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Biogenesis of theGram-Negative BacterialOuter 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
Copyright c 2007 by Annual Reviews. All rights reserved
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 membranes, the inner and the outer membrane, that are separated by
the periplasm. The outer membrane consists of phospholipidslipopolysaccharides, integral membrane proteins, and lipoproteins
These components are synthesized in the cytoplasm or at the inne
leaflet of the inner membrane and have to be transported across theinner 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.
191
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IM: innermembrane
OM: outermembrane
Periplasm:compartment between the innerand outer
membranesPeptidoglycan:bacterial cell wallconsisting of sugarpolymers covalently connected viaoligopeptides
Lipopolysaccharide(LPS): glycolipidconstituting theouter layer of thebacterial outermembrane
β-barrel: commonstructure of outermembrane proteins,consisting of amphipathicantiparallelβ-strands
Contents
INTRODUCTION... . . . . . . . . . . . . . . 192INTEGRAL OUTER MEMBRANE
PROTEINS . . . . . . . . . . . . . . . . . . . . . 193Passage Across the IM and
Through the Periplasm . . . . . . . . 193
Assembly into the OM . . . . . . . . . . . . 194Role of LPS in OMP Biogenesis . . 199 Model for OMP Biogenesis . . . . . . . 200
LIPOPROTEINS . . . . . . . . . . . . . . . . . . . 200LIPOPOLYSACCHARIDE . . . . . . . . . 202
Structure and Biosynthesis . . . . . . . . 202
Transport to the Cell Surface . . . . . 202 Models for LPS Transport . . . . . . . . 205
PHOSPHOLIPIDS . . . . . . . . . . . . . . . . . 206PERS PECT IV ES . . . . . . . . . . . . . . . . . . . 2 0 6
INTRODUCTION
The cell envelope of gram-negative bacteriaconsists 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 membraneshave 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 theintegral membrane proteins. Whereas inte-
gral IM proteins typically span the membranein the form of hydrophobic α -helices, inte-
gral OM proteins (OMPs) generally consist of antiparallelamphipathicβ-strandsthatfold
into cylindrical β-barrels with a hydrophilicinterior and hydrophobic residues pointing
outward to face the membrane lipids (49).Both membranes also contain lipoproteins,
which are anchored to the membranes viaan N-terminal N -acyl-diacylglycerylcysteine,
with the protein moiety usually facing theperiplasm in the case of Escherichia coli . In
other gram-negative bacteria, however, the
protein moiety of OM lipoproteins may alsoextend 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 barrierthat protects the bacteria from harmful com-pounds, such as antibiotics, in the environ-
ment. Unlike the IM, the OM is not energizedby 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 allowthe 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. Suchprocesses are dependent on complex energy-
coupling systems, such as the TonB system
which couples the proton-motive force of theIM to receptor-mediated uptake processes in
the OM (110). Whereas the composition, structure, and
function of the OM have been known fordecades, 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 throughthe periplasm to reach their destination and
to assemble into the OM. Recently, many
new components involved in these processeshave been described, and in this review we
focus on these recent developments. Apartfrom studies in the classical model organisms
E. coli and Salmonella enterica, much progressin 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 truefor E. coli is not necessarily true for other
bacteria.
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INTEGRAL OUTER MEMBRANEPROTEINS
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 translocationacross the IM. Two translocation machinerieshave been identified: the Sec system for the
translocation of unfolded proteins (23) and
the Tat system, which transports proteinsfolded in the cytoplasm (57). However, to
our knowledge, all OMPs studied to date aretransported 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 theOMPs 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 containsdecreased amounts of OMPs (16). Anotherperiplasmic chaperone, SurA, was initially
identified as a protein required for survivalduring 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). Theprotein has peptidyl-prolyl cis / trans isomerase
(PPIase) activity (56, 62), which nevertheless
appears to be dispensable for the chaperonefunction (5). Unlike most cytoplasmic chaper-ones, SurA is selective andpreferentially binds
nonnative OMPs over other proteins (5). By screening peptide libraries, Hennecke et al.
