applications in biomedicine and nanotechnology. MVs were first discovered to
originate from controlled blebbing of the outer membrane of Gram-negative bacteria and are
therefore often called outer-membrane vesicles (OMVs). However, recent work has shown that
Gram-positive bacteria can produce MVs, that different types of MVs besides OMVs exist and
that, in addition to membrane blebbing, MVs can also be formed by endolysin-triggered cell lysis.
In this Review, we provide an overview of the structures and compositions of the various vesicle
types and discuss novel formation routes, which may lead to distinct vesicle types that serve
particular functions.

1
Department of Life and
Environmental Sciences,
University of Tsukuba,
Tsukuba, Japan.
2
Department of Plant and
Microbial Biology, University
of Zurich, Zurich, Switzerland.
*e-mail: toyofuku.masanori.
gf@u.tsukuba.ac.jp;
leberl@botinst.uzh.ch
https://doi.org/10.1038/
s41579-018-0112-2
Bacteria release membrane vesicles (MVs) with sizes
ranging from 20 to 400 nm in diameter that affect
diverse biological processes, including virulence, hori-
zontal gene transfer, export of cellular metabolites, phage
infection and cell-to-cell communication1–3. Microbial
MVs also have immunomodulatory activities and are
therefore used as vaccines and show great potential for
the development of anticancer drugs and for applica-
tions in nanotechnology4,5. Bacterial MVs are abundant
in coastal and open-ocean sea water, which implies that
these structures are important for carbon cycling in the
marine ecosystem6. MVs are also abundant in micro-
bial biofilms, where they are an integral constituent of
the biofilm matrix and can protect biofilm cells from
certain antibiotics7–10.
MVs were first found to be produced through
controlled blebbing of the outer membrane of Gram-
negative bacteria and are therefore often referred to as
outer- membrane vesicles (OMVs). The composition
of OMVs and their biological functions, as well as vari-
ous OMV formation mechanisms and the underlying
environmental triggers, have been summarized in a
number of excellent articles1,3,11–13. This Review focuses
on recent work that has demonstrated that other types of
MVs exist in addition to OMVs (FIG. 1), including outer-
inner membrane vesicles (OIMVs), cytoplasmic mem-
brane vesicles (CMVs) and tube-shaped membranous
structures (TSMSs), and that MVs can also be formed as
a consequence of phage endolysin-triggered cell lysis14,15.
On the basis of these studies, we suggest that different
formation routes lead to distinct types of MVs and that
their structure and composition reflect the routes by
which they were formed. We summarize the published
evidence showing that MVs that originate from cell lysis
are different from MVs originating from membrane
blebbing of living cells and that this has consequences
for their composition and content and possibly for
their function.
Types of membrane vesicles
Recent work has shown that MVs can differ in their
structure and composition, and some of these differ-
ences might be explained by the different routes by
which the MVs were formed (FIGS 1,2; TABLE 1).
Outer-membrane vesicles. OMVs are the archetypal
bacterial MV. They are spherical particles that consist
of an outer leaflet of lipopolysaccharide (LPS) and an
inner leaflet of phospholipid, which is derived from the
outer membrane of Gram-negative bacteria1,11–13. Classic
OMVs originate from blebbing of the outer mem-
brane and are therefore enriched for outer-membrane
proteins, show specific lipid compositions and differ in
the amount and content of cargo molecules depending
on growth conditions3,16–19. Many studies have shown
that they contain periplasmatic and cytosolic proteins,
DNA and RNA and that they transport virulence fac-
tors14,20–24. OMVs are therefore considered to represent
a specialized bacterial secretion pathway25.
However, despite many years of research, the sorting
mechanism required for cargo selection is unknown,
and the presence of cytoplasmic contents within

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a c e

CMV

Phage tail

Phage head

b Peptidoglycan d f

Inner membrane Outer membrane

Fig. 1 | Types of vesicles. a | Outer-membrane vesicles (OMVs) are produced by blebbing of the outer membrane
and thus consist of a single membrane bilayer (green), as shown in an electron cryotomographic (ECT) image (left) and
a reconstructed 3D model (right) of an OMV isolated from an Acinetobacter baumannii culture (scale bar = 50 nm).
b | By contrast, outer-inner membrane vesicles (OIMVs) have two membrane bilayers derived from the outer membrane
(green) and the inner membrane (purple), and in between the membranes there is peptidoglycan (OIMV from
A. baumannii; scale bar = 100 nm). c | Gram-positive bacteria can produce cytoplasmic membrane vesicles (CMVs),
as exemplified here by an ECT image of a CMV produced by Bacillus subtilis. Notably, the vesicle carries PBSX phage
particles. PBSX encodes endolysin, and its expression leads to the formation of holes in the peptidoglycan layer through
which the cytoplasmic membrane protrudes, forming CMVs (scale bar = 100 nm). d | Tube-shaped membranous
structures are considered a specialized type of membrane vesicle. B. subtilis forms intercellular nanotubes that connect
neighbouring cells (scale bar = 1 µm). e | Myxococcus xanthus forms OMV chains, which can connect cells in biofilms (scale
bar = 100 nm). f | Vibrio vulnificus forms segmented tubes, from which OMVs can be pinched off (scale bar = 200 nm).
Parts a and b reproduced from Koning, R. I. et al. Cryo-electron tomography analysis of membrane vesicles from
Acinetobacter baumannii ATCC19606 T. Research in Microbiology 2013; 164(5):397–405. Copyright © 2013 Elsevier
Masson SAS. All rights reserved. Part c adapted from REF.15, CC-BY-4.0. Part d adapted with permission from REF.50,
Elsevier. Part e reproduced with permission from REF.41, Wiley-VCH. Part f adapted from REF.48, CC-BY-4.0.

