Biogenesis of the gram negative Nieznany

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Biogenesis of the
Gram-Negative Bacterial
Outer Membrane

Martine P. Bos, Viviane Robert,
and Jan Tommassen

Department of Molecular Microbiology and Institute of Biomembranes,
Utrecht University, 3584 CH Utrecht, The Netherlands; email: m.bos@uu.nl,
vivianerobert@hotmail.com, j.p.m.tommassen@uu.nl

Annu. Rev. Microbiol. 2007. 61:191–214

First published online as a Review in Advance on
June 7, 2007

The Annual Review of Microbiology is online at
micro.annualreviews.org

This article’s doi:
10.1146/annurev.micro.61.080706.093245

2007 by Annual Reviews.

0066-4227/07/1013-0191$20.00

Key Words

lipopolysaccharide, outer membrane proteins, lipoproteins,
phospholipids

Abstract

The cell envelope of gram-negative bacteria consists of two mem-
branes, the inner and the outer membrane, that are separated by
the periplasm. The outer membrane consists of phospholipids,
lipopolysaccharides, integral membrane proteins, and lipoproteins.
These components are synthesized in the cytoplasm or at the inner
leaﬂet of the inner membrane and have to be transported across the
inner membrane and through the periplasm to assemble eventually
in the correct membrane. Recent studies in Neisseria meningitidis and
Escherichia coli have led to the identiﬁcation of several machineries
implicated in these transport and assembly processes.

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IM:

inner

membrane

OM:

outer

membrane

Periplasm:

compartment
between the inner
and outer
membranes

Peptidoglycan:

bacterial cell wall
consisting of sugar
polymers covalently
connected via
oligopeptides

Lipopolysaccharide
(LPS):

glycolipid

constituting the
outer layer of the
bacterial outer
membrane

-barrel:

common

structure of outer
membrane proteins,
consisting of
amphipathic
antiparallel β-strands

Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 192
INTEGRAL OUTER MEMBRANE

PROTEINS . . . . . . . . . . . . . . . . . . . . . 193
Passage Across the IM and

Through the Periplasm . . . . . . . . 193

Assembly into the OM . . . . . . . . . . . . 194
Role of LPS in OMP Biogenesis . . 199
Model for OMP Biogenesis . . . . . . . 200

LIPOPROTEINS. . . . . . . . . . . . . . . . . . . 200
LIPOPOLYSACCHARIDE . . . . . . . . . 202

Structure and Biosynthesis . . . . . . . . 202
Transport to the Cell Surface . . . . . 202
Models for LPS Transport . . . . . . . . 205

PHOSPHOLIPIDS . . . . . . . . . . . . . . . . . 206
PERSPECTIVES . . . . . . . . . . . . . . . . . . . 206

INTRODUCTION

The cell envelope of gram-negative bacteria
consists of two membranes, the inner mem-
brane (IM) and the outer membrane (OM),
that are separated by the periplasm containing
the peptidoglycan layer. The two membranes
have an entirely different structure and com-
position. Whereas the IM is a phospholipid
bilayer, the OM is an asymmetrical bilayer,
consisting of phospholipids and lipopolysac-
charides (LPS) in the inner and outer leaﬂet,
respectively. Additionally, these membranes
differ with respect to the structure of the
integral membrane proteins. Whereas inte-
gral IM proteins typically span the membrane
in the form of hydrophobic α-helices, inte-
gral OM proteins (OMPs) generally consist
of antiparallel amphipathic β-strands that fold
into cylindrical β-barrels with a hydrophilic
interior and hydrophobic residues pointing
outward to face the membrane lipids (49).
Both membranes also contain lipoproteins,
which are anchored to the membranes via
an N-terminal N-acyl-diacylglycerylcysteine,
with the protein moiety usually facing the
periplasm in the case of Escherichia coli. In

other gram-negative bacteria, however, the
protein moiety of OM lipoproteins may also
extend into the extracellular medium; ex-
amples are the LbpB and TbpB compo-
nents of the lactoferrin and transferrin re-
ceptor, respectively, of Neisseria meningitidis
(70).

The OM functions as a selective barrier

that protects the bacteria from harmful com-
pounds, such as antibiotics, in the environ-
ment. Unlike the IM, the OM is not energized
by a proton gradient and ATP is not avail-
able in the periplasm. In the absence of read-
ily available energy sources, nutrients usually
pass the OM by passive diffusion via an abun-
dant class of trimeric OMPs called porins (66).
Porins form water-ﬁlled channels that allow
the passage of small hydrophilic solutes with
molecular weights up to

∼600 Da. Never-

theless, energy-requiring transport processes
in the OM have also been described. Such
processes are dependent on complex energy-
coupling systems, such as the TonB system,
which couples the proton-motive force of the
IM to receptor-mediated uptake processes in
the OM (110).

Whereas the composition, structure, and

function of the OM have been known for
decades, its assembly in the absence of en-
ergy sources has remained largely enigmatic.
All the components of the OM are syn-
thesized in the cytoplasm or at the cyto-
plasmic face of the IM, and they have to
be transported across the IM and through
the periplasm to reach their destination and
to assemble into the OM. Recently, many
new components involved in these processes
have been described, and in this review we
focus on these recent developments. Apart
from studies in the classical model organisms,
E. coli and Salmonella enterica, much progress
in this ﬁeld was reached by studying N. menin-
gitidis, and a comparison between these sys-
tems reveals important differences in these
fundamental processes. Hence, what is true
for E. coli is not necessarily true for other
bacteria.

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Robert

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INTEGRAL OUTER MEMBRANE
PROTEINS

Passage Across the IM and Through
the Periplasm

Integral OMPs are synthesized in the cyto-
plasm as precursors with an N-terminal signal
sequence, which is required for translocation
across the IM. Two translocation machineries
have been identiﬁed: the Sec system for the
translocation of unfolded proteins (23) and
the Tat system, which transports proteins
folded in the cytoplasm (57). However, to
our knowledge, all OMPs studied to date are
transported via the Sec system, indicating that
they reach the periplasm in an unfolded state.

After transport across the IM, the nascent

OMPs are accessible to periplasmic chaper-
ones (30). These chaperones have been stud-
ied extensively, mainly in E. coli. One of
these chaperones, Skp (seventeen-kilodalton-
protein), immediately interacts with the
OMPs as soon as they emerge from the
Sec channel (37). The crystal structure of
this homotrimeric protein resembles a three-
pronged grasping forceps that could bind a
nonnative OMP between the prongs and pro-
tect it and prevent it from aggregating dur-
ing passage through the periplasm (52, 109).
An skp mutant of E. coli is viable but contains
decreased amounts of OMPs (16). Another
periplasmic chaperone, SurA, was initially
identiﬁed as a protein required for survival
during the stationary phase (102). Mutants in
surA display an OMP assembly defect (56),
and speciﬁcally the conversion of unfolded
monomers of OMPs into folded monomers
appears affected in such mutants (77). The
protein has peptidyl-prolyl cis/trans isomerase
(PPIase) activity (56, 62), which nevertheless
appears to be dispensable for the chaperone
function (5). Unlike most cytoplasmic chaper-
ones, SurA is selective and preferentially binds
nonnative OMPs over other proteins (5). By
screening peptide libraries, Hennecke et al.
(38) demonstrated that SurA binds peptides
rich in aromatic residues and preferentially
those containing Ar-Ar or Ar-X-Ar motifs

Lipoprotein:

protein attached to
the bacterial inner or
outer membrane via
an N-terminal lipid
moiety

Porin:

protein that

forms water-ﬁlled
channels in the outer
membrane

Sec system:

general

protein export
apparatus

Chaperone:

protein

that guides another
molecule to its
destination, being
the folded state or
the right cellular
compartment

(where Ar is an aromatic residue and X is any
residue). Transmembrane β-strands of OMPs
are particularly enriched with such motifs.
Like skp mutants, surA mutants are viable, but
the combination of an skp and a surA muta-
tion results in a synthetically lethal phenotype
(75). Therefore, it was suggested that Skp and
SurA are functionally redundant and that they
operate in parallel pathways for chaperone ac-
tivity (75). However, this is not the only pos-
sible explanation for the synthetic phenotype.
We favor the possibility that Skp and SurA act
sequentially in the same pathway (Figure 1).
Skp may act as a holding chaperone, prevent-
ing aggregation of nonnative OMPs in the
periplasm, and SurA may act as a folding chap-
erone. The demand for the holding chaper-
one may be limited when the subsequent fold-
ing and assembly steps are efﬁcient. Under
those conditions, the OMPs have only little
chance to aggregate and an skp null mutation

Periplasm

Sec

Omp85

Skp

SurA

Porin

OMP

YfgL

YfiO NlpB

Figure 1

Model for OMP biogenesis. As soon as an OMP emerges from the Sec
translocon, it is bound by the chaperone Skp, which may prevent
aggregation in the periplasm. The C-terminal signature sequence of the
OMP functions as a targeting signal and binds the periplasmic domain of
Omp85. Other chaperones and folding catalysts, such as SurA, may act on
the OMP. After folding, the OMP inserts into the lipid bilayer, possibly in
between the Omp85 subunits. Oligomerization of certain OMPs, such as
porins, may occur after insertion. The exact function of the accessory
lipoproteins YfgL, YﬁO, and NlpB is not known. Another accessory
lipoprotein recently identiﬁed, SmpA (84), is not indicated here.

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PPIase:

peptidyl-prolyl
cis/trans isomerase

alternative

sigma factor that
guides RNA
polymerase to the
promoters of genes
involved in relieving
periplasmic stress
conditions

has only a limited effect. The demand for such
a chaperone will increase when the subsequent
folding and assembly processes are hampered,
e.g., in a surA mutant. Hence, it is not possible
to construct the skp surA double mutant.

Apart from SurA, the periplasm contains

three other PPIases, PpiA (also known as
RotA), PpiD, and FkpA. PpiA is by far the
most active of them, because its inactivation
leads to barely detectable residual PPIase ac-
tivity (47). However, a ppiA null mutation had
no detectable effect on OMP assembly (47). In
contrast, a ppiD null mutation led to an overall
reduction in the level and folding of OMPs,
and a combination of ppiD and surA mutations
was lethal (19), suggesting functional redun-
dancy. However, PpiD is anchored in the IM,
whereas OMP folding is presumably initiated
after targeting to the OM, with which, indeed,
a proportion of the SurA molecules cofrac-
tionated (38). Later, it was reported that ppiD
mutants, like ppiA and fkpA mutants and even
a ppiD ppiA fkpA triple mutant, did not show
different overall OMP patterns or OM per-
meability compared with the wild type and
that a ppiD surA double mutant could be con-
structed, which had the same phenotype as a
surA single mutant (44). Overall, there is little
evidence for a direct role of PpiA, PpiD, or
FkpA in OMP biogenesis.