(38) demonstrated that SurA binds peptidesrich in aromatic residues and preferentially
those containing Ar-Ar or Ar-X-Ar motifs
Lipoprotein:protein attached tothe bacterial inner orouter membrane viaan N-terminal lipidmoiety
Porin: protein that forms water-filledchannels in the outermembrane
Sec system: generaprotein export apparatus
Chaperone: proteinthat guides anothermolecule to itsdestination, beingthe folded state orthe right cellularcompartment
(where Ar is an aromatic residue and X is any
residue). Transmembraneβ-strands of OMPsare 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 theperiplasm, andSurA mayact 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. Underthose conditions, the OMPs have only little
chance to aggregate and an skp null mutation
Periplasm
OM
IMSec
Omp85
Skp
SurA
C
PorinOMP
YfgL YfiO NlpB
C
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 theOMP functions as a targeting signal and binds the periplasmic domain of Omp85. Other chaperones and folding catalysts, such as SurA, may act onthe OMP. After folding, the OMP inserts into the lipid bilayer, possibly inbetween the Omp85 subunits. Oligomerization of certain OMPs, such asporins, 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-prolylcis / trans isomerase
σE: alternativesigma factor that
guides RNA polymerase to thepromoters of genesinvolved in relievingperiplasmic stressconditions
has only a limited effect. The demand for such
achaperonewillincreasewhenthesubsequent folding and assembly processes are hampered,
e.g., in a surA mutant. Hence, it is not possibleto construct the skp surA double mutant.
Apart from SurA, the periplasm containsthree other PPIases, PpiA (also known as
RotA), PpiD, and FkpA. PpiA is by far themost active of them, because its inactivationleads to barely detectable residual PPIase ac-
tivity (47). However, a ppiA null mutation hadnodetectableeffectonOMPassembly(47).In
contrast, a ppiD null mutation led to an overallreduction 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 evena 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 littleevidence 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 andDsbC, 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 observationshowed that the disulfide bond was formed
duringperiplasmictransitandthatatleastpar-tial folding occurs prior to OM insertion (30).
DegP is a protease that degrades unfolded ormisfolded proteins in the periplasm but also
has chaperone activity, as was shown in vitroon the soluble substrates MalS andcitrate 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 itsfunction as a chaperone in this respect (13).
Assembly into the OM
The insertion of OMPs into the OM has longremained 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 requiredfor OMP insertion, i.e., a protein designated
Omp85 in N. meningitidis . Omp85 was foundto be essential for the viability of the bacteriaand homologues of the omp85 gene were
found in all gram-negative bacteria for whichthe genome sequence was available (106)
suggesting its involvement in an importantprocess. 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 proteaseinvolved in the σE-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 examinedincluding porins, a siderophore receptor, an
enzyme, and a secretin involved in type IVpili assembly, accumulated as aggregates in
the periplasm (105, 106). The role of Omp85in 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 temperatureresulted 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 outermembrane 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 cellsof either N. meningitidis or E. coli . Whereasunfolded OMPs accumulated as aggregates
in such cells of N. meningitidis (105), Omp85depletion in E. coli primarily led to severely
reduced amounts of detectable OMPs (112,114). The difference is presumably caused by
the absence of the σE-dependent periplasmic
stress response in N. meningitidis . In E. coli ,this stress response is induced upon accumu-
lation of unfolded OMPs in the periplasm (fora 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 theprotease domain of DegS and RseP, result-
ing in the cleavage of the antisigma factor
RseA and the release of σE in the cytoplasm(Figure 2). The released σE then binds the
RNA polymerase core enzyme, resulting inthe transcription of the genes for periplasmic
D e g S
P D Z
P D Z
R s e A
R s e
B
R s e P
D e g S
R s e A
R s e
B
R s e P
Periplasm
Periplasmic
stress response
Stress
OM
IM
C
RNA
polymerase
sRNAsskp
surA
degP
σE
σE
P r o t e a s
e
P r o t e a s e
OMPs
OMP
Figure 2
Periplasmic stressresponse. Uponaccumulation of unfolded OMPs inthe periplasm, astress response isinitiated that starts with the recognitionof the C termini of the OMPs by the
PDZ domain of theprotease DegS. Thisrecognition initiatesa proteolytic cascaderesulting in therelease of thealternative sigmafactor σE, whichbinds RNA polymerase and leadsto enhancedtranscription of genes encoding
periplasmicchaperones, such asSkp and SurA, andthe protease DegP. The σE response alsoleads to productionof sRNAs that negatively regulateOMP expression.