Endolysins
Hydrolytic enzymes that are
produced by bacteriophages
to degrade the cell wall of the
bacterial host during the final
stage of the lytic cycle.
Autolysins
Peptidoglycan hydrolases that
are produced by bacteria for
peptidoglycan turnover and to
complete cell division by
separating the daughter cell
from the mother cell.
OMVs has remained unexplained26. It is possible that
previous reports, which found nucleic acids or cyto-
solic proteins in OMVs, might not have analysed bona
fide OMVs, which are formed through the blebbing of
the outer membrane and therefore do not have direct
access to cytoplasmic contents26,27. It is worth noting,
however, that OMVs that are formed through cell
lysis of a small subpopulation of the cultured bacteria
can contain RNA, chromosomal DNA and endolysins14
(see below).
Outer-inner membrane vesicles. Kadurugamuwa and
Beveridge reported in 1995 that MVs from Pseudomonas
aeruginosa contain DNA, confirming earlier stud-
ies demonstrating the presence of DNA and RNA in
Gram-negative MVs28–31. As none of the blebbing models
at the time could convincingly explain how DNA
passes through the inner membrane and enters OMVs,
they proposed a new formation mechanism. In their
model, the peptidoglycan layer of the bacterial cell is
weakened by autolysins such that the inner membrane
protrudes into the periplasm, allowing cytoplasmic con-
tents such as DNA to enter the vesicle, which is even-
tually pinched off from the cell surface together with
a surrounding outer membrane. The first experimen-
tal evidence for MVs consisting of both the outer and
the inner membranes was provided by a transmission
electron cryomicroscopy study of Shewanella vesiculosa
M7 supernatant32, which unambiguously demonstrated
the production of double bilayered MVs, which were
originally named O-IMVs (referred to as OIMVs in the
following sections). The study also showed that although
this bacterium produces both OMVs and OIMVs, most
of the DNA is contained in OIMVs. Subsequent work
demonstrated that diverse bacteria produce OIMVs
and that DNA is specifically packed into this type of
MV32–34. The fraction of OIMVs produced seems to
be highly variable, ranging from 0.1% of total MVs in
S. vesiculosa M7 to 49% in Pseudoalteromonas
marina32,35. Although the proposed blebbing model
could explain how plasmid DNA enters OIMVs, it is
difficult to understand how DNA fragments of the
bacterial chromosome are packaged into MVs with-
out assuming that the producing cell dies. Indeed, the
presence of chromosomal DNA in MVs is considered
to be indicative of cell lysis26,27. ‘Explosive cell lysis’ is
another possible route for the formation of MVs con-
taining chromosomal DNA, as cell lysis is triggered by
DNA damage and results in cell envelope fragments that
re-circularize and thereby enclose the released DNA (see
below). Notably, this would also explain why vesicles
can be enriched for endolysins, as peptidoglycan deg-
radation is the key process in explosive cell lysis14,36,37.
In support of this hypothesis, a recent study showed

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Gram-negative bacteria
Outer membrane Peptidoglycan Gram-positive bacteria
Endolysin
Inner membrane Cytoplasmic molecule Peptidoglycan Cytoplasmic membrane

Membrane DNA
molecule

Hydrophobic Phage
molecule Endolysin

Imbalanced
cell wall Explosive Bubbling
Intercalation cell lysis cell death
biosynthesis

OMV OIMV EOMV CMV

Fig. 2 | Different routes lead to the formation of distinct membrane vesicle types. Gram-negative bacteria have two
main routes for vesicle formation: blebbing of the outer membrane and explosive cell lysis. Blebbing leads to the production
of outer-membrane vesicles (OMVs) and occurs as a result of cell envelope disturbances, which can be caused by either
imbalance of peptidoglycan biosynthesis or intercalation of hydrophobic molecules into the outer membrane. As the inner
membrane remains intact, cytoplasmic components have no direct access to these OMVs. Explosive cell lysis is triggered by
phage-derived endolysin that degrades the peptidoglycan cell wall. Once the peptidoglycan is degraded, the cell rounds
up and explodes, and the shattered membrane fragments round up and self-assemble into outer-inner membrane vesicles
(OIMVs) and explosive outer-membrane vesicles (EOMVs). In contrast to OMVs formed by blebbing, EOMVs randomly
contain cytoplasmic components. Endolysin also triggers ‘bubbling cell death’ in Gram-positive bacteria, in which it gives
rise to cytoplasmic membrane vesicles (CMVs). CMVs can contain membrane and cytoplasmic components. Other
peptidoglycan-damaging enzymes and treatments may have similar consequences as endolysin. In Gram-negative bacteria,
CMVs, arising from the inner membrane, have also been reported, but the underlying mechanism is not understood.

that treatment of Stenotrophomonas maltophilia with
ciprofloxacin not only induces the SOS response and
consequently cell lysis (BOX 1) but also stimulates the
production of OIMVs38.
Cytoplasmic membrane vesicles. Given that MVs were
originally thought to originate solely from the pinching-
off of outer-membrane material from Gram-negative
bacteria, it came as a surprise when vesicle formation
was observed in Gram- positive bacteria. Over the
past two decades, vesicle formation has been demon-
strated in numerous Gram-positive bacteria (reviewed
previously2). Given the lack of an outer membrane, we
suggest that MVs produced by Gram-positive bacteria
be named CMVs. Although it has been demonstrated
that CMVs can arise from dying cells15,39 (see below), it is
conceivable that conservative blebbing mechanisms exist
that are independent of cell death. Large intracellular
vesicles have also been observed in the Gram-negative
bacterium Acidiphilium cryptum JF-5 when grown under
anaerobic conditions40. The strain also formed blebs at
the cell surface and tube-shaped projections, similar to
the nanotubes discussed below. Electron micrographs
of thin sections showed morphological continuity
between the inner membrane (or cytoplasmic membrane),
the blebs and the projections, indicating that the mem-
branes of the blebs and projections originated from the
inner membrane.
Tube- shaped membranous structures. Several dif-
ferent bacteria produce TSMSs, often referred to as
nanotubes, nanowires or nanopods. These structures
are tube-like protrusions of the cytoplasmic membrane
of Gram-positive bacteria or of the outer membrane of
Gram-negative bacteria and are considered to be spe-
cialized types of MVs. They often decorate the surface
of the producing cell and form bridges between cells that
enable the exchange of various cellular components.
The simplest version of these structures is a chain of
OMVs, which was first observed in Myxococcus xan-
thus41,42. Using various imaging techniques, including
3D focused ion beam scanning electron microscopy, a
study41 showed that these structures range between 30
and 60 nm in width and up to 5 μm in length and form
an extensive membrane-enclosed network that seems
to connect cells within biofilms at the level of the peri-
plasmic space. It has been suggested that these vesicular
connections constitute intercellular bridges that enable
cytoplasmic molecular trade between biofilm cells to
coordinate social activities43,44.
The metal- reducing microorganism Shewanella
oneidensis MR-1 produces OMV chains that can elongate
and turn into smooth filaments45. The most remarka-
ble property of these tubular structures is their electri-
cal conductivity and their ability to connect separate
S. oneidensis MR-1 cells. They are therefore considered
a special type of microbial nanowire, which is normally