Other proteins that may play a role at

the periplasmic stage of OMPs are DsbA and
DsbC, two enzymes involved in the forma-
tion and isomerization, respectively, of disul-
ﬁde bonds. DsbA catalyzed the formation of
a disulﬁde bond between two cysteines engi-
neered in OM porin PhoE at positions not
accessible from the periplasm once the porin
is inserted into the OM. This observation
showed that the disulﬁde bond was formed
during periplasmic transit and that at least par-
tial folding occurs prior to OM insertion (30).
DegP is a protease that degrades unfolded or
misfolded proteins in the periplasm but also
has chaperone activity, as was shown in vitro
on the soluble substrates MalS and citrate syn-
thase (87). A combination of surA and degP
mutations was synthetically lethal, suggesting

a role for DegP in OMP assembly (75). How-
ever, the role of DegP as a protease, remov-
ing misfolded or unfolded OMPs from the
periplasm, may be more important than its
function as a chaperone in this respect (13).

Assembly into the OM

The insertion of OMPs into the OM has long
remained enigmatic and has been considered
a spontaneous process (98). However, recent
work has identiﬁed a proteinaceous machin-
ery that is essential for this process.

Omp85, an essential component of the
OMP assembly machinery.

Recently, we

(105) identiﬁed a ﬁrst component required
for OMP insertion, i.e., a protein designated
Omp85 in N. meningitidis. Omp85 was found
to be essential for the viability of the bacteria,
and homologues of the omp85 gene were
found in all gram-negative bacteria for which
the genome sequence was available (106),
suggesting its involvement in an important
process. Moreover, in many of these genomes,
including those of N. meningitidis and E. coli,
the omp85 gene is ﬂanked by the skp gene,
which encodes the periplasmic chaperone in-
volved in OMP biogenesis, and rseP (formally
designated yaeL), which encodes a protease
involved in the σ

-dependent stress response

of E. coli that is induced upon accumulation of
unfolded OMPs in the periplasm (see below).
All these features are consistent with a vital
role of Omp85 in OMP assembly. Upon de-
pletion of Omp85 in a genetically engineered
strain, unfolded forms of all OMPs examined,
including porins, a siderophore receptor, an
enzyme, and a secretin involved in type IV
pili assembly, accumulated as aggregates in
the periplasm (105, 106). The role of Omp85
in OMP assembly was conﬁrmed in E. coli,
in which the corresponding gene, designated
yaeT, was also an essential gene. Either deple-
tion of Omp85 or growth of a temperature-
sensitive mutant at the restrictive temperature
resulted in severe OMP assembly defects
(27, 112, 114). Even more interestingly, a

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homolog of Omp85 was essential for the
assembly of β-barrel proteins into the outer
membrane of mitochondria, a eukaryotic cell
organelle of endosymbiont origin (33, 54,
69). Apparently, the OMP assembly pathway
is highly conserved during evolution.

A difference was observed with respect to

the fate of OMPs in Omp85-depleted cells
of either N. meningitidis or E. coli. Whereas
unfolded OMPs accumulated as aggregates
in such cells of N. meningitidis (105), Omp85
depletion in E. coli primarily led to severely
reduced amounts of detectable OMPs (112,
114). The difference is presumably caused by

the absence of the σ

-dependent periplasmic

stress response in N. meningitidis. In E. coli,
this stress response is induced upon accumu-
lation of unfolded OMPs in the periplasm (for
a review, see Reference 79). Such OMPs are
sensed by the PDZ domain of the membrane-
bound protease DegS, upon which a prote-
olytic cascade is initiated that involves the
protease domain of DegS and RseP, result-
ing in the cleavage of the antisigma factor
RseA and the release of σ

in the cytoplasm

(Figure 2). The released σ

then binds the

RNA polymerase core enzyme, resulting in
the transcription of the genes for periplasmic

DegS

PDZ

RseA

RseB

RseP

DegS

RseA

RseB

RseP

Periplasm

Periplasmic
stress response

Stress

RNA

polymerase

sRNAs

skp

surA

degP

Protease

OMPs

OMP

Figure 2

Periplasmic stress
response. Upon
accumulation of
unfolded OMPs in
the periplasm, a
stress response is
initiated that starts
with the recognition
of the C termini of
the OMPs by the
PDZ domain of the
protease DegS. This
recognition initiates
a proteolytic cascade,
resulting in the
release of the
alternative sigma
factor σ

, which

binds RNA
polymerase and leads
to enhanced
transcription of
genes encoding
periplasmic
chaperones, such as
Skp and SurA, and
the protease DegP.
The σ

response also

leads to production
of sRNAs that
negatively regulate
OMP expression.

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Outer Membrane Biogenesis

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sRNA:

small

regulatory RNA

chaperones, such as Skp and SurA, and for
the potent protease DegP. Together these
chaperones and protease relieve the periplas-
mic stress. Moreover, the σ

response results

in the production of small regulatory RNAs
(sRNAs) in Enterobacteriaceae that prevent
OMP expression at the translational level (31,
43, 68). Thus, in E. coli, misfolded OMPs
are rescued or degraded in the periplasm and
OMP synthesis is inhibited during the stress
period. Although chaperones such as Skp and
SurA are present in N. meningitidis, many es-
sential components of the signal transduction
pathway are lacking. Whereas E. coli contains
three genes for the related proteases DegP,
DegS, and DegQ (107), only a homologue of
DegQ can be found in N. meningitidis (en-
coded by the NMB0532 locus in the N. menin-
gitidis strain MC58). Moreover, homologues
of RseA and RseB appear to be absent. Al-
though there is an alternative σ factor belong-
ing to the ECF (extracytoplasmic factor) fam-
ily (to which σ

also belongs) encoded on the

meningococcal chromosome (i.e., NMB2144
in strain MC58), it seems to have an entirely
different function. Its inactivation in the re-
lated bacterium Neisseria gonorrhoeae did not
affect global gene expression as analyzed by
microarray analysis, whereas its overexpres-
sion affected only very few genes, including
those for methionine sulfoxide reductase (35).
Thus, the σ

-dependent periplasmic stress re-

sponse appears to be absent in the pathogenic
Neisseriaceae. As a result, when there is an
OMP assembly defect, OMPs will continue to
be synthesized and they will accumulate in the
periplasm, where they will form aggregates.

Omp85 is part of a multisubunit complex.

Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (PAGE) analysis under non-
denaturing conditions revealed that Omp85
of N. meningitidis is present in a high-
molecular-weight complex (105). The only
other protein identiﬁed in this complex was
the RmpM protein ( J. Geurtsen, R. Voulhoux
& J. Tommassen, unpublished results), which
has a peptidoglycan-binding motif and largely

resides in the periplasm but is ﬁrmly associ-
ated with many different integral OMPs via
an N-terminal peptide (71). RmpM proba-
bly anchors the OM to the underlying pep-
tidoglycan layer (88), and it has no particular
function in OMP biogenesis. Cross-linking
experiments revealed that the Omp85 ho-
molog of E. coli forms a complex with three
lipoproteins, YfgL, YﬁO, and NlpB (114). A
similar complex was identiﬁed upon a pro-
teomic analysis of OMP complexes resolved
by blue-native PAGE (93). Copuriﬁcation ex-
periments using strains with mutations in the
genes for various components of the complex
indicated that YfgL and YﬁO directly interact
with Omp85, whereas NlpB is associated with
the complex via YﬁO (58).

The yfgL gene was originally identiﬁed

in a screen for suppressors that restored the
OM permeability defect of a partial loss-of-
function mutation in the imp gene (29, 78),
which encodes an OMP involved in LPS
biogenesis (see below). Although yfgL is not
an essential gene, null mutations create an
OM permeability defect and result in reduced
amounts of OMPs (67, 78, 114), consistent
with a role in OMP assembly. Remarkably, al-
though the yfgL gene is widely disseminated
among gram-negative bacteria, we could not
identify a homolog in the sequenced genomes
of N. meningitidis and N. gonorrhoeae.

The yﬁO gene is essential in E. coli (67),

but a mutant with a transposon insertion near
the 3

end of the gene was viable (114). This

mutant showed increased OM permeability
and reduced amounts of OMPs, and a yﬁO
depletion strain showed a phenotype similar
to that of the yaeT/omp85 depletion strain of
E. coli (58). In N. gonorrhoeae, a transposon
insertion in the middle of the yﬁO homolog
was described (32). This mutant was viable,
showed a reduced cell size, and was transfor-
mation deﬁcient; therefore, the gene was des-
ignated comL. However, attempts to introduce
other comL truncations into the chromosome
failed (32), and we have not yet succeeded
to generate a complete comL deletion in the
chromosome of N. meningitidis (E. Volokhina,

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M.P. Bos & J. Tommassen, unpublished re-
sults). Hence, also in the Neisseriaceae, YﬁO
(ComL) appears essential for viability and the
N-terminal half of the protein appears sufﬁ-
cient for partial functionality. The ComL pro-
tein is covalently linked to the peptidoglycan,
both in N. gonorrhoeae and when expressed
in E. coli (32). Furthermore, yfgL mutations
affected peptidoglycan synthesis, possibly by
regulating the activity of lytic transglycosy-
lases (29). Thus, both YfgL and YﬁO might
have a role in modulating the peptidoglycan
to facilitate the passage of OMPs through this
layer.

Whereas yﬁO, like yfgL and omp85, is

widely disseminated among gram-negative
bacteria, this is less so for nlpB. Null mutants
in nlpB of E. coli are viable; they show only
moderate OM permeability defects and no
obvious defects in OMP assembly (67, 114).
However, because an nlpB surA double knock-
out mutant has a synthetic lethal phenotype
(67), it is clear that NlpB also has a direct role
in OMP assembly, possibly redundant to that
of the periplasmic chaperone SurA.

Structure of Omp85.

On the basis of

the sequence, we (105) have proposed
that Omp85 consists of two domains, a
C-terminal β-barrel embedded in the OM
and an N-terminal domain extending into
the periplasm. Support for this model was
obtained in proteolytic digestion experi-
ments, which resulted in the degradation of
the N-terminal domain and left the predicted
membrane-embedded β-barrel domain intact
(76, 91). The periplasmic extension contains
repeats of a conserved domain, POTRA
(polypeptide transport associated), suggested
to have chaperone-like qualities (81). Such
POTRA domains were further identiﬁed in
other members of the Omp85 superfamily,
which includes the Omp85 homologs of mi-
tochondria, the Toc75 OM component of the
chloroplast protein import machinery, and
the OM-localized TpsB component of the
two-partner secretion (TPS) systems of gram-
negative bacteria, and in members of the

POTRA:

polypeptide
transport associated

FtsQ/DivIB family of IM proteins involved in
cell division. The bacterial Omp85 homologs
are unique in having ﬁve of these POTRA do-
mains, whereas the number of such domains
in the other proteins with known functions
is restricted to one or two. Another member
of the Omp85 superfamily that is widely
disseminated among gram-negative bacteria
(encoded by the ytfM gene in E. coli and the
NMB2134 locus in N. meningitidis strain
MC58) contains three POTRA domains.
The function of this protein is unknown (92).

To study its structure and function in

more detail, we produced the Omp85 protein
of E. coli in inclusion bodies, which were
isolated, and refolded the protein into the
native conformation in vitro (76). The re-
folded protein formed oligomers, presumably
tetramers, similarly as reported for another
member of the Omp85 superfamily, i.e.,
HMW1B, the TpsB component of a TPS
system of Haemophilus inﬂuenzae (95). Of
note, the interactions between the subunits in
this homo-oligomeric complex are not stable;
blue-native PAGE indicated equilibrium
between monomeric and oligomeric forms
(76). Consistently, the hetero-oligomeric
Omp85/YﬁO/YfgL/NlpB complex identiﬁed
in vivo appeared to consist of one copy of
each subunit (93).