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sRNA: smallregulatory RNA
chaperones, such as Skp and SurA, and for
the potent protease DegP. Together thesechaperones and protease relieve the periplas-
mic stress. Moreover, the σE response resultsin 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 OMPsare rescued or degraded in the periplasm andOMP synthesis is inhibited during the stress
period. Although chaperones such as Skp andSurA are present in N. meningitidis , many es-
sential components of the signal transductionpathway 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, homologuesof RseA and RseB appear to be absent. Al-
though there is an alternativeσ factor belong-
ing to the ECF (extracytoplasmic factor) fam-ily (to which σE also belongs) encoded on the
meningococcal chromosome (i.e., NMB2144in 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, includingthose for methionine sulfoxide reductase (35).
Thus, theσE-dependent periplasmicstressre-
sponse appears to be absent in the pathogenic Neisseriaceae. As a result, when there is an
OMP assembly defect, OMPs will continue tobe 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 viaan 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-linkingexperiments revealed that the Omp85 ho-
molog of E. coli forms a complex with threelipoproteins, YfgL, YfiO, and NlpB (114). Asimilar complex was identified upon a pro-
teomic analysis of OMP complexes resolvedby blue-native PAGE (93). Copurification ex-
periments using strains with mutations in thegenes 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 theOM permeability defect of a partial loss-of-
function mutation in the imp gene (29, 78)
which encodes an OMP involved in LPSbiogenesis (see below). Although yfgL is not
an essential gene, null mutations create anOM 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 disseminatedamong gram-negative bacteria, we could not
identify a homolog in the sequenced genomesof 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 permeabilityand reduced amounts of OMPs, and a yfiO
depletion strain showed a phenotype similarto that of the yaeT / omp85 depletion strain of
E. coli (58). In N. gonorrhoeae, a transposoninsertion in the middle of the yfiO homolog
was described (32). This mutant was viableshowed 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 succeededto 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. TheComL pro-
tein is covalently linked to the peptidoglycan,both in N. gonorrhoeae and when expressed
in E. coli (32). Furthermore, yfgL mutationsaffected 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 thislayer.
Whereas yfiO, like yfgL and omp85 , is widely disseminated among gram-negative
bacteria, this is less so for nlpB. Null mutantsin nlpB of E. coli are viable; they show only
moderate OM permeability defects and noobvious 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 proposedthat Omp85 consists of two domains, a
C-terminal β
-barrel embedded in the OMand an N-terminal domain extending into
the periplasm. Support for this model wasobtained in proteolytic digestion experi-
ments, which resulted in the degradation of
the N-terminal domain and left the predictedmembrane-embeddedβ-barrel domain intact
(76, 91). The periplasmic extension containsrepeats of a conserved domain, POTRA
(polypeptide transport associated), suggestedto have chaperone-like qualities (81). Such
POTRA domains were further identified inother members of the Omp85 superfamily,
which includes the Omp85 homologs of mi-tochondria, the Toc75 OM component of the
chloroplast protein import machinery, andthe OM-localized TpsB component of the
two-partner secretion (TPS) systems of gram-
negative bacteria, and in members of the
POTRA:polypeptidetransport associated
FtsQ/DivIB family of IM proteins involved in
cell division. The bacterial Omp85 homologsare unique in having five of these POTRA do-
mains, whereas the number of such domainsin the other proteins with known functions
is restricted to one or two. Another memberof the Omp85 superfamily that is widely
disseminated among gram-negative bacteria(encoded by the ytfM gene in E. coli and theNMB2134 locus in N. meningitidis strain
MC58) contains three POTRA domains. The function of this protein is unknown (92).
To study its structure and function inmore detail, we produced the Omp85 protein
of E. coli in inclusion bodies, which wereisolated, and refolded the protein into the
native conformation in vitro (76). The re-folded protein formed oligomers, presumably
tetramers, similarly as reported for anothermember 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 identifiedin 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 envelopesformed 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 aninteraction would affect the channel activity.