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formed by electrically conductive proteins46. Whether
redox-functionalized membrane extensions represent
a general strategy for long-range extracellular electron
transport between bacteria remains to be shown, but it
is worth noting that, in addition to OMV chains, nano-
tubes that connect M. xanthus cells in biofilms have been
observed41,42. Francisella tularensis, the causative agent of
the zoonotic disease tularaemia, produces OMVs and
TSMSs in liquid media, which contribute to cell–cell
communication and the exchange of molecules without
forming bridges between cells47. A recent study48 nicely
illustrated the interplay between OMVs and nanotubes.
Cryo-electron microscopy showed that in cultures of
capsulated Vibrio vulnificus strains, individual OMVs
were pinched off from nanotubes, creating an intrigu-
ing ‘beads-on-a-string’ pattern. This suggests that, in
addition to strings of OMVs fusing to form nanotubes,
as observed for M. xanthus, nanotubes, in turn, can also
disintegrate into OMVs.
Nanotubes that originate from extensions of the
outer membrane may enable the intercellular transfer of
periplasmic metabolites, membrane proteins and lipids
but not of cytoplasmic contents. This is in contrast to
nanotubes formed by the Gram- positive bacterium
Bacillus subtilis, which the bacterium uses to exchange
cytoplasmic contents such as proteins and plasmid
DNA49,50. Electron microscopic analyses showed that
these multilayered structures consist of cell wall material,
membrane components and cytoplasmic contents43.
The texture of the nanotubes resembles the bacterial cell
surface, suggesting similar exterior composition and
surface continuity.
Routes of vesicle formation
Evidence has accumulated that suggests that two prin-
cipal routes for the formation of MVs exist: blebbing
of membrane material of living cells that gives rise
to classic OMVs, and endolysin- triggered cell lysis

Table 1 | Proposed vesicle types, their routes of formation and their compositions
Formation routes Outer- Cytoplasmic (or Plasmids RNA and Endolysinsa Virulence Hydrophobic Phagesa
membrane inner) membrane chromosomal factors molecules
proteins proteins DNA
Outer-membrane vesicle (Gram-negative)
Outer-membrane blebbing + – (+)b + – (+)c – + – –
(imbalanced peptidoglycan
turnover, phospholipid
accumulation or deacylation
of lipid A)
Intercalation of hydrophobic + – (+)b ? – (+)c – + +d –
molecules (such as bacterial
signalling molecules,
antibiotics and hydrophobic
substrates) into the outer
membrane
Shedding of + – ? – – ? – –
lipopolysaccharide
from flagella
Explosive outer-membrane vesicle (Gram-negative)
OMVs generated through + + + + + + + +
explosive cell lysis (either
directly or through
rearrangement of OIMVs)
Outer-inner membrane vesicle (Gram-negative)
Explosive cell lysis and + + + + + + + +
cell budding
Cytoplasmic membrane vesicle (Gram-positivee)
‘Bubbling cell death’ and NA + + + + + – +
bacterial autolysins
Tube-shaped membranous structure (Gram-negative)
Mechanism unknown + – – – ? – – –
Tube-shaped membranous structure (Gram-positive)
Mechanism unknown NA – + + ? ? – –
+, present; –, absent; ?, unknown; CMV, cytoplasmic membrane vesicle; EOMV, explosive outer-membrane vesicle; NA , not applicable; OIMV, outer-inner membrane
vesicle; OMV, outer-membrane vesicle; TSMS, tube-shaped membranous structure. aMembrane vesicles (MVs) originating from cell lysis are intrinsically associated
with endolysins and sometimes entire phages, whereas they are expected to be absent from MVs originating through blebbing mechanisms. OMVs can adsorb
phages on their surface but would not carry phages inside. bOccasionally , cytoplasmic proteins have been observed in OMVs, but it is not known how these proteins
are packaged into OMVs. cAlthough several reports have demonstrated the presence of plasmid DNA in OMVs, the presence of chromosomal DNA and RNA is
considered an indication of cell lysis. We therefore speculate that these nucleic acids are mainly associated with OIMVs and EOMVs. dHydrophobic molecules that
can induce vesicle formation through blebbing will stay associated with the vesicles. eVesicles originating from the inner (or cytoplasmic) membrane have also been
reported in Gram-negative bacteria, but the underlying formation route is unclear.