Interaction of Omp85 with its substrate
OMPs.

When reconstituted in planar lipid

bilayers, both in vitro refolded Omp85 and
Omp85 extracted from E. coli cell envelopes
formed narrow ion-conductive channels (76,
91). This property was used to study the in-
teraction between Omp85 and its substrate
OMPs, emanating from the idea that such an
interaction would affect the channel activity.
Indeed, nonnative OMPs drastically enhance
Omp85 channel activities (76), showing that
these substrates interact directly with Omp85.
Furthermore, using mutant OMPs and syn-
thetic oligopeptides in this assay, we demon-
strated that Omp85 speciﬁcally recognizes a
C-terminal motif in its substrates (76) that was
shown to be required for efﬁcient assembly

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of these proteins into the OM in vivo (94).
This C-terminal signature motif, which con-
sists of a phenylalanine (or tryptophan) at the
C-terminal position, a tyrosine or hydropho-
bic residue at position 3 from the C terminus,
and also hydrophobic residues at positions 5,
7, and 9 from the C terminus, is present in
most bacterial OMPs, including porins, re-
ceptors, enzymes, and autotransporters. It is
interesting to note that the same signature
sequence is recognized by the PDZ domain
of DegS when unfolded OMPs accumulate
in the periplasm (108), thereby initiating the
σ

-dependent periplasmic stress response (see

above).

In the planar lipid bilayer experiments an

OMP of N. meningitidis, in contrast to E. coli
OMPs, did not enhance the activity of the E.
coli Omp85 channels (76). Consistently, high-
level expression of neisserial OMPs in E. coli
is often lethal and leads to their misassembly,
suggesting that the E. coli OMP assembly ma-
chinery cannot deal efﬁciently with neisserial
proteins. Indeed, expression of E. coli omp85
cannot complement an omp85 mutation in N.
meningitidis and vice versa (E. Volokhina, V.
Robert, M.P. Bos & J. Tommassen, unpub-
lished observations). Although the C termini
of neisserial OMPs do contain the signature
sequence with the features outlined above,
they are further characterized by the presence
of a positively charged residue at the penulti-
mate position, which could inhibit the inter-
action with E. coli Omp85. Indeed substitu-
tion of this positively charged residue in the N.
meningitidis porin PorA drastically improved
PorA assembly into the E. coli OM in vivo (76).
Thus, although the OMP assembly machin-
ery is highly conserved among gram-negative
bacteria, species-speciﬁc adaptations seem to
have occurred during evolution.

Of note, the C-terminal signature se-

quence of OMPs is not absolutely essential
for assembly into the OM. Thus, whereas
the high-level expression of a mutant form of
the E. coli porin PhoE lacking the C-terminal
phenylalanine was lethal and resulted in its
periplasmic aggregation (21, 94), its low-level

expression was tolerated and allowed for its
assembly into the OM (21). Similarly, several
studies reporting the assembly of neisserial
OMPs into the E. coli OM in vivo (113) may
be explained by low expression levels. Pulse-
chase experiments in E. coli overexpressing
PhoE revealed the existence of two assem-
bly pathways: approximately half of the PhoE
molecules assembled within the 30-s pulse
period into their native conformation in the
OM, whereas the other half of the molecules
followed much slower kinetics and allowed
for the detection of several assembly inter-
mediates (42). In these assays, the mutant
PhoE lacking the C-terminal phenylalanine
followed only the slower kinetic pathway. This
study underscores the role of the C-terminal
signature sequence as a targeting signal and
indicates that there must be alternative, less
efﬁcient targeting signals in OMPs. Further-
more, kinetic partitioning between OM incor-
poration and aggregation of periplasmic in-
termediates may explain the observation that
OMPs with defective signature sequences are
still assembled into the OM at low expression
levels, when the kinetics of aggregation is low
and hence the time span for assembly into the
OM is elongated (76).

What could be the nature of the al-

ternative targeting sequences in OMPs? Of
note, the C-terminal signature sequence con-
tributes an amphipathic β-strand to the β-
barrel in the folded protein. In the absence
of the C-terminal phenylalanine, Omp85
may recognize, though less efﬁciently, in-
ternal β-strands, many of which also end
with an aromatic residue. In some classes
of OMPs, the C-terminal signature motif
could not be discerned (94). Perhaps, in
these cases also, Omp85 recognizes an in-
ternal β-strand. One of the major OMPs of
E. coli, OmpA, consists of two domains, an
N-terminal OM-embedded β-barrel and a C-
terminal periplasmic extension. The signature
motif was found in this case at the end of the β-
barrel domain (94) and its importance in OM
targeting was demonstrated in a deletion anal-
ysis (48). Although this example demonstrates

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that the targeting motif can be located inter-
nally in the primary structure of an OMP, this
appears to be a rather exceptional case be-
cause the C-terminal extension of an OMP
sequence with, for example, a His tag usually
results in severe assembly defects (91).

Another class of OMPs that lack the C-

terminal signature motif is constituted by
TolC and its homologs, which are involved in
type I protein secretion and drugs extrusion.
In its native trimeric structure, TolC forms a
β-barrel in the OM, to which each monomer
contributes four β-strands, whereas the ma-
jor portion of the protein extends as long
α-helices into the periplasm (53). TolC is de-
pendent on Omp85 for its assembly (112);
hence, possibly also in this case, an inter-
nal β-strand is recognized. In contrast, Wza,
an OMP involved in the export of capsu-
lar polysaccharides, has an entirely different
structure; the membrane-embedded portion
of this octameric protein consists of a barrel
of eight amphipathic α-helices, to which each
monomer contributes one helix (28). It is con-
ceivable that this protein assembles entirely
independently of the Omp85 machinery. The
secretins form a class of OMPs involved in di-
verse processes, including type II and type III
protein secretion, type IV pili biogenesis, and
the extrusion of ﬁlamentous phages (7). The
structure of these large multimeric proteins is
not known at atomic resolution, but they ap-
pear to be rather poor in β-sheet content (14),
which could indicate that they also follow a
different assembly pathway. However, at least
one secretin, PilQ of N. meningitidis, depends
on Omp85 for its assembly (105). Perhaps, the
binding of these proteins and other OMPs
that lack the C-terminal signature motif is
indirect and requires accessory factors, such
as the lipoproteins YﬁO, YfgL, and NlpB, or
speciﬁc chaperones, such as the pilotins in the
case of the secretins (3).

Role of LPS in OMP Biogenesis

Since the observation that the amounts of sev-
eral OMPs are severely decreased in deep-

rough mutants of E. coli and Salmonella ty-
phimurium, which contain truncated LPS
molecules (2, 51), a role of LPS in OMP
assembly has been suggested. Consistently,
the OM porin PhoE of E. coli could be con-
verted in vitro into a folded monomeric form
in the presence of LPS, detergent, and diva-
lent cations. This folded monomer appeared
to be an assembly intermediate because it
could subsequently be converted into its na-
tive trimeric OM-inserted form (22). Also
for other OMPs, LPS-dependent folding in
vitro has been reported (83). However, LPS-
independent in vitro folding conditions were
later established for many OMPs, including
PhoE (42), arguing against a speciﬁc role of
LPS in OMP folding. Moreover, an lpxA mu-
tant of N. meningitidis, which is completely
defective in LPS biosynthesis, appeared to be
viable (90), and all OMPs examined, including
porins, were correctly assembled in vivo into
the LPS-free OM of this mutant (89). Nev-
ertheless, species-speciﬁc differences with re-
spect to the LPS dependency of OMP assem-
bly cannot yet be excluded at this stage.

The crystal structure of E. coli Skp re-

vealed a putative LPS-binding site (109), con-
sistent with the earlier description of the pro-
tein in Salmonella minnesota as an LPS-binding
protein (34). Thus, LPS may exert its role
in OMP biogenesis via Skp. Furthermore, it
was demonstrated in protease-digestion as-
says that binding of LPS and phospholipids
could inversely modulate the structure of Skp
in vitro (20). Thus, after binding nonnative
OMPs at the IM, a conformational change
triggered by LPS binding may release the
cargo at the OM. Note that LPS is present
only in the outer leaﬂet of the OM. Hence,
Skp should bind to LPS molecules that have
not yet reached their destination. Consis-
tently, OMP biogenesis is heavily affected by
cerulinin, a drug that inhibits lipid synthe-
sis (8). However, the putative LPS-binding
site in E. coli Skp is largely conserved in
N. meningitidis Skp, where OMP biogene-
sis is independent of LPS. Hence, the role
of this putative LPS-binding site remains to

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Imp:

increased

membrane
permeability

be determined in site-directed mutagenesis
experiments.

We speculate that the role of LPS in OMP

biogenesis is restricted to late stages after
the insertion into the OM, such as the sta-
bilization of porin trimers (55), which pre-
sumably requires some LPS-mediated rear-
rangements in the cell surface-exposed loops
of the proteins. The severely reduced amounts
of OMPs in deep-rough mutants may be ex-
plained by the induction of the σ

response

in such mutants, resulting in the production
of sRNAs that inhibit the synthesis of many
OMPs (see above). Changes in LPS structure
induce the σ

response in E. coli by a hitherto

unknown mechanism (97). At least one OMP
of E. coli, TolC, appears totally unaffected by
LPS structure (111). Presumably, the expres-
sion of TolC is unaffected by the sRNAs. The
assembly of this lipid-independent OMP is in-
dependent of the accessory component YfgL
of the Omp85 assembly machinery (15). Of
note, a homolog of the yfgL gene is lacking in
N. meningitidis, in which the assembly of all
OMPs is independent of LPS. Hence, there
may be an additional role for LPS in the as-
sembly of lipid-dependent OMPs in E. coli,
and YfgL may play a role speciﬁcally in the
assembly of this class of OMPs. In this re-
spect, it may indeed be relevant that the yfgL
mutants were initially picked up as suppres-
sors of a partial loss-of-function imp mutant
(29), and the imp gene product is speciﬁcally
involved in LPS biogenesis (9) (see below).

Model for OMP Biogenesis

We propose the following model for OMP
biogenesis (Figure 1). After their transport
through the Sec translocon, nascent OMPs
are immediately bound by Skp, which may as-
sist their release from the IM and prevent their
aggregation in the periplasm. The Skp/OMP
complex is targeted to the Omp85 complex in
the OM, whereby the C-terminal signature
motif of the OMPs functions as the primary
targeting signal that binds directly to Omp85,
presumably to its N-terminal POTRA do-

mains. Binding initiates folding, which results
in the release of Skp (20) and may be assisted
by the presumed chaperone activities of the
POTRA domains and by periplasmic proteins
such as SurA and DsbA. Binding of an OMP
also results in a conformational change in the
C-terminal domain of Omp85, which is re-
ﬂected by the increased pore activity observed
in the planar lipid bilayer experiments (76).
This conformational change allows the OMPs
to insert into the OM, possibly in between the
Omp85 subunits. Dissociation of the Omp85
subunits releases the assembled OMPs into
the OM, where ﬁnal conformation changes in
the cell surface-exposed loops may be induced
upon interaction with LPS.