Indeed, nonnative OMPs drastically enhanceOmp85 channel activities (76), showing that
these substratesinteract directly withOmp85.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 isinteresting to note that the same signature
sequence is recognized by the PDZ domainof DegS when unfolded OMPs accumulate
in the periplasm (108), thereby initiating the
σE-dependentperiplasmicstressresponse (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 terminiof 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-
tionof this positively charged residue in the N.
meningitidis porin PorA drastically improved
PorA assembly into the E. coli OMinvivo(76). Thus, although the OMP assembly machin-
ery is highly conserved among gram-negativebacteria, species-specific adaptations seem to
have occurred during evolution.Of note, the C-terminal signature se-
quence of OMPs is not absolutely essentialfor assembly into the OM. Thus, whereas
the high-level expression of a mutant form of
the E. coli porin PhoE lacking the C-terminalphenylalanine 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, severalstudies reporting the assembly of neisserial
OMPs into the E. coli OM in vivo (113) maybe explained by low expression levels. Pulse-
chase experiments in E. coli overexpressingPhoE revealed the existence of two assem-
bly pathways: approximately half of the PhoEmolecules assembled within the 30-s pulse
period into their native conformation in the
OM, whereas the other half of the moleculesfollowed much slower kinetics and allowed
for the detection of several assembly inter-mediates (42). In these assays, the mutant
PhoE lacking the C-terminal phenylalaninefollowedonlytheslowerkineticpathway.This
study underscores the role of the C-terminalsignature sequence as a targeting signal and
indicates that there must be alternative, lessefficient targeting signals in OMPs. Further-
more,kineticpartitioningbetweenOMincor-poration and aggregation of periplasmic in-
termediates may explain the observation that
OMPs with defective signature sequences arestill assembled into the OM at low expression
levels, when the kinetics of aggregation is lowand 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? Ofnote, 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, Omp85may recognize, though less efficiently, in-
ternal β-strands, many of which also end
with an aromatic residue. In some classesof OMPs, the C-terminal signature motif
could not be discerned (94). Perhaps, inthese 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-embeddedβ-barrel and a C-terminal periplasmicextension. The signature
motifwasfoundinthiscaseattheendofthe β-barrel domain (94) and its importance in OM
targeting wasdemonstrated 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, thisappears to be a rather exceptional case be-
cause the C-terminal extension of an OMPsequence 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 intype 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 barrelof 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. Thesecretins form a class of OMPs involved in di-
verse processes, including type II and type IIIprotein secretion, type IV pili biogenesis, and
the extrusion of filamentous phages (7). Thestructure of these large multimeric proteins is
not known at atomic resolution, but they ap-pear to be rather poor inβ-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, thebinding of these proteins and other OMPs
that lack the C-terminal signature motif isindirect and requires accessory factors, such
as the lipoproteins YfiO, YfgL, and NlpB, orspecific 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 LPSmolecules (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 formin the presence of LPS, detergent, and diva-
lent cations. This folded monomer appearedto be an assembly intermediate because it could subsequently be converted into its na-
tive trimeric OM-inserted form (22). Alsofor other OMPs, LPS-dependent folding in
vitro has been reported (83). However, LPS-independent in vitro folding conditions were
later established for many OMPs, includingPhoE (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-teinin Salmonella minnesota as an LPS-binding
protein (34). Thus, LPS may exert its rolein 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 nonnativeOMPs at the IM, a conformational change
triggered by LPS binding may release thecargo 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: increasedmembranepermeability
be determined in site-directed mutagenesis
experiments. We speculate that the role of LPS in OMP
biogenesis is restricted to late stages afterthe 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 loopsof theproteins. Theseverely reduced amountsof OMPs in deep-rough mutants may be ex-
plained by the induction of the σE responsein such mutants, resulting in the production
of sRNAs that inhibit the synthesis of many OMPs (see above). Changes in LPS structure
induce the σE response in E. coli by a hithertounknown 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. Theassembly 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 allOMPs 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 theassembly of this class of OMPs. In this re-
spect, it may indeed be relevant that the yfgLmutants 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-sisttheirreleasefromtheIMandpreventtheir
aggregation in the periplasm. The Skp/OMPcomplex 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 assistedby the presumed chaperone activities of the
POTRA domains and by periplasmic proteinssuch as SurA and DsbA. Binding of an OMP
also results in a conformational change in theC-terminal domain of Omp85, which is re-
flected by the increased pore activity observedin the planar lipid bilayer experiments (76) This conformational change allows the OMPs
to insert into the OM, possibly in between theOmp85 subunits. Dissociation of the Omp85
subunits releases the assembled OMPs intothe OM, where final conformation changes in
the cell surface-exposed loops may be inducedupon 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 roleis less crucial, although YfiO is definitely es-
sential, at least in E. coli (67). These proteinsmay play a role in the recognition of OMPs
that do not display the C-terminal signaturemotif, which includes the essential protein
Imp (increased membrane permeability), inmodulating 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 OMPsthat 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 witha feedback inhibition on their synthesis via
σE-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 tostudy the role of other assembly factors.