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Box 1 | SOS response in bacteria
The SOS response is a global regulatory system that allows bacteria to respond to DNA
damage. It was first identified in Escherichia coli, in which it was shown to control
prophage induction, cell filamentation and mutation upon exposure to ultraviolet
radiation125. Since then, it has become a paradigm of a general stress response system
that is conserved and widespread among bacteria. Two regulatory proteins modulate
the expression of genes involved in the SOS response126: the LexA repressor, which
inhibits expression of the SOS genes during normal cell growth by binding to specific
DNA sequences (SOS boxes) upstream of target genes and thus blocking their
transcription127; and the RecA protein, which binds to single-stranded DNA generated
by DNA-damaging agents or inhibition of replication. The binding of RecA to single-
stranded DNA, which is assisted by the components of the RecFOR and RecBCD
proteins of the two major recombinational repair pathways in E.coli128, activates RecA
and stimulates autocatalytic cleavage of LexA129. Cleaved LexA is no longer able to bind
to SOS boxes, and thus, expression of SOS genes is derepressed. In E. coli and many
other bacteria, the SOS regulon comprises genes of three DNA repair pathways,
namely, homologous recombination, nucleotide excision repair and translesion DNA
synthesis130. Translesion DNA synthesis often introduces mutations, as this pathway
involves error-prone DNA polymerases, and thus increases the occurrence of antibiotic-
resistant strains131,132. The SOS response also affects cell division and blocks septum
formation, leading to filamentation133. Expression of lexA is negatively autoregulated,
and thus, SOS gene expression is repressed once the DNA damage has been
repaired134,135. Importantly, RecA also activates self-cleavage of other transcriptional
repressor proteins, most notably phage repressors, and therefore, phage production is
typically induced upon DNA damage.
that leads to the formation of OIMVs, explosive outer-
membrane vesicles (EOMVs) (discussed below) and
CMVs (FIG. 2; TABLE 1).
Vesicle formation through membrane blebbing.
Membrane blebbing results from cell envelope distur-
bances, which can be caused by either the unbalanced
biosynthesis of cell wall components or the intercalation
Intercalation of hydrophobic molecules into the outer membrane of
The reversible insertion of Gram-negative bacteria. Although the release of OMVs
molecules into materials with occurs during normal cell growth without affecting
layered structures such as the
cell viability, growth conditions can have a profound
cellular membrane.
effect on vesiculation51. Several blebbing models have
Turgor pressure been proposed, including localized membrane remod-
Turgor is the force that pushes elling, altered turgor pressure of the periplasmic space,
the cytoplasmic membrane anionic charge repulsion between LPS molecules and
against the cell wall as a result
of the osmotic flow of water.
loss of linkage between the peptidoglycan and the
outer membrane.
Quorum sensing The common assumption of these models is that
A cell-to-cell communication OMVs protrude from regions of the outer membrane,
mechanism in bacteria by
in which disrupted crosslinking between peptide gly-
which gene regulation is
controlled in a population- can and the outer membrane leads to dissociation
dependent manner through of the outer membrane from the peptidoglycan layer,
the production and perception for example, at the site of cell division52–54. Mutants
of signal molecules. with defects in crosslinking produce more OMVs
B-band LPS
than wild-type strains55–59. However, as some of these
Pseudomonas aeruginosa mutants show substantial leakage of intracellular com-
synthesizes two types of ponents, it cannot be ruled out that at least some of
lipopolysaccharide (LPS) the increase in vesiculation is due to cell lysis. More
referred to as A-band and
recently, an Escherichia coli nlpI mutant with a subtle
B-band LPS. The A-band LPS
contains a conserved defect in crosslinking (a reduction of approximately
O-polysaccharide region 40% compared with wild- type cells) was shown to
composed of d-rhamnose hypervesiculate without detectable cell lysis59,60. NlpI
(homopolymer), whereas the is an outer-membrane lipoprotein that is involved in
B-band O-antigen
(heteropolymer) structure
modulating peptidoglycan dynamics, and it was pos-
varies among different tulated that an imbalance in peptidoglycan turnover
serotypes. prevented crosslinking and thus stimulated OMV
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formation in the mutant. A recent study showed that
phospholipid accumulation in the outer leaflet of the
outer membrane of Haemophilus influenzae promotes
the release of OMVs without compromising mem-
brane integrity61. Phospholipids accumulated when
the VacJ (also known as MlaA) and Yrb ATP-binding
cassette (ABC) transport system, a proposed phospho-
lipid transporter, is either mutated or its expression is
downregulated under iron-limiting conditions. Given
that inactivation of this transporter increased vesicu-
lation in phylogenetically diverse bacteria, it has been
suggested that this OMV biogenesis pathway could be
universal in Gram-negative bacteria16. In Salmonella
enterica subsp. enterica serovar Typhimurium, deacyl-
ation of LPS was shown to affect the level of OMV prod-
uction: expression of the lipid A deacylase PagL led to
overvesiculation with deacylated lipid A accumulation
exclusively in OMVs18.
Another mechanism for OMV formation through
blebbing is the accumulation of peptidoglycan fragments
or misfolded proteins in the periplasmic space59,62,63.
This is thought to create a turgor stress on the outer
membrane that eventually leads to OMV release.
Another route for OMV formation through bleb-
bing involves integration of molecules that induce
membrane curvature into the cell envelope of bacteria.
The best investigated example for this ‘bilayer-couple’
model of OMV formation comes from P. aeruginosa:
the quorum-sensing molecule Pseudomonas quinolone
signal (PQS) mediates its own packaging and transport
by stimulating OMV formation through intercalation
into the outer membrane64–66. The accumulation of PQS
in the outer leaflet is thought to change the membrane
curvature, providing the initial driving force for OMV
formation. PQS was shown to increase anionic repul-
sions between LPS molecules by sequestering divalent
cations, which are important to stabilize the negatively
charged B- band LPS molecules67. The bilayer- couple
model may not be limited to bacterial signal molecules,
as previous studies have demonstrated that antibiotics
that cause membrane perturbations, such as gentamicin
and the cationic antimicrobial peptides polymyxin and
colistin, induce OMV formation8,31,68,69 (BOX 2).
Another mechanism for OMV formation has been
demonstrated in Aliivibrio fischeri. This bacterium
assembles flagella that are surrounded by a sheath
derived from the outer membrane. Membrane blebs
are commonly found along the sheathed flagella, and
these are released when the flagella rotate 70,71. The
LPS that is associated with these OMVs is an impor-
tant trigger for morphogenesis of the light organ of its
symbiotic host, the Hawaiian bobtail squid, Euprymna