The speciﬁc role of the accessory lipopro-

teins YfgL, YﬁO, and NlpB has not been ad-
dressed experimentally so far. Of note, no ho-
mologs of these proteins have been implicated
in the assembly of β-barrel proteins into the
mitochondrial OM, suggesting that their role
is less crucial, although YﬁO is deﬁnitely es-
sential, at least in E. coli (67). These proteins
may play a role in the recognition of OMPs
that do not display the C-terminal signature
motif, which includes the essential protein
Imp (increased membrane permeability), in
modulating the peptidoglycan layer, and/or
they may function as chaperones in the fold-
ing of OMPs. YfgL may have a role in coor-
dinating the assembly of a subclass of OMPs
that require LPS for their assembly, but such a
role is difﬁcult to assess in E. coli, in which as-
sembly defects of OMPs are associated with
a feedback inhibition on their synthesis via
σ

-induced sRNAs, whereas N. meningitidis,

which does not display such a feedback inhibi-
tion, does not have a YfgL homolog. The ab-
sence of this feedback inhibition mechanism
makes N. meningitidis a favorable organism to
study the role of other assembly factors.

LIPOPROTEINS

Bacterial

lipoproteins

are

membrane

attached

via

N-terminal

N-acyl-

diacylglycerylcysteine. Lipidation and folding

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of these proteins take place after their translo-
cation over the IM via the Sec machinery.
Prior to cleavage of the signal sequence, a
diacylglycerol group is transferred by the
enzyme Lgt from phosphatidylglycerol to
the sulfhydryl group of the cysteine that is
invariably present at the

+1 position relative

to the processing site (82). Subsequently, the
diacylglycerylprolipoprotein is processed by a
dedicated signal peptidase, signal peptidase II
(24), after which the free α-amino group of
the cysteine is acylated by the enzyme Lnt
(36), yielding the mature lipoprotein.

Lipoproteins are sorted to the IM or OM

according to a sorting signal that comprises
the amino acids ﬂanking the lipidated cysteine
in the mature protein (101). Lipoproteins
lacking an IM retention signal, usually an
aspartate at the

+2 position of the mature

protein, are transported to the OM by the
Lol system (Figure 3). The ﬁrst component
of the Lol system was identiﬁed in an in vitro
system, whereby lipoproteins were synthe-
sized in a radioactive form in spheroplasts of
E. coli (60). The mature forms of the newly
synthesized lipoproteins were retained at
the surface of the spheroplasts, from which
they could be released upon addition of a
periplasmic extract. The active component
of the periplasmic extract was identiﬁed and
designated LolA. Furthermore, a complex of
the three proteins LolC, LolD, and LolE,
which constitutes an ATP-binding cassette
(ABC) transporter in the IM, is required
for lipoprotein transport (65). The integral
membrane components of this transporter,
LolC and LolE, show considerable sequence
similarity and may function as a heterodimer.
Of note, N. meningitidis contains only one of
these proteins (encoded by locus NMB1235
in strain MC58), which may form a homod-
imer. The current model (Figure 3) states
that lipoproteins destined for the OM are ﬁrst
bound by LolC/E in an ATP-independent
manner. This binding results in an increase in
the afﬁnity of LolD for ATP. Subsequently,
ATP binding to LolD causes a conformational
change in the LolC/E moiety that results in a

Periplasm

- X

-X

C-X

- X

- D

- D/X

Sec

Sorting

LolA

LolB

LolC

LolD

LolE

LolD

ATP ADP ATP ADP

Figure 3

Lipoprotein transport through the bacterial cell envelope. After their
transport via the Sec system and subsequent lipidation at the N-terminal
cysteine (C), lipoproteins bind to the ABC-transporter LolCDE, provided
they do not possess a Lol-avoidance motif, which usually is an aspartate
(D) ﬂanking the N-terminal C (C-D). Lipoproteins with another amino
acid at the

+2 position (X) are destined for the OM. Energy from ATP

hydrolysis by LolD is transferred to LolC and LolE and utilized to open
the hydrophobic LolA cavity to accommodate the lipoprotein. When the
LolA-lipoprotein complex interacts with the OM receptor LolB, the
lipoprotein is transferred to LolB and inserted into the OM. Further
transport to the outer leaﬂet of the OM, if applicable, occurs through
unknown mechanisms.

Spheroplasts:

bacterial cells of
which the outer
membrane and the
peptidoglycan layer
have been disrupted
by treatment with
EDTA and lysozyme

ABC:

ATP-binding

cassette

decrease in lipoprotein binding afﬁnity. ATP
hydrolysis is then required for transfer of the
lipoprotein from LolC/E to the periplasmic
chaperone LolA (41). The LolA-lipoprotein
complex crosses the periplasm and interacts
with an OM receptor, LolB (61). The lipopro-
tein is then transferred to LolB according
to the afﬁnity difference between LolA and
LolB. LolA and LolB are structurally similar;
both contain a hydrophobic cavity. The
LolA cavity is composed mostly of aromatic
residues, whereas the cavity in LolB is made
mostly of leucine and isoleucine residues,
which is likely to explain the difference in
lipoprotein-binding afﬁnity (64, 96).

In E. coli, all known lipoproteins face the

periplasm, but in other bacteria, most no-
tably in members of the spirochetes (12), cell
surface-exposed lipoproteins also are present.

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Whether lipoprotein transport over the OM
occurs through an extension of the Lol sys-
tem or by an unrelated transport system
is presently unclear. The only cell surface-
exposed lipoprotein studied in this respect is
the starch-disbranching enzyme pullulanase
of Klebsiella oxytoca (72). Pullulanase contains
an aspartate at position

+2, indicating that

it is not a substrate for the Lol system. In-
deed the transport of pullulanase to the cell
surface requires a type II protein secretion
apparatus. In the absence of such an appa-
ratus, pullulanase is retained in the IM (72).
Thus, in K. oxytoca, sorting between periplas-
mically and cell surface-exposed lipoproteins
takes place at the IM level. However, this is
not necessarily always the case. N. meningitidis
contains several cell surface-exposed lipopro-
teins, including LbpB and TbpB, but the se-
quenced genomes do not reveal the presence
of a type II secretion apparatus (104). Further-
more, LbpB and TbpB contain an isoleucine
and a leucine, respectively, at the

+2 posi-

tion, suggesting they are substrates for the
Lol system. Thus, these lipoproteins may be
transported to the cell surface via an exten-
sion of the Lol system, which remains to be
identiﬁed.

LIPOPOLYSACCHARIDE

Structure and Biosynthesis

LPS is a complex glycolipid exclusively
present in the outer leaﬂet of the OM of gram-
negative bacteria. It consists of a hydrophobic
membrane anchor, lipid A, substituted with
an oligosaccharide core region, which in some
bacteria (e.g., in most E. coli strains, but not in
the laboratory strain E. coli K-12 and also not
in N. meningitidis) is extended with a repeating
oligosaccharide, the O-antigen. These differ-
ent LPS constituents are synthesized at the cy-
toplasmic leaﬂet of the IM. The lipid A moi-
ety of LPS is rather well conserved among
gram-negative bacteria. It usually consists of a
β-1,6-linked d-glucosamine disaccharide car-
rying ester- and amide-linked 3-hydroxy fatty

acids at the 2, 3, 2

, and 3

positions and phos-

phate groups at the 1 and 4

positions. The

primary 3-hydroxy fatty acids may be substi-
tuted with secondary fatty acids. The lipid A
biosynthetic pathway, also known as the Raetz
pathway (74), has been characterized in detail,
mostly in E. coli and Salmonella typhimurium,
but it appears to be highly conserved among
other gram-negative bacteria (74). Remark-
ably, although LPS has so far only been de-
tected in gram-negative bacteria, homologs of
the genes encoding the lipid A biosynthetic
enzymes have also been found in sequenced
plant genomes (74).

The core oligosaccharide is much more

variable between bacterial species. In addi-
tion, a huge amount of LPS heterogeneity is
created by numerous modiﬁcations of both
the lipid A part and the core part of LPS
(73). The modifying enzymes, which are lo-
calized in different cellular compartments, are
not generally conserved; so different species
can express uniquely modiﬁed types of LPS
(103). The O-antigen, if present, is the most
variable part of LPS and shows even a high
degree of variability between different strains
of the same species.

Transport to the Cell Surface

In contrast to the understanding of its biosyn-
thesis, the mechanism of the transport of LPS
from its site of synthesis to its ﬁnal destina-
tion forms a much less complete picture (25).
It is clear that the lipid A–core moiety and
the O-antigen subunits, if present, are trans-
ported separately over the IM. The O-antigen
subunits are transferred over the IM by one
of three different routes: the Wzy-, ABC-
transporter-, or synthase-dependent pathway
(74). After polymerization, the subsequent lig-
ation of the O-antigen to the lipid A–core
moiety at the periplasmic side of the IM is
an incompletely understood process that in-
volves at least the WaaL ligase (45).

The translocation of the lipid A–core moi-

ety over the IM is mediated by an ABC fam-
ily transporter called MsbA, as inferred from

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the accumulation of LPS in the IM of a
temperature-sensitive E. coli msbA mutant at
the restrictive temperature (116). The LPS
accumulated was not modiﬁed by periplas-
mic enzymes, demonstrating that it was not
transported to the periplasmic leaﬂet of the
IM (26). More evidence for a role of MsbA
in LPS transport came from a study of an
msbA mutant of N. meningitidis. The viabil-
ity of LPS-deﬁcient N. meningitidis mutants
makes this organism well suited for the study
of LPS biogenesis, because clean knockouts
of genes involved in this process can be con-
structed. Indeed, MsbA is a nonessential pro-
tein in N. meningitidis (99). A neisserial msbA
mutant produced only low amounts of LPS, a
feature indicative of a defect in LPS transport
in this species. In N. meningitidis, biosynthe-
sis and transport of LPS are coupled in such a
way that synthesis is reduced under conditions
in which transport is halted (9, 99).

The subsequent steps in LPS transport

to the exterior of the bacterium have long
remained obscure. However, an OM compo-
nent required for the appearance of LPS at the
bacterial cell surface was identiﬁed recently.
This component is an OMP known as Imp
or OstA (organic solvent tolerance), because
E. coli strains expressing mutant versions of
this protein showed altered membrane per-
meability (1, 80). Imp is an essential protein in
E. coli. In a conditional imp mutant, correctly
folded OMPs accumulate in aberrant mem-
branes with an increased density, indicative
of an altered lipid/protein ratio (11). The
precise role of Imp was demonstrated in
N. meningitidis. Imp was not essential in this
bacterial species, allowing the construction
of an imp deletion mutant. The phenotype
of this mutant demonstrated a role for Imp
in LPS biogenesis: It produced less than
10% of wild-type levels of LPS, which were
not accessible to LPS-modifying enzymes
recombinantly expressed in the OM or added
to the extracellular medium (9). Therefore,
Imp appears to function in the transport of
LPS over the OM to the cell surface. This
role of Imp was conﬁrmed in an E. coli imp

depletion strain. Upon depletion of Imp,
newly synthesized LPS was not accessible to
LPS-modifying enzymes in the OM, showing
that it did not reach the outer leaﬂet of the
OM (115). In E. coli, another OM component
was identiﬁed to play a role in LPS transport,
i.e., the essential lipoprotein RlpB. Depletion
of this lipoprotein resulted in a phenotype
similar to that expressed upon depletion of
Imp. Moreover, Imp and RlpB exist in a com-
plex. Depletion of Imp or RlpB resulted in
an increased total cellular LPS content (115),
indicating that in E. coli, in contrast to N.
meningitidis, defective transport does not lead
to feedback inhibition on LPS biosynthesis.