LIPOPROTEINS
Bacterial lipoproteins are membraneattached via an 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 theenzyme Lgt from phosphatidylglycerol to
the sulfhydryl group of the cysteine that isinvariably present at the +1 position relative
to the processing site (82). Subsequently, thediacylglycerylprolipoprotein is processed by adedicated 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 comprisesthe amino acids flanking the lipidated cysteine
in the mature protein (101). Lipoproteinslacking an IM retention signal, usually an
aspartate at the +
2 position of the matureprotein, are transported to the OM by the
Lol system (Figure 3). The first component
of the Lol system was identified in an in vitrosystem, 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 aperiplasmic extract. The active component
of the periplasmic extract was identified anddesignated 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 integralmembrane components of this transporter,
LolC and LolE, show considerable sequencesimilarity 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 inthe affinity of LolD for ATP. Subsequently,
ATP binding to LolD causes a conformational
change in the LolC/E moiety that results in a
Periplasm
OM
IM
C - X C
- X
C -X
C-X
C
- X
C - X
C - X
C - D
C - D / X
Sec
Sorting
LolA
LolA
LolB
LolC
LolD
LolE
LolD
?
ATP ADP ATP ADP
Figure 3
Lipoprotein transport through the bacterial cell envelope. After theirtransport via the Sec system and subsequent lipidation at the N-terminalcysteine (C), lipoproteins bind to the ABC-transporter LolCDE, providedthey do not possess a Lol-avoidance motif, which usually is an aspartate(D) flanking the N-terminal C (C-D). Lipoproteins with another aminoacid at the +2 position (X) are destined for the OM. Energy from ATPhydrolysis by LolD is transferred to LolC and LolE and utilized to openthe hydrophobic LolA cavity to accommodate the lipoprotein. When theLolA-lipoprotein complex interacts with the OM receptor LolB, thelipoprotein is transferred to LolB and inserted into the OM. Furthertransport to the outer leaflet of the OM, if applicable, occurs throughunknown mechanisms.
Spheroplasts:bacterial cells of which the outermembrane and thepeptidoglycan layerhave been disruptedby treatment withEDTA and lysozyme
ABC: ATP-bindingcassette
decrease in lipoprotein binding affinity. ATPhydrolysis is then required for transfer of the
lipoprotein from LolC/E to the periplasmic
chaperone LolA (41). The LolA-lipoproteincomplex 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 andLolB. LolA and LolB are structurally similar;
both contain a hydrophobic cavity. TheLolA cavity is composed mostly of aromatic
residues, whereas the cavity in LolB is mademostly of leucine and isoleucine residues,
which is likely to explain the difference inlipoprotein-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 pullulanaseof 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 secretionapparatus. 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 lipoproteinstakes 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 isoleucineand a leucine, respectively, at the +2 posi-
tion, suggesting they are substrates for theLol 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
presentintheouterleafletoftheOMofgram-negative bacteria. It consists of a hydrophobic
membrane anchor, lipid A, substituted withan oligosaccharide core region, which in some
bacteria (e.g., in most E. coli strains, but not inthe laboratory strain E. coli K-12 and also not
in N. meningitidis ) is extended with a repeatingoligosaccharide, the O-antigen. These differ-
entLPSconstituentsaresynthesized at thecy-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-linkedd-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. Theprimary 3-hydroxy fatty acids may be substi-
tuted with secondary fatty acids. The lipid Abiosynthetic pathway, also known as the Raetz
pathway (74), has been characterized in detailmostly in E. coli and Salmonella typhimurium
but it appears to be highly conserved amongother gram-negative bacteria (74). Remark-ably, although LPS has so far only been de-
tected in gram-negative bacteria, homologs ofthe genes encoding the lipid A biosynthetic
enzymes have also been found in sequencedplant genomes (74).