scolopes71,72. Likewise, Vibrio cholerae was shown to use
its sheathed flagella to release OMVs carrying LPS70,
which can trigger inflammation and an immune
response in the host. Flagellar motility is an important
virulence trait of the pathogens Brucella melitensis and
Helicobacter pylori, whose sheathed flagella have also
been shown to carry membrane blebs73,74, suggesting
that the phenomenon of flagellar-mediated LPS release
through OMVs may be widespread among bacteria
with sheathed flagella.
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Box 2 | Antibiotics and bacterial vesicles
Treatment of bacteria with sublethal concentrations of certain antibiotics is a well-
established trigger of bacterial vesicle formation. At least three mechanisms by which
antibiotics stimulate membrane vesicle (MV) formation can be distinguished: cell
envelope stress caused by antibiotics, induction of the SOS response and inhibition of
cell wall biosynthesis (see the figure).
Polymyxin Ciprofloxacin β-lactams
Outer-membrane DNA Cell wall
stress damage biosynthesis
SOS
response
OMVs OIMVs EOMVs CMVs
Antibiotics that weaken the cell wall, such as β-lactams, stimulate cytoplasmic
membrane vesicle (CMV) formation in Gram-positive bacteria15,136,137. This treatment
will generate holes in the peptidoglycan layer through which cytoplasmic membrane
material can protrude into the extracellular space and be released as CMVs. It has been
well established that some antibiotics, particularly quinolones such as ciprofloxacin,
induce the cellular SOS response (BOX 1), which in turn has been shown to stimulate
vesicle production14,75,138,139. As the SOS response triggers expression of endolysins
encoded by prophages, these antibiotics stimulate vesicle formation through lysis.
This pathway seems to be the main route for outer-inner membrane vesicle
(OIMV) production38.
Antibiotics that cause cell envelope stress, such as polymyxin or gentamicin, will cause
outer-membrane vesicle (OMV) formation through blebbing of the outer membrane8,140.
A particularly interesting case is the biosynthesis of linearmycin by Streptomyces sp. Mg1
(REF.141). This hydrophobic compound, which has antifungal and antibacterial properties,
is released and transported by OMVs, and it induces OMV formation, possibly by
affecting membrane curvature.
The fact that some antibiotics stimulate vesicle formation has important implications
for the choice of antibiotics for the treatment of infections. For example, Escherichia coli
strains O157:H7 and O104:H4 secrete Shiga toxin (Stx), the major virulence factor
of these bacteria, through MVs118,119. The synthesis of Stx is associated with the
induction of the Stx prophage that carries the toxin gene115. Hence, antibiotics that
induce the SOS response not only induce Stx production but also trigger explosive
cell lysis and thus dispersal of the toxin through MVs. For these reasons, the use of
ciprofloxacin for the treatment of patients infected with enterohaemorrhagic E. coli has
been discouraged142.
As a consequence of their formation routes, OIMVs, explosive outer-membrane
vesicles (EOMVs) and CMVs carry endolysins, which can lyse and kill bacteria. They may
also carry the antibiotics that triggered the SOS response, and these may also contribute
to the toxicity of these MVs. Antibiotics that intercalate into the outer membrane induce
OMV formation through blebbing. These OMVs carry the inducing antibiotics and
therefore can kill susceptible bacteria68,96,143. Classic OMVs that are produced owing to
outer-membrane blebbing can function as decoys for envelope-targeting antibiotics
and thereby protect the bacteria8.
Vesicle formation through endolysin-triggered cell
death. Recent work has unravelled novel vesicle for-
mation routes that are based on the enzymatic action of
endolysins, which are typically used by double-stranded
DNA phages to lyse their hosts so that the phage prog-
eny can be released. In the Gram-negative bacterium
P. aeruginosa, DNA-damaging stress induces expres-
sion of an endolysin, which is part of a pyocin bio-
synthesis gene cluster, leading to degradation of the
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peptidoglycan layer 14. As a consequence, the cells
round up and explode, which is why this phenomenon
was named explosive cell lysis. The remaining shattered
membrane fragments round up and self-assemble into
MVs. Explosive cell lysis was shown to be stimulated
in biofilms and under anoxic conditions14,75. Another
study showed that, similar to P. aeruginosa, expression
of an endolysin encoded by a defective prophage trig-
gers vesicle formation in the Gram-positive bacterium
B. subtilis15. Although the enzymatic activities of the
endolysins weaken peptidoglycan in both organisms,
the consequences are different: whereas P. aeruginosa
cells round up and explode, B. subtilis cells pro-
trude cytoplasmic membrane material through holes
in the peptidoglycan, which is released as CMVs.
Furthermore, whereas P. aeruginosa cells completely
disintegrate, the thick Gram- positive cell wall of
B. subtilis is not entirely hydrolysed, although most cells
die owing to the loss of membrane integrity, as evi-
denced by the formation of ghost cells and intracellular
CMVs. This phenomenon has therefore been named
‘bubbling cell death’. The endolysins released from
dying B. subtilis cells were shown to trigger CMV for-
mation in neighbouring cells, as they hydrolyse the cell
wall from outside15. Similarly, MVs originating from
explosive cell lysis of Gram-negative bacteria also carry
endolysins and therefore may lyse other cells37,68, which
in turn could generate MVs. These studies suggest that
phage-triggered cell death may be an underestimated
route for vesicle formation in nature. In addition to
phage- derived endolysins, bacterial autolysins have
been reported to be involved in vesicle formation in
some bacteria76,77.
Conceivably, explosive cell lysis can generate
OIMVs, as supported by the finding that ciprofloxacin
treatment of S. maltophilia triggered phage expression,
cell lysis and OIMV formation38. However, it cannot be
ruled out that during cell explosion the inner and outer
membranes of the cell envelope fragments become sep-
arated and spontaneously re-circularize to form OMVs
or possibly CMVs. Incidentally, Koning et al.78 reported
that in some OIMVs of Acinetobacter baumannii the
inner membrane was partially detached from the pep-
tidoglycan layer, which remained associated with the
outer membrane. Moreover, the authors also observed
OIMVs with partial inner membranes and OMV for-
mation from OIMVs. Hence, explosive cell lysis will
also lead to the formation of OMVs (FIG. 2). However,
as these vesicles likely differ from classic OMVs with
respect to their cargo, we refer to them as EOMVs.
Tube- shaped membranous structure formation.
Very little is known about the mechanisms of TSMS
formation. It was suggested that local lysis of the
Gram- positive cell wall causes bulging of the cyto-
plasmic membrane, which, in turn, initiates nanotube
formation49. This is reminiscent of the early stage of
endolysin- triggered CMV formation, during which
material from the cytoplasmic membrane protrudes
through holes in the peptidoglycan layer15. However,
whereas membrane blebs are released during CMV
formation, these membrane extrusions elongate during
www.nature.com/nrmicro