Imp and RlpB homologs are widely dis-

seminated among gram-negative bacteria,
suggesting that the mechanism of LPS trans-
port is highly conserved. Imp is predicted to
contain a β-barrel domain embedded in the
OM, with a long N-terminal domain and a
short C-terminal domain extending into the
periplasm (Figure 4). In the conserved do-
main database at the National Center for
Biotechnology Information (59), the β-barrel
domain is recognized as a conserved domain,
designated OstA-C, in all Imp homologs. An-
other conserved domain, COG1934, is recog-
nized in the N-terminal periplasmic extension
of many but not all Imp homologs (Figure 4).
Nevertheless, all Imp homologs show se-
quence similarity over the entire length, and
also when the COG1934 domain is not
recognized.

Additional components putatively in-

volved in LPS translocation were identiﬁed
by Sperandeo et al. (86), who discovered sev-
eral new essential genes in E. coli, some of
which appeared to play a role in cell enve-
lope biogenesis. Depletion of recombinant
bacteria for the proteins encoded by two of
these genes resulted in similar phenotypes as
described for Imp- and RlpB-depleted cells:
The bacteria exhibited an altered OM density
and an increased cellular LPS content. The
genes, which form an operon, were designated
lptA and lptB (LPS transport) (85). Unfortu-
nately, we cannot use the same designations in

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Imp

YrbK

102

195

319

214

LptA/
NMB0355

Acidobacteria bacterium
Ellin345

Nostoc

sp. PCC712

Acinetobacter

sp. ADP1

COG1934

COG1934

COG1934

COG3117

OstA_C

Figure 4

Conserved protein domains present in LPS transport components. The Conserved Domain Database at
the National Center for Biotechnology Information was searched in December 2006 with the proteins
shown at the left. The type and number (#) of retrieved architectures are shown. The species expressing
the most unusual architectures are indicated at the right. A topology model for Imp, based on manual
inspection for putative β-strands, is shown at the top right-hand corner.

N. meningitidis, for which the acronym lptA is
used for a gene encoding an LPS phospho-
ethanolamine transferase (17).

Also the neisserial homolog of E. coli

LptA, encoded by the NMB0355 locus in
N. meningitidis strain MC58, plays a role in
LPS transport: In these bacteria, the cor-
responding gene is not essential, and its
deletion results in severely decreased lev-
els of LPS (10). The LPS is accessible to
periplasmic LPS-modifying enzymes in this
mutant, indicating that the LptA/NMB0355
protein acts at a step after translocation by
MsbA (10). The LptA protein of E. coli was
found in the soluble periplasmic fraction (85),
but we found the majority of the corre-
sponding neisserial protein (NMB0355) in
the membrane fraction, although it has no
obvious membrane-spanning segments (M.P.
Bos & J. Tommassen, unpublished obser-
vations). LptA largely consists of the con-
served domain COG1934, the same domain
found in the N-terminal periplasmic domain

of Imp (Figure 4), indicative of a common
function.

LptB is a 27-kDa protein present in a

140-kDa IM complex; unfortunately, no in-
teracting partners were identiﬁed (93). The
protein possesses the typical features of an
ABC protein but has no obvious membrane-
spanning segments.

Thus, the current data suggest the involve-

ment of a novel ABC transporter in LPS trans-
port. LptB is the ABC component of this
transporter, but the cognate integral mem-
brane component remains to be identiﬁed.
The protein encoded by the yrbK gene, which
is located immediately upstream of the lptAB
operon and which is also essential for viability
in E. coli (86), may be a part of the transporter.
The genetic organization of the yrbK-lptA-
lptB locus is highly conserved among gram-
negative bacteria, and the observation that a
conserved protein domain present in YrbK
is sometimes present in one polypeptide to-
gether with conserved domains from Imp or

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LptA (Figure 4) is suggestive for a role of
YrbK in LPS transport. However, secondary
structure predictions of YrbK show only one
putative transmembrane helix, making it un-
likely that this protein functions as the integral
membrane component of the ABC trans-
porter, as such components of ABC trans-
porters usually contain multiple transmem-
brane helices (6).

Models for LPS Transport

Two models for LPS transport through the
cell envelope have been considered, and
with the current situation, no model can be
considered deﬁnitively proven or discounted
(Figure 5). One possibility is that LPS passes

through the periplasm in a soluble complex
with a chaperone that shields its hydrophobic
moiety, similar to the Lol system for lipopro-
tein transport (Figure 3). Indeed, the recent
identiﬁcation of a novel ABC transporter in-
volved in LPS transport may suggest sim-
ilarities with the lipoprotein transport sys-
tem. The LptA protein, which is a soluble
periplasmic protein in E. coli (86), may func-
tion as the LPS chaperone, as LolA does for
lipoproteins. Furthermore, while LolA passes
its cargo to the structurally related OM recep-
tor LolB, LptA may pass the LPS molecules
to the periplasmic N-terminal domain of Imp,
which shows sequence similarity to LptA. The
β-barrel of Imp may form a channel for fur-
ther transport to the cell surface. Secondary

Periplasm

Membrane contact sites

Lol system-like

Imp

MsbA

Imp

RlpB

LptA

LptB

YrbK

RlpB

ATP

ADP

ATP

ADP

ATP

ADP

ATP

ADP

Figure 5

Models for LPS transport through the bacterial cell envelope. LPS is synthesized at the inner leaﬂet of
the IM and transported over the IM by the ABC transporter MsbA. LPS then travels through the
periplasm by an unknown mechanism that could resemble the Lol system (right). LptB together with an
unknown (?) membrane protein and possibly YrbK may form an ABC transporter that delivers LPS to
the periplasmic chaperone LptA. LPS may then be transferred from LptA to the periplasmic domain of
Imp, which may have a structure similar to LptA. Alternatively, LPS transport may take place at contact
sites between the two membranes (left). YrbK, LptA, LptB, and an unknown transmembrane domain may
function in the formation of these contact sites. Finally, Imp and RlpB are required to transfer LPS to the
cell surface, perhaps by acting as a ﬂippase complex.

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structure predictions for LptA show many
β-strands, possibly forming a soluble beta-
barrel, resembling LolA (10, 96). The LptB
protein could be the functional LolD ho-
molog of the LPS transport system. As ex-
plained above, the putative LolC/E compo-
nents of such an LPS transport system remain
to be found.

The other model postulates that LPS ac-

tually never leaves its membranous environ-
ment and that it is transported at contact
sites between the IM and OM (Figure 5).
The ﬁrst indication for the existence of such
sites, known as zones of adhesion or Bayer
junctions, came from electron microscopy
studies (4), although ﬁxation procedures may
have affected the results (46). Later, M ¨uhlradt
et al. (63) reported that newly synthesized
LPS appears in patches in the OM, close
to the membrane contact sites. Membrane
fractionation studies showed the existence
of a minor fraction, designated OM

, that

contains IM, peptidoglycan, and OM. This
membrane fraction, which contained pepti-
doglycan biosynthesis activity, may represent
membrane contact sites. Pulse-chase exper-
iments combined with fractionation proce-
dures showed that newly synthesized LPS
transiently passed through this fraction on its
way to the OM (40). Furthermore, when a
similar approach was used that led to the iden-
tiﬁcation of LolA (see above), newly synthe-
sized LPS could not be released from sphero-
plasts upon addition of periplasmic extracts.
Rather, LPS transport from IM to OM con-
tinued in the spheroplasts, suggesting that this
process does not involve a soluble periplas-
mic component and proceeds via contact sites
(100). In this model, the LptA, LptB, and
YrbK components may have a role in the for-
mation of these contact sites (Figure 5).

PHOSPHOLIPIDS

The major OM phospholipids of E. coli are
phosphatidylethanolamine and phosphatidyl-
glycerol. Phospholipids are synthesized at the
cytoplasmic side of the IM (18, 39). Then, in

order to reach the OM, they ﬁrst need to ro-
tate (ﬂip-ﬂop) over the membrane. It is not
clear whether a dedicated ﬂippase is neces-
sary for this process. The LPS transporter
MsbA was also implicated in phospholipid
transport because the conditional E. coli msbA
mutant accumulated both LPS and phospho-
lipids in the IM under restrictive conditions
(116). However, an msbA mutant of N. menin-
gitidis appeared viable and still made a dou-
ble membrane, showing that at least in this
bacterium MsbA is not required for phospho-
lipid transport (99). Another distant msbA ho-
molog in N. meningitidis, i.e., the NMB0264
locus in strain MC58, could be disrupted
without causing any obvious phenotype (M.P.
Bos, unpublished observations). Moreover,
various α-helical membrane-spanning pep-
tides, but curiously not MsbA, induced phos-
pholipid translocation in synthetic lipid bi-
layers (50). Thus, ﬂip-ﬂop of phospholipids
may not require a speciﬁc transporter but
merely the presence of the typical α-helical
membrane-spanning segments of some IM
proteins. The next steps in phospholipid bio-
genesis, i.e., transport through the periplasm
and incorporation into the inner leaﬂet of the
OM, remain obscure. Unlike LPS transport,
the transport of phospholipids was halted in
spheroplasts, and unlike lipoproteins, newly
synthesized phospholipids could not be re-
leased from the spheroplasts upon addition of
a periplasmic extract (100). Thus, the trans-
port mechanism appears different from those
of LPS and lipoproteins. Any components in-
volved in phospholipid transport remain to be
identiﬁed.

PERSPECTIVES

In the past few years, much progress has
been made in the ﬁeld of OM biogenesis
with the identiﬁcation of many new compo-
nents involved in the process. The ﬁeld will
rapidly move forward, gaining mechanistic
insights to which structural analysis of the
newly identiﬁed components by X-ray crystal-
lography will make important contributions.

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The major players required for OMP assem-
bly have likely been identiﬁed. Provided that
no energy-coupling system is required and
that protein folding and partitioning into the
hydrophobic environment of the membrane
are the driving forces, it may be possible to
set up an in vitro system for OMP assembly
with puriﬁed components. For LPS, a major
issue remains how it is transported through
the periplasm. Studying the binding of LPS
to the components together with immuno-
gold electron microscopy studies to deter-
mine whether these components are associ-
ated with the contact sites between IM and
OM will help to address these questions. For
lipoproteins, an important issue is how such
molecules are transported to the cell surface.
Research on the transport of phospholipids
to the OM has to start more or less from the
beginning.

Importantly, much progress in the ﬁeld has

been reached by studies in two model organ-
isms, E. coli and N. meningitidis. These stud-

ies have revealed similarities as well as differ-
ences. For example, whereas an LPS transport
defect in N. meningitidis results in feedback in-
hibition of its synthesis, this is not the case in
E. coli. For OMP assembly, the reverse is true:
An OMP assembly defect leads to feedback
inhibition in E. coli, but not in N. meningitidis.
Such differences make it attractive to study
speciﬁc aspects of OM biogenesis in different
organisms. In addition, considering the dif-
ferences already observed between these two
model organisms, it is likely that studies in
other bacteria will uncover new, unanticipated
features.

Further studies in this ﬁeld will remain

important, because they will uncover funda-
mental biological processes. In addition, the
knowledge gained from these studies may be
useful for medical applications: The essential
nature of the bacterial machineries involved
and their surface localization make them at-
tractive targets for the development of new
antimicrobial drugs and vaccines.

SUMMARY POINTS

1. OMPs and lipoproteins are transported across the IM via the Sec system.

2. Assembly of bacterial outer membrane proteins requires the outer membrane protein

Omp85, which is evolutionary conserved and found even in the OM of mitochondria.

3. Omp85 recognizes its substrate OMPs by virtue of their C-terminal signature

sequences.

4. Other proteins involved in OMP transport and assembly are the periplasmic chap-

erones Skp and SurA, and the OM-associated lipoproteins YﬁO, YfgL, NlpB, and
SmpA, the function of which remains to be determined.

5. Transport of lipoproteins to the OM depends on the Lol system, which consists of

an ABC transporter in the IM, a soluble periplasmic chaperone, and an OM-attached
receptor.

6. MsbA is an ABC transporter required for the transport of LPS across the IM.

7. Further transport of LPS to the cell surface requires, at least, the ABC protein LptB,

the periplasmic protein LptA, the OM-attached lipoprotein RlpB, and the integral
OMP Imp.

8. Nothing is known regarding the transport of phospholipids to the OM.

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DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of
this review.

ACKNOWLEDGMENTS

The work in our laboratory is supported by grants from the Netherlands Research Council for
Earth and Life Sciences (ALW) and the Netherlands Research Council for Chemical Sciences
(CW) with ﬁnancial aid from the Netherlands Organization for Scientiﬁc Research (NWO).

LITERATURE CITED

1. Abe S, Okutsu T, Nakajima H, Kakuda N, Ohtsu I, et al. 2003. n-Hexane sensitivity

of Escherichia coli due to low expression of imp/ostA encoding an 87 kDa minor protein
associated with the outer membrane. Microbiology 149:1265–73

2. Ames GFL, Spudich EN, Nikaido H. 1974. Protein composition of the outer membrane

of Salmonella typhimurium: effect of lipopolysaccharide mutations. J. Bacteriol. 117:406–16

3. Bayan N, Guilvout I, Pugsley AP. 2006. Secretins take shape. Mol. Microbiol. 60:1–4
4. Bayer ME. 1968. Areas of adhesion between wall and membrane of Escherichia coli. J. Gen.

Microbiol. 53:395–404

5. Behrens S, Maier R, de Cock H, Schmid FX, Gross CA. 2001. The SurA periplasmic

PPIase lacking its parvulin domains functions in vivo and has chaperone activity. EMBO
J. 20:285–94

6. Biemans-Oldehinkel E, Doeven MK, Poolman B. 2006. ABC transporter architecture

and regulatory roles of accessory domains. FEBS Lett. 580:1023–35

7. Bitter W. 2003. Secretins of Pseudomonas aeruginosa: large holes in the outer membrane.

Arch. Microbiol. 179:307–14

8. Bolla JM, Lazdunski C, Pag`es JM. 1988. The assembly of the major outer membrane

protein OmpF of Escherichia coli depends on lipid synthesis. EMBO J. 7:3595–99

9. First
demonstration that
an integral OMP,
Imp, is required for
the transport of
LPS to the cell
surface.

9. Bos MP, Tefsen B, Geurtsen J, Tommassen J. 2004. Identification of an outer

membrane protein required for lipopolysaccharide transport to the bacterial cell
surface. Proc. Natl. Acad. Sci. USA 101:9417–22

10. Bos MP, Tommassen J. 2006. Identiﬁcation of LPS transport components in Neisseria

meningitidis. In Fifteenth Int. Pathogenic Neisseria Conf., ed. J Davies, M Jennings, pp. 33.
West Leederville, Australia: Cambridge Publ.

11. Braun M, Silhavy TJ. 2002. Imp/OstA is required for cell envelope biogenesis in Es-

cherichia coli. Mol. Microbiol. 45:1289–302

12. Casjens S. 2000. Borrelia genomes in the year 2000. J. Mol. Microbiol. Biotechnol. 2:401–10
13. CastilloKeller M, Misra R. 2003. Protease-deﬁcient DegP suppresses lethal effects of a

mutant OmpC protein by its capture. J. Bacteriol. 185:148–54

14. Chami M, Guilvout I, Gregorini M, Remigy HW, M ¨uller SA, et al. 2005. Structural

insights into the secretin PulD and its trypsin-resistant core. J. Biol. Chem. 280:37732–41

15. Charlson ES, Werner JN, Misra R. 2006. Differential effects of yfgL mutation on Es-

cherichia coli outer membrane proteins and lipopolysaccharide. J. Bacteriol. 188:7186–94

16. Chen R, Henning U. 1996. A periplasmic protein (Skp) of Escherichia coli binds a class of

outer membrane proteins. Mol. Microbiol. 19:1287–94

17. Cox AD, Wright JC, Li J, Hood DW, Moxon ER, Richards JC. 2003. Phosphorylation

of the lipid A region of meningococcal lipopolysaccharide: identiﬁcation of a family of

208

Bos

Robert

Tommassen

Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org

by CNRS-multi-site on 10/11/07. For personal use only.

ANRV322-MI61-11

ARI

6 August 2007

16:59

transferases that add phosphoethanolamine to lipopolysaccharide. J. Bacteriol. 185:3270–
77

18. Cronan JE. 2003. Bacterial membrane lipids: Where do we stand? Annu. Rev. Microbiol.

57:203–24

19. Dartigalongue C, Raina S. 1998. A new heat-shock gene, ppiD, encodes a peptidyl-prolyl

isomerase required for folding of outer membrane proteins in Escherichia coli. EMBO J.
17:3968–80

20. De Cock H, Sch¨afer U, Potgeter M, Demel R, M ¨uller M, et al. 1999. Afﬁnity of the

periplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides and
non-native outer membrane proteins. Role of Skp in the biogenesis of outer membrane
protein. Eur. J. Biochem. 259:96–103

21. De Cock H, Struyv´e M, Kleerebezem M, van der Krift T, Tommassen J. 1997. Role of

the carboxy-terminal phenylalanine in the biogenesis of outer membrane protein PhoE
of Escherichia coli K-12. J. Mol. Biol. 269:473–78

22. De Cock H, Tommassen J. 1996. Lipopolysaccharides and divalent cations are involved

in the formation of an assembly-competent intermediate of outer membrane protein
PhoE of E. coli. EMBO J. 15:5567–73

23. De Keyzer J, van der Does C, Driessen AJM. 2003. The bacterial translocase: a dynamic

protein channel complex. Cell Mol. Life Sci. 60:2034–52

24. Dev IK, Ray PH. 1984. Rapid assay and puriﬁcation of a unique signal peptidase that

processes the prolipoprotein from Escherichia coli B. J. Biol. Chem. 259:11114–20

25. Doerrler WT. 2006. Lipid trafﬁcking to the outer membrane of gram-negative bacteria.

Mol. Microbiol. 60:542–52

26. Doerrler WT, Gibbons HS, Raetz CRH. 2004. MsbA-dependent translocation of lipids

across the inner membrane of Escherichia coli. J. Biol. Chem. 276:45102–9

27. Doerrler WT, Raetz CRH. 2005. Loss of outer membrane proteins without inhibition

of lipid export in an Escherichia coli YaeT mutant. J. Biol. Chem. 280:27679–87

28. Dong C, Beis K, Nesper J, Brunkan-Lamontagne AL, Clarke BR, et al. 2006. Wza the

translocon for E. coli capsular polysaccharides deﬁnes a new class of membrane protein.
Nature 444:226–29

29. Eggert US, Ruiz N, Falcone BV, Branstrom AA, Goldman RC, et al. 2001. Genetic basis

for activity differences between vancomycin and glycolipid derivatives of vancomycin.
Science 294:361–64

30. Eppens EF, Nouwen N, Tommassen J. 1997. Folding of a bacterial outer membrane

protein during passage through the periplasm. EMBO J. 16:4295–301

31. Figueroa-Bossi N, Lemire S, Maloriol D, Balbontin R, Casadesus J, et al. 2006. Loss

of Hfq activates the σ

-dependent envelope stress response in Salmonella enterica. Mol.

Microbiol. 62:838–52

32. Fussenegger M, Facius D, Meier J, Meyer TF. 1996. A novel peptidoglycan-linked

lipoprotein (ComL) that functions in natural transformation competence of Neisseria
gonorrhoeae. Mol. Microbiol. 19:1095–105

33. Gentle I, Gabriel K, Beech P, Waller R, Lithgow T. 2004. The Omp85 family of proteins

is essential for outer membrane biogenesis in mitochondria and bacteria. J. Cell Biol.
164:19–24

34. Geyer R, Galanos C, Westphal O, Golecki JR. 1979. A lipopolysaccharide-binding cell-

surface protein from Salmonella minnesota. Isolation, partial characterization and occur-
rence in different Enterobacteriaceae. Eur. J. Biochem. 98:27–38

www.annualreviews.org

•

Outer Membrane Biogenesis

209

Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org

by CNRS-multi-site on 10/11/07. For personal use only.

ANRV322-MI61-11

ARI

6 August 2007

16:59

35. Gunesekere IC, Kahler CM, Ryan CS, Snyder LA, Saunders NJ, et al. 2006. Ecf, an

alternative sigma factor from Neisseria gonorrhoeae, controls expression of msrAB, which
encodes methionine sulfoxide reductase. J. Bacteriol. 188:3463–69

36. Gupta SD, Gan K, Schmid MB, Wu HC. 1993. Characterization of a temperature-

sensitive mutant of Salmonella typhimurium defective in apolipoprotein N-acyltransferase.
J. Biol. Chem. 268:16551–56

37. Harms N, Koningstein G, Dontje W, Muller M, Oudega B, et al. 2001. The early inter-

action of the outer membrane protein PhoE with the periplasmic chaperone Skp occurs
at the cytoplasmic membrane. J. Biol. Chem. 276:18804–11

38. Hennecke G, Nolte J, Volkmer-Engert R, Schneider-Mergener J, Behrens S. 2005. The

periplasmic chaperone SurA exploits two features characteristic of integral outer mem-
brane proteins for selective substrate recognition. J. Biol. Chem. 280:23540–48

39. Huijbrechts RPH, de Kroon AIPM, de Kruijff B. 2000. Topology and transport of mem-

brane lipids in bacteria. Biochim. Biophys. Acta 1469:43–61

40. Ishidate K, Creeger E, Zrike J, Deb S, Glauner B, et al. 1986. Isolation of differentiated

membrane domains from Escherichia coli and Salmonella typhimurium, including a fraction
containing attachment sites between the inner and outer membrane and the murein
skeleton of the cell envelope. J. Biol. Chem. 261:428–43

41. Ito Y, Kanamaru K, Taniguchi N, Miyamoto S, Tokuda H. 2006. A novel ligand bound

ABC transporter, LolCDE, provides insights into the molecular mechanisms underlying
membrane detachment of bacterial lipoproteins. Mol. Microbiol. 62:1064–75