Iron limitation Outer-
membrane
blebbing
Signalling molecules
(PQS)
Hydrophobic compounds
Explosive
Antibiotics cell lysis
DNA-damaging agents
Peptidoglycan- Bubbling
degrading enzymes cell death
Fig. 3 | Different triggers that induce membrane vesicle formation. Certain growth
conditions, such as iron limitation, and antibiotics can influence the lipid homeostasis in
Gram-negative bacteria and trigger the blebbing of the outer membrane. Blebbing is
also triggered by intercalation of hydrophobic molecules including signalling molecules,
such as the Pseudomonas quinolone signal (PQS), and by membrane-targeting
antibiotics. Antibiotics and DNA-damaging agents can induce the expression of phage-
derived endolysin through the SOS response, and this can trigger vesicle formation
through explosive cell lysis in Gram-negative bacteria and ‘bubbling cell death’ in Gram-
positive bacteria. Other peptidoglycan-degrading enzymes might also trigger explosive
cell lysis and bubbling cell death. β-lactam antibiotics affect the integrity of the cell wall
and thus can induce the formation of membrane vesicles, which is similar to what
ultimately happens in bubbling cell death. See also BOX 2 for the effects of antibiotics
on vesicle formation.
nanotube formation. It is possible that the size of the
hole in the peptidoglycan layer determines the fate of
the membrane protrusion. The enzymatic activity of
endolysins may create large and more quickly expand-
ing holes, which will lead to cell lysis, whereas the holes
for nanotube formation may instead be confined to
small punctures in the peptidoglycan layer.
Triggers of vesicle formation
Certain stimuli trigger specific outer- membrane
vesicle formation routes. Evidence has accumulated
that vesicle production not only depends on the genetic
background of the producing strain but also is strongly
influenced by the growth conditions. A plethora of envi-
ronmental factors affect the rate of vesicle formation,
including media composition, growth phase, temper-
ature, iron and oxygen availability, exposure to anti-
biotics and genotoxic stress3,11. In the following sections,
we summarize the evidence that suggests that some
conditions and stimuli trigger certain routes of vesicle
formation (FIG. 3).
Factors triggering vesicle formation through bleb-
bing. Forward genetic screens in several bacterial
species have identified several genes that affect OMV
production through blebbing. In most cases, these genes
either affect envelope crosslinking, the accumulation of
peptidoglycan fragments or misfolded proteins in the
periplasmic space, or the lipid composition of the outer
membrane, which determines membrane curvature and
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REVIEWS
fluidity3,13,16. Recent work has shown that deacylation
of lipid A and the accompanying outer- membrane
remodelling in S. Typhimurium and phospholipid
accumulation in the outer leaflet of the outer mem-
brane of H. influenzae and V. cholerae lead to hyper-
vesiculation18,61. Iron limitation has been suggested
to control OMV formation by affecting expression of
phospholipid transporter genes in V. cholera, E. coli and
H. influenzae61, but very little is known about the envi-
ronmental stimuli that affect expression of the other
genes involved in OMV formation.
Growth of bacteria on highly hydrophobic carbon
sources, such as hexadecane or phenanthrene, which
likely interact with the outer membrane, was also
reported to stimulate vesiculation 79,80. OMVs can
also detoxify cells from membrane- targeting com-
pounds, as has been shown for Pseudomonas putida
IH-2000 (REF.81). In this example, toluene, a solvent, inter-
calates into hydrophobic regions of the outer membrane,
causing a change in membrane curvature and the pro-
duction of OMVs. Toluene was found to be enriched in
OMVs compared to the cell envelope, suggesting that
the OMVs are used to dispose of the toxic compound
and thus protect the cell.
Factors triggering vesicle formation through
endolysin- triggered cell death. Vesicle formation
through explosive cell lysis or bubbling cell death
depends on the expression of phage-derived endolysins.
Double-stranded DNA phages use these peptidoglycan-
hydrolysing enzymes to ensure the release of newly
packaged phage particles82. To reach the peptidoglycan,
endolysins depend on small hydrophobic proteins called
holins. These proteins oligomerize in the cytoplasmic
membrane and form pores that allow the endolysins to
access the peptidoglycan83.
Whereas lytic phages immediately enter a produc-
tive cycle, temperate phages can integrate into the
bacterial host genome and are then transmitted as
prophages to daughter cells at each cell division. In this
lysogenic state, the prophage does not promote cell
death or the production of phage particles. The pro-
phage can enter the lytic cycle again when the cell is
stressed, typically upon exposure to DNA-damaging
agents or ultraviolet radiation, which are conditions
that activate the SOS response (BOX 1). The prophage
then induces expression of lytic genes that promote
DNA replication, phage particle assembly, DNA
packaging, host DNA degradation and bacterial lysis.
Even in the absence of an obvious stressor, a low rate
of spontaneous induction of lytic production occurs.
In P. aeruginosa and E. coli, less than 1% of the popu-
lation induces the SOS response when grown under
optimal conditions in liquid medium14,84. However, a
considerably higher proportion of the population was
found to be induced when the cells were growing in
biofilms85,86. This is in agreement with previous work
that has shown that MVs are major components of
biofilm matrices9,10. It is noteworthy that cell lysis will
also lead to the release of extracellular DNA, which
is known to be an important structural component of
bacterial biofilms87.
VOLUME 17 | JANUARY 2019 | 19
REVIEWS
Susceptible cell
Infection
Phage
Phage receptor
Bacteriocins
Protein or peptide toxins
produced by bacteria to kill or
inhibit growth of bacteria that
are closely related to the
producer.
20 | JANUARY 2019 | VOLUME 17
Peptidoglycan Cytoplasmic membrane
MV formation
Endolysin
Fig. 4 | Roles of membrane vesicles during phage infection. Phages specifically infect cells that carry matching phage
receptors. When the phage expresses endolysin in an infected cell, the resulting membrane vesicles (MVs) also carry the
phage receptors. On the one hand, these MVs can adsorb extracellular phages and function as a decoy that protects other
cells from infection. On the other hand, when such MVs fuse with a previously resistant cell, they can transfer phage
receptors to make the cells susceptible to the phage.
Endolysin- triggered cell lysis is required for the
release of not only phages but also phage tail- like
bacteriocins in Gram-negative bacteria, as has been most
intensively investigated for the R-type and F-type pyo-
cins of P. aeruginosa88. The initial report on explosive
cell lysis as a novel route for vesicle formation was based
on the observation that SOS-dependent expression of
the holin (PA0614) and endolysin (PA0629) genes not
only caused cell death but also greatly increased the
number of MVs produced14,75.
In conclusion, enzymes that degrade peptidoglycan
are widespread among bacteria and can serve a wide
variety of functions, including bacterial killing, release
of phages and nutrient acquisition. Independently of
their eventual purpose, these enzymes will lyse bacteria
and thereby trigger vesicle formation. It is of note that
these processes are induced under certain environmen-
tal conditions, and thus, the cargo of the vesicle reflects
the physiological state of the cell at the time of death
rather than being the consequence of a specific sort-
ing mechanism. As the SOS response is normally acti-
vated in only a physiologically distinct subpopulation,
it may, at least partly, explain why MVs have a different
protein and lipid profile from the outer membrane of
living cells.
Functional consequences
Given their different compositions, it is possible that
some MV types have certain functions. For example,
two traits that are typically associated with MVs, namely,
DNA transfer 30,69,89–96 and bacterial killing 37,68,96–98,
depend on the presence of DNA and enzymes that
hydrolyse peptidoglycan in MVs. As discussed above,
these constituents seem to be mainly associated with
MVs originating from cell lysis, that is, EOMVs
and OIMVs and in Gram- positive bacteria CMVs.
Exposure of Acinetobacter baylyi to sublethal concen-
trations of gentamicin stimulated MV production and
Decoy
Resistant cell
Receptor
transfer
increased both the amount of DNA associated with
the MVs and the transfer efficiency of plasmid DNA69.
MVs of several bacterial species were shown to lyse
other bacteria37, and it has been suggested that these
‘predatory MVs’ provide a fitness benefit to the vesicle-
producing population. Moreover, some bacterivorous
bacteria that thrive on the degradation of macromol-
ecules secrete a cocktail of antibiotics and bacteriolytic
enzymes to lyse other microbial cells99,100. A particularly
interesting case is Lysobacter sp. XL1, which secretes
five bacteriolytic enzymes into its surroundings to
kill prey bacteria101. At least one of these enzymes, the
endopeptidase L5, has been shown to be exclusively
secreted through OMVs and was suggested to also be
involved in OMV formation. However, the mecha-
nism by which L5 affects OMV formation remains to
be elucidated102,103.
Phage- triggered cell lysis releases not only phage
progeny but also OIMVs from Gram-negative bacteria
and CMVs from Gram-positive bacteria. Recent work
has unravelled a complex interplay between phages, bac-
teria and their MVs (FIG. 4). For example, B. subtilis cells
that are sensitive to the phage SPP1 release CMVs that
carry phage receptors104. When these CMVs fuse with
phage-resistant cells, they render the cells sensitive to
the phage. This mechanism allows phages to infect non-
host species and thus promotes phage spread and inva-
sion in mixed natural communities. As this facilitates
transduction and consequently horizontal gene transfer
among phylogenetically diverse bacteria, this phenom-
enon will accelerate bacterial evolution 104. Another
aspect of this interplay is the finding that OMVs can
function as decoys that can neutralize phages8,104–107.
Similar to phages, some antibiotics stimulate OMV for-
mation, although by different routes (BOX 2), and these
OMVs can neutralize environmental agents that target
the outer membrane of Gram-negative bacteria, such as
antimicrobial peptides and bacteriophages8.
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As mentioned above, the signal molecule PQS stim-
ulates OMV production through intercalation into the
outer membrane. The resulting OMVs transport this
molecule within a population65. Recent reports suggest
a general role of MVs for the release and dispersal of
hydrophobic signal molecules, such as long-chain N-
acylhomoserine lactones (AHLs)34,108. For Paracoccus
denitrificans, it was shown that the amount of AHLs car-
ried by a single vesicle suffices to trigger the quorum-
sensing response of a target cell. In contrast to the
classic quorum- sensing model, which assumes free
diffusion of signal molecules to synchronize bacterial
activities, vesicle-mediated signalling is binary (that is,
cells either receive a vesicle or not) and would lead to
physiologically heterogeneous populations108. At pres-
ent, it is not known whether long-chain AHL signal
molecules can stimulate vesicle formation through
intercalation into the outer membrane or whether they
are just packed into MVs for their release and pro-
tection. It is also possible that they constitute specific
signals that trigger a regulatory pathway controlling
vesiculation, as has recently been suggested for the
diffusible signal factor (DSF) of S. maltophilia and
Xylella fastidiosa109,110.
Conclusions
We hope that this Review will raise awareness that dif-
ferent types of MVs exist and provide a guide for their
identification. We have summarized evidence that sug-