42. Jansen C, Heutink M, Tommassen J, de Cock H. 2000. The assembly pathway of outer

membrane protein PhoE of Escherichia coli. Eur. J. Biochem. 267:3792–800

43. Johansen J, Rasmussen AA, Overgaard M, Valentin-Hansen P. 2006. Conserved small

noncoding RNAs that belong to the σ

regulon: role in down-regulation of outer mem-

brane proteins. J. Mol. Biol. 364:1–8

44. Justice SS, Hunstad DA, Harper JR, Duguay AR, Pinkner JS, et al. 2005. Periplasmic

peptidyl prolyl cis-trans isomerases are not essential for viability, but SurA is required for
pilus biogenesis in Escherichia coli. J. Bacteriol. 187:7680–86

45. Kaniuk NA, Vinogradov E, Whitﬁeld C. 2004. Investigation of the structural require-

ments in the lipopolysaccharide core acceptor for ligation of O antigens in the genus
Salmonella: WaaL “ligase” is not the sole determinant of acceptor speciﬁcity. J. Biol.
Chem. 279:36470–80

46. Kellenberger E. 1990. The ‘Bayer bridges’ confronted with results from improved elec-

tron microscopy methods. Mol. Microbiol. 4:697–705

47. Kleerebezem M, Heutink M, Tommassen J. 1995. Characterization of an Escherichia coli

rotA mutant, affected in periplasmic peptidyl-prolyl cis/trans isomerase. Mol. Microbiol.
18:313–20

48. Klose M, Schwarz H, MacIntyre S, Freudl R, Eschbach ML, et al. 1988. Internal deletions

in the gene for an Escherichia coli outer membrane protein deﬁne an area possibly important
for recognition of the outer membrane by this polypeptide. J. Biol. Chem. 263:13291–96

49. Koebnik R, Locher KP, Van Gelder P. 2000. Structure and function of bacterial outer

membrane proteins: barrels in a nutshell. Mol. Microbiol. 37:239–53

50. Kol M, van Dalen A, de Kroon AIPM, de Kruijff B. 2003. Translocation of phospholipids

is facilitated by a subset of membrane-spanning proteins of the bacterial cytoplasmic
membrane. J. Biol. Chem. 278:24586–93

51. Koplow J, Goldﬁne H. 1974. Alterations in the outer membrane of the cell envelope of

heptose-deﬁcient mutants of Escherichia coli. J. Bacteriol. 181:527–43

210

Bos

Robert

Tommassen

Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org

by CNRS-multi-site on 10/11/07. For personal use only.

ANRV322-MI61-11

ARI

6 August 2007

16:59

52. Kornd ¨orfer IP, Dommel MK, Skerra A. 2004. Structure of the periplasmic chaperone

Skp suggests functional similarity with cytosolic chaperones despite different architecture.
Nat. Struct. Mol. Biol. 11:1015–20

53. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C. 2000. Crystal structure of the

bacterial membrane protein TolC central to multidrug efﬂux and protein export. Nature
405:914–19

54. Kozjak V, Wiedemann N, Milenkovic D, Lohaus C, Meyer HE, et al. 2003. An essential

role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer
membrane. J. Biol. Chem. 278:48520–23

55. Laird MW, Kloser AW, Misra R. 1994. Assembly of LamB and OmpF in deep rough

lipopolysaccharide mutants of Escherichia coli K-12. J. Bacteriol. 176:2259–64

56. Lazar SW, Kolter R. 1996. SurA assists the folding of Escherichia coli outer membrane

proteins. J. Bacteriol. 178:1770–73

57. Lee PA, Tullman-Ercek D, Georgiou G. 2006. The bacterial twin-arginine translocation

pathway. Annu. Rev. Microbiol. 60:373–95

58. Malinverni JC, Werner J, Kim S, Sklar JG, Kahne D, et al. 2006. YﬁO stabilizes the YaeT

complex and is essential for outer membrane protein assembly in Escherichia coli. Mol.
Microbiol. 61:151–64

59. Marchler-Bauer A, Anderson JB, Cherukuri PF, DeWeese-Scott C, Geer LY, et al.

2005. CDD: a Conserved Domain Database for protein classiﬁcation. Nucleic Acids Res.
33:D192–96

60. Matsuyama S, Tajima T, Tokuda H. 1995. A novel carrier protein involved in the sorting

and transport of Escherichia coli lipoproteins destined for the outer membrane. EMBO J.
14:3365–72

61. Matsuyama S, Yokota N, Tokuda H. 1997. A novel outer membrane lipoprotein, LolB

(HemM), involved in the LolA (p20)-dependent localization of lipoproteins to the outer
membrane of Escherichia coli. EMBO J. 16:6947–55

62. Missiakas D, Betton JM, Raina S. 1996. New components of protein folding in extracy-

toplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH. Mol. Microbiol.
21:871–84

63. M ¨uhlradt PF, Menzel J, Golecki JR, Speth V. 1973. Outer membrane of Salmonella. Sites

of export of newly synthesised lipopolysaccharide on the bacterial surface. Eur. J. Biochem.
35:471–81

64. Narita S, Matsuyama S, Tokuda H. 2004. Lipoprotein trafﬁcking in Escherichia coli. Arch.

Microbiol. 182:1–6

65. Narita S, Tokuda H. 2006. An ABC transporter mediating the membrane detachment of

bacterial lipoproteins depending on their sorting signals. FEBS Lett. 580:1164–70

66. Nikaido H. 2003. Molecular basis of bacterial outer membrane permeability revisited.

Microbiol. Mol. Biol. Rev. 67:593–656

67. Onufryk C, Crouch ML, Fang FC, Gross CA. 2005. Characterization of six lipoproteins

in the σ

regulon. J. Bacteriol. 187:4552–61

68. Papenfort K, Pfeiffer V, Mika F, Lucchini S, Hinton JCD, et al. 2006. σ

-dependent

small RNAs of Salmonella respond to membrane stress by accelerating global omp mRNA
decay. Mol. Microbiol. 62:1674–88

69. Paschen SA, Waizenegger T, Stan T, Preuss M, Cyrklaff M, et al. 2003. Evolutionary

conservation of biogenesis of β-barrel membrane proteins. Nature 426:862–66

70. Pettersson A, Poolman JT, van der Ley P, Tommassen J. 1997. Response of Neisseria

meningitidis to iron limitation. Antonie van Leeuwenhoek 71:129–36

www.annualreviews.org

•

Outer Membrane Biogenesis

211

Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org

by CNRS-multi-site on 10/11/07. For personal use only.

ANRV322-MI61-11

ARI

6 August 2007

16:59

71. Prinz T, Tommassen J. 2000. Association of iron-regulated outer membrane proteins of

Neisseria meningitidis with the RmpM (class 4) protein. FEMS Microbiol. Lett. 183:49–53

72. Pugsley AP. 1993. The complete general secretory pathway in gram-negative bacteria.

Microbiol. Rev. 57:50–108

73. Raetz CRH, Reynolds CM, Trent MS, Bishop RE. 2007. Lipid A modiﬁcation systems

in gram-negative bacteria. Annu. Rev. Biochem. 76:295–329

74. Raetz CRH, Whitﬁeld C. 2002. Lipopolysaccharide endotoxins. Annu. Rev. Biochem.

71:635–700

75. Rizzitello AE, Harper JR, Silhavy TJ. 2001. Genetic evidence for parallel pathways of

chaperone activity in the periplasm of Escherichia coli. J. Bacteriol. 183:6794–800

76. Demonstrates
that the C-terminal
signature sequence
of OMPs functions
as a targeting
factor, recognized
by assembly factor
Omp85.

76. Robert V, Volokhina EB, Senf F, Bos MP, Van Gelder P, et al. 2006. Assembly

factor Omp85 recognizes its outer membrane protein substrates by a species-
specific C-terminal motif. PLoS Biol. 4:1984–95

77. Rouvi`ere PE, Gross CA. 1996. SurA, a periplasmic protein with peptidyl-prolyl isomerase

activity, participates in the assembly of outer membrane porins. Genes Dev. 10:3170–87

78. Ruiz N, Falcone B, Kahne D, Silhavy TJ. 2005. Chemical conditionality: a genetic strategy

to probe organelle assembly. Cell 121:307–17

79. Ruiz N, Silhavy TJ. 2005. Sensing external stress: watchdogs of the Escherichia coli cell

envelope. Curr. Opin. Microbiol. 8:122–26

80. Sampson BA, Misra R, Benson SA. 1989. Identiﬁcation and characterization of a new gene

of Escherichia coli K-12 involved in outer membrane permeability. Genetics 122:491–501

81. Sanchez-Pulido L, Devos D, Genevrois S, Vincente M, Valencia A. 2003. POTRA: a

conserved domain in the FtsQ family and a class of β-barrel outer membrane proteins.
Trends Biochem. Sci. 28:523–26

82. Sankaran K, Wu HC. 1994. Lipid modiﬁcation of bacterial prolipoprotein. Transfer of

diacylglyceryl moiety from phosphatidylglycerol. J. Biol. Chem. 269:19701–6

83. Sen K, Nikaido H. 1990. In vivo trimerization of OmpF porin secreted by spheroplasts

of Escherichia coli. Proc. Natl. Acad. Sci. USA 87:743–47

84. Sklar JG, Wu T, Gronenberg LS, Malinverni JC, Kahne D, et al. 2007. Lipoprotein

SmpA is a component of the YaeT complex that assembles outer membrane proteins in
Escherichia coli. Proc. Natl. Acad. Sci. USA 104:6400–5

85. Identifies an
ABC protein and a
periplasmic protein
as new factors
required for the
transport of LPS to
the cell surface.

85. Sperandeo P, Cescutti R, Villa R, Di Benedetto C, Candia D, et al. 2007. Char-

acterization of lptA and lptB, two essential genes implicated in lipopolysaccharide
transport to the outer membrane of Escherichia coli. J. Bacteriol. 189:244–53

86. Sperandeo P, Pozzi C, Deho G, Polissi A. 2006. Non-essential KDO biosynthesis and

new essential cell envelope biogenesis genes in the Escherichia coli yrbG-yhbG locus. Res.
Microbiol. 157:547–58

87. Spies C, Beil A, Ehrmann M. 1999. A temperature-dependent switch from chaperone to

protease in a widely conserved heat shock protein. Cell 97:339–47

88. Steeghs L, Berns M, ten Hove J, de Jong A, Roholl P, et al. 2002. Expression of foreign

LpxA acyltransferases in Neisseria meningitidis results in modiﬁed lipid A with reduced
toxicity and retained adjuvant activity. Cell. Microbiol. 4:599–611

89. Steeghs L, de Cock H, Evers E, Zomer B, Tommassen J, et al. 2001. Outer membrane

composition of a lipopolysaccharide-deﬁcient Neisseria meningitidis mutant. EMBO J.
20:6937–45

90. First
demonstration that
N. meningitidis is
viable without LPS,
making this
organism suitable
for further studies
on LPS transport.

90. Steeghs L, den Hartog R, den Boer A, Zomer B, Roholl P, et al. 1998. Meningitis

bacterium is viable without endotoxin. Nature 392:449–50

91. Stegmeier JF, Andersen C. 2006. Characterization of pores formed by YaeT (Omp85)

from Escherichia coli. J. Biochem. 140:275–83

212

Bos

Robert

Tommassen

Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org

by CNRS-multi-site on 10/11/07. For personal use only.