gests that two principal vesicle formation routes exist,
namely, membrane blebbing and endolysin-triggered
cell lysis. These two routes give rise to various types of
MVs that differ in their compositions and possibly in
their functions (FIG. 2; TABLE 1). This information may
stimulate future research that aims at investigating the
abundance and ecological role of different vesicle types
in the environment. Recent work has shown that MVs
have important physiological roles in coral reefs34 and
a so far underestimated effect on nutrient cycles across
the biosphere6,111. Even so, many aspects of vesicle func-
tions, including cell detoxification, killing of bacteria,
DNA transfer and signal molecule release, in natural
environments remain to be explored and appear to
be highly valuable fields for future research. It would be
particularly interesting to elucidate which function is
associated with a particular vesicle type.
Although endolysin- triggered vesicle formation
requires the death of a small subpopulation of cells, this
may provide a benefit to the surviving population. For
example, some antibiotics induce the SOS response,
cell lysis and vesicle formation in lysogenic bacteria.
The remaining bacterial population, however, is now
protected by the produced OMVs, as they can neutral-
ize environmental agents that target the outer mem-
brane of Gram- negative bacteria, including phages,
membrane- targeting antibiotics and eukaryotic host
defence factors8,105,112–114.
It is also worth noting that expression of phage endo-
lysins is often controlled by the SOS response, which is
a genetically hardwired regulatory cascade (BOX 1). This
means that cells, in which the SOS response has been
induced before lysis, are in a special physiological state
NATURE REVIEWS | MICROBIOLOGY
REVIEWS
and also show specific protein expression patterns. For
example, it is well documented that SOS induction trig-
gers virulence in some bacteria by inducing the produc-
tion of phage-encoded toxins, such as the cholera toxin
in V. cholera and the Shiga toxin in pathogenic E. coli
strains115–117. Intriguingly, these toxins were shown to
be secreted through MVs, which deliver them to the
eukaryotic host cells118,119. Hence, the SOS response not
only induces production of the toxins but also triggers
explosive cell lysis and thus ensures that the toxins are
secreted and delivered by MVs.
As some stimuli can trigger certain vesicle for-
mation routes (FIG. 3), it is likely that environmental
niches select for particular vesicle types. Recent DNA
and RNA-sequencing analyses have provided evidence
that a considerable proportion of the MVs present
in natural habitats likely originate from cell lysis by
demonstrating that MVs can harbour complete viral
genomes120, that DNA associated with MVs isolated
from open-ocean samples is highly enriched for viral
sequences6,121,122 and that ultraviolet radiation of water
samples, a trigger for prophage induction, greatly stim-
ulates vesicle formation123. Given that phages are the
most abundant form of life on Earth, exceeding bacte-
ria in number by tenfold124, phage-triggered cell lysis
appears to be an important and so far underestimated
route for vesicle formation in nature. However, the
sequencing of environmental MVs may overestimate
the role of endolysin-triggered cell death for vesicle for-
mation in the environment, as RNA and DNA appear
to be mostly associated with OIMVs and CMVs, which
are mainly produced through cell lysis routes. In this
context, it will be important to unravel to what extent
explosive cell lysis triggers the formation of OMVs
(which we refer to as EOMVs), and whether this occurs
directly or from OIMVs78, as we predict that they will
differ from OMVs formed through blebbing mecha-
nisms (TABLE 1). EOMVs can carry cytosolic contents,
including chromosomal DNA and endolysins that can
kill bacteria. By contrast, classic OMVs formed by bleb-
bing through conventional mechanisms are unlikely to
carry chromosomal DNA or cytosolic proteins27 but
can be enriched for hydrophobic molecules, including
bacterial signals and membrane-targeting antibiotics,
and may show specific lipid compositions and carry
particular cargo molecules, which are often virulence
factors. In bacteria carrying phage endolysins, cell
lysis of a subpopulation of the cells is inevitable, even
under optimal exponential growth conditions. It can
therefore not be ruled out that previous studies report-
ing the presence of RNA and DNA in OMVs have been
biased by the presence of small amounts of EOMVs
and OIMVs.
The isolation and biochemical and structural charac-
terization of MVs from environmental samples will be
required to determine which types of vesicle prevail in
each particular niche. This in turn may shed light on the
environmental conditions that the cells encounter and
may allow conclusions to be made about the possible
functions of the different types of vesicles.
Published online 5 November 2018
VOLUME 17 | JANUARY 2019 | 21
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Acknowledgements
M.T. was supported by the Ministry of Education, Culture,
Sports, Science and Technology of Japan (MEXT) (project
16H06189), N.N. was supported by the Japan Science and
Technology Agency (JST) (ERATO project JPMJER1502),
and L.E. was supported by the Swiss National Science
Foundation (SNSF) (Project 31003A_169307). The authors
acknowledge K. Agnoli-Antkowiak for valuable comments on
the manuscript.
Author contributions
M.T. and L.E. researched data for the article and contributed
substantially to discussion of the content. All authors wrote
the article and reviewed and edited the manuscript before
submission.
Competing interest
The authors declare no competing interests.
Publisher’s note
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Reviewer information
Nature Reviews Microbiology thanks S. Schild, S. N. Wai and
the other anonymous reviewer(s) for their contribution to the
peer review of this work.
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