ANRV322-MI61-11

ARI

6 August 2007

16:59

92. Stegmeier JF, Gl ¨uck A, Sukumaran S, M¨antele W, Andersen C. 2007. Characterization

of YtfM, a second member of the Omp85 family in Escherichia coli. Biol. Chem. 388:37–46

93. Stenberg F, Chovanec P, Maslen SL, Robinson CV, Ilag LL, et al. 2005. Protein com-

plexes of the Escherichia coli cell envelope. J. Biol. Chem. 280:34409–19

94. Struyv´e M, Moons M, Tommassen J. 1991. Carboxy-terminal phenylalanine is essential

for the correct assembly of a bacterial outer membrane protein. J. Mol. Biol. 218:141–48

95. Surana NK, Grass S, Hardy GG, Li H, Thanassi DG, et al. 2004. Evidence for conserva-

tion of architecture and physical properties of Omp85-like proteins throughout evolution.
Proc. Natl. Acad. Sci. USA 101:14497–503

96. Provides
high-resolution
structures of LolA
and LolB,
providing insights
into the lipoprotein
transport
mechanism.

96. Takeda K, Miyatake H, Yokota N, Matsuyama S, Tokuda H, et al. 2003. Crystal

structures of bacterial lipoprotein localization factors, LolA and LolB. EMBO J.
22:3199–209

97. Tam C, Missiakas D. 2005. Changes in lipopolysaccharide structure induce the σ

dependent response of Escherichia coli. Mol. Microbiol. 55:1403–12

98. Tamm LK, Arora A, Kleinschmidt JH. 2001. Structure and assembly of β-barrel mem-

brane proteins. J. Biol. Chem. 276:32399–402

99. Tefsen B, Bos MP, Beckers F, Tommassen J, de Cock H. 2005. MsbA is not required for

phospholipid transport in Neisseria meningitidis. J. Biol. Chem. 280:35961–66

100. Tefsen B, Geurtsen J, Beckers F, Tommassen J, de Cock H. 2005. Lipopolysaccharide

transport to the bacterial outer membrane in spheroplasts. J. Biol. Chem. 280:4504–9

101. Terada M, Kuroda T, Matsuyama S, Tokuda H. 2001. Lipoprotein-sorting signals eval-

uated as the LolA-dependent release of lipoproteins from the cytoplasmic membrane of
Escherichia coli. J. Biol. Chem. 276:47690–94

102. Tormo A, Almir ´on M, Kolter R. 1990. surA, an Escherichia coli gene essential for survival

in stationary phase. J. Bacteriol. 172:4339–47

103. Trent MS, Stead CM, Tran AX, Hankins JV. 2006. Diversity of endotoxin and its impact

on pathogenesis. J. Endotoxin Res. 12:205–23

104. Van Ulsen P, Tommassen J. 2006. Protein secretion and secreted proteins in pathogenic

Neisseriaceae. FEMS Microbiol. Rev. 30:292–319

105. First
demonstration that
an integral OMP,
Omp85, is required
for the assembly of
OMPs.

105. Voulhoux R, Bos MP, Geurtsen J, Mols M, Tommassen J. 2003. Role of a highly

conserved bacterial protein in outer membrane protein assembly. Science 299:262–
65

106. Voulhoux R, Tommassen J. 2004. Omp85, an evolutionarily conserved bacterial protein

involved in outer-membrane-protein assembly. Res. Microbiol. 155:129–35

107. Waller PR, Sauer RT. 1996. Characterization of degQ and degS, Escherichia coli genes

encoding homologs of the DegP protease. J. Bacteriol. 178:1146–53

108. Demonstrates
that the C-terminal
signature sequence
of OMPs triggers
the σ

-dependent

periplasmic stress
response when
unfolded OMPs
accumulate in the
periplasm.

108. Walsh NP, Alba BM, Bose B, Gross CA, Sauer RT. 2003. OMP peptide signals

initiate the envelope-stress response by activating DegS protease via relief of in-
hibition mediated by its PDZ domain. Cell 113:61–71

109. Walton TA, Sousa MC. 2004. Crystal structure of Skp, a prefoldin-like chaperone that

protects soluble and membrane proteins from aggregation. Mol. Cell 15:367–74

110. Wandersman C, Delepelaire P. 2004. Bacterial iron sources: from siderophores to

hemophores. Annu. Rev. Microbiol. 58:611–47

111. Werner J, Augustus AM, Misra R. 2003. Assembly of TolC, a structurally unique and

multifunctional outer membrane protein of Escherichia coli K-12. J. Bacteriol. 185:6540–
47

112. Werner J, Misra R. 2005. YaeT (Omp85) affects the assembly of lipid-dependent and

lipid-independent outer membrane proteins of Escherichia coli. Mol. Microbiol. 57:1450–
59

www.annualreviews.org

•

Outer Membrane Biogenesis

213

Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org

by CNRS-multi-site on 10/11/07. For personal use only.

ANRV322-MI61-11

ARI

6 August 2007

16:59

113. White DA, Barlow AK, Clarke IN, Heckels JE. 1990. Stable expression of meningococcal

class I protein in an antigenically reactive form in outer membranes of Escherichia coli.
Mol. Microbiol. 4:769–76

114. Identifies
three additional
components of the
Omp85 complex
required for OMP
assembly.

114. Wu T, Malinverni J, Ruiz N, Kim S, Silhavy TJ, et al. 2005. Identification of a

multicomponent complex required for outer membrane biogenesis in Escherichia
coli. Cell 121:235–45

115. Identifies
RlpB as an
additional
component of the
Imp complex
required for
transport of LPS to
the cell surface.

115. Wu T, McCandlish AC, Gronenberg LS, Chng SS, Silhavy TJ, et al. 2006. Identi-

fication of a protein complex that assembles lipopolysaccharide in the outer mem-
brane of Escherichia coli. Proc. Natl. Acad. Sci. USA 103:11754–59

116. Zhou Z, White K, Polissi A, Georgopoulos C, Raetz CRH. 1998. Function of Escherichia

coli MsbA, an essential ABC family transporter, in lipid A and phospholipid biosynthesis.
J. Biol. Chem. 273:12466–75

214

Bos

Robert

Tommassen

Annu. Rev. Microbiol. 2007.61:191-214. Downloaded from arjournals.annualreviews.org

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Annual Review of
Microbiology

Volume 61, 2007

Contents

Frontispiece

Margarita Salas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p xiv

40 Years with Bacteriophage

Margarita Salas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1

The Last Word: Books as a Statistical Metaphor for Microbial Communities

Patrick D. Schloss and Jo Handelsman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 23

The Mechanism of Isoniazid Killing: Clarity Through the Scope

of Genetics

Catherine Vilchèze and William R. Jacobs, Jr. p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 35

Development of a Combined Biological and Chemical Process for

Production of Industrial Aromatics from Renewable Resources

F. Sima Sariaslani p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 51

The RNA Degradosome of Escherichia coli: An mRNA-Degrading

Machine Assembled on RNase E

Agamemnon J. Carpousis p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 71

Protein Secretion in Gram-Negative Bacteria via the Autotransporter

Pathway

Nathalie Dautin and Harris D. Bernstein p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 89

Chlorophyll Biosynthesis in Bacteria: The Origins of Structural and

Functional Diversity

Aline Gomez Maqueo Chew and Donald A. Bryant p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113

Roles of Cyclic Diguanylate in the Regulation of Bacterial Pathogenesis

Rita Tamayo, Jason T. Pratt, and Andrew Camilli p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p131

Aggresomes and Pericentriolar Sites of Virus Assembly:

Cellular Defense or Viral Design?

Thomas Wileman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p149

As the Worm Turns: The Earthworm Gut as a Transient Habitat for

Soil Microbial Biomes

Harold L. Drake and Marcus A. Horn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p169

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Biogenesis of the Gram-Negative Bacterial Outer Membrane

Martine P. Bos, Viviane Robert, and Jan Tommassen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p191

SigB-Dependent General Stress Response in Bacillus subtilis and

Related Gram-Positive Bacteria

Michael Hecker, Jan Pané-Farré, and Uwe Völker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p215

Ecology and Biotechnology of the Genus Shewanella

Heidi H. Hau and Jeffrey A. Gralnick p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237

Nonhomologous End-Joining in Bacteria: A Microbial Perspective

Robert S. Pitcher, Nigel C. Brissett, and Aidan J. Doherty p p p p p p p p p p p p p p p p p p p p p p p p p p p p259

Postgenomic Adventures with Rhodobacter sphaeroides

Chris Mackenzie, Jesus M. Eraso, Madhusudan Choudhary, Jung Hyeob Roh,
Xiaohua Zeng, Patrice Bruscella, Ágnes Puskás, and Samuel Kaplan p p p p p p p p p p p p p p p p p283

Toward a Hyperstructure Taxonomy

Vic Norris, Tanneke den Blaauwen, Roy H. Doi, Rasika M. Harshey,
Laurent Janniere, Alfonso Jim´enez-S´anchez, Ding Jun Jin,
Petra Anne Levin, Eugenia Mileykovskaya, Abraham Minsky,
Gradimir Misevic, Camille Ripoll, Milton Saier, Jr., Kirsten Skarstad,
and Michel Thellier p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p309

Endolithic Microbial Ecosystems

Jeffrey J. Walker and Norman R. Pace p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p331

Nitrogen Regulation in Bacteria and Archaea

John A. Leigh and Jeremy A. Dodsworth p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p349

Microbial Metabolism of Reduced Phosphorus Compounds

Andrea K. White and William W. Metcalf p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p379

Bioﬁlm Formation by Plant-Associated Bacteria

Thomas Danhorn and Clay Fuqua p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p401

Heterotrimeric G Protein Signaling in Filamentous Fungi

Liande Li, Sara J. Wright, Svetlana Krystofova, Gyungsoon Park,
and Katherine A. Borkovich p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p423

Comparative Genomics of Protists: New Insights into the Evolution

of Eukaryotic Signal Transduction and Gene Regulation

Vivek Anantharaman, Lakshminarayan M. Iyer, and L. Aravind p p p p p p p p p p p p p p p p p p p p453

Lantibiotics: Peptides of Diverse Structure and Function

Joanne M. Willey and Wilfred A. van der Donk p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p477

The Impact of Genome Analyses on Our Understanding of

Ammonia-Oxidizing Bacteria

Daniel J. Arp, Patrick S.G. Chain, and Martin G. Klotz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503

Contents

vii

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Morphogenesis in Candida albicans

Malcolm Whiteway and Catherine Bachewich p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529

Structure, Assembly, and Function of the Spore Surface Layers

Adriano O. Henriques and Charles P. Moran, Jr. p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p555

Cytoskeletal Elements in Bacteria

Peter L. Graumann p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p589

Indexes

Cumulative Index of Contributing Authors, Volumes 57–61

p p p p p p p p p p p p p p p p p p p p p p p p619

Cumulative Index of Chapter Titles, Volumes 57–61

p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p622

Errata

An online log of corrections to Annual Review of Microbiology articles may be found
at http://micro.annualreviews.org/

viii

Contents

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