The core oligosaccharide is much more variable between bacterial species. In addi-
tion, a huge amount of LPS heterogeneity iscreated 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 speciescan 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 strainsof 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 andthe O-antigen subunits, if present, are trans-
ported separately overthe IM. The O-antigensubunits are transferred over the IM by one
of three different routes: the Wzy-, ABC-transporter-, or synthase-dependent pathway
(74).After polymerization, the subsequentlig-ation of the O-antigen to the lipid A–core
moiety at the periplasmic side of the IM isan 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 theIM (26). More evidence for a role of MsbA
in LPS transport came from a study of anmsbA 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 proteinin
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). Theprecise role of Imp was demonstrated in
N. meningitidis . Imp was not essential in thisbacterial species, allowing the construction
of an imp deletion mutant. The phenotypeof this mutant demonstrated a role for Imp
in LPS biogenesis: It produced less than10% of wild-type levels of LPS, which were
not accessible to LPS-modifying enzymesrecombinantly 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 toLPS-modifying enzymes in the OM, showing
that it did not reach the outer leaflet of theOM (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 phenotypesimilar to that expressed upon depletion of
Imp. Moreover, Imp and RlpB exist in a com-
plex. Depletion of Imp or RlpB resulted inan 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 tocontain a β-barrel domain embedded in the
OM, with a long N-terminal domain and ashort C-terminal domain extending into the
periplasm (Figure 4). In the conserved do-
main database at the National Center forBiotechnology 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 theN-terminal periplasmic extension
of many but not all Imp homologs (Figure 4).Nevertheless, all Imp homologs show se-
quence similarity over the entire length, andalso 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. Thegenes, 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
1
1
1
LptA/
NMB0355
Acidobacteria bacterium
Ellin345
Nostoc sp. PCC712
Acinetobacter sp. ADP1
COG1934
COG1934
COG1934
C O G 1 9 3 4
COG1934 COG1934
COG3117
COG3117
COG3117
COG3117
OstA_C
OstA_C
OstA_C
Figure 4Conserved 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 proteinsshown at the left. The type and number (#) of retrieved architectures are shown. The species expressingthe most unusual architectures are indicated at the right. A topology model for Imp, based on manualinspection 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 inLPS transport: In these bacteria, the cor-
responding gene is not essential, and itsdeletion results in severely decreased lev-
els of LPS (10). The LPS is accessible toperiplasmic LPS-modifying enzymes in this
mutant, indicating that the LptA/NMB0355protein acts at a step after translocation by
MsbA (10). The LptA protein of E. coli wasfound in the soluble periplasmic fraction (85),
but we found the majority of the corre-
sponding neisserial protein (NMB0355) inthe 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 commonfunction.
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-
mentofanovelABCtransporterinLPStrans-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, whichis 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 YrbKis 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-likelythatthisproteinfunctionsastheintegral
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 thecell envelope have been considered, and
with the current situation, no model can beconsidered 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 hydrophobicmoiety, 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 solubleperiplasmic protein in E. coli (86), may func-tion as the LPS chaperone, as LolA does for
lipoproteins. Furthermore, while LolA passesits cargo to the structurally related OM recep-
tor LolB, LptA may pass the LPS moleculesto theperiplasmic N-terminal domainof 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
OM
Membrane contact sites Lol system-like
IM
Imp
MsbA MsbA
Imp
RlpB
LptA
LptA
LptB LptB
YrbK YrbK
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 theperiplasm by an unknown mechanism that could resemble the Lol system (right ). LptB together with anunknown (?) membrane protein and possibly YrbK may form an ABC transporter that delivers LPS tothe 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 thecell 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 suchsites, 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, closeto the membrane contact sites. Membrane
fractionation studies showed the existence
of a minor fraction, designated OML, 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 LPStransiently passed through this fraction on its
way to the OM (40). Furthermore, when asimilar 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 notclear 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 thisbacterium 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 butmerely the presence of the typical α -helical
membrane-spanning segments of some IMproteins. The next steps in phospholipid bio-
genesis, i.e., transport through the periplasmand incorporation into the inner leaflet of the
OM, remain obscure. Unlike LPS transportthe transport of phospholipids was halted in
spheroplasts, and unlike lipoproteins, newlysynthesized phospholipids could not be re-
leased from the spheroplasts upon addition ofa 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 hasbeen 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 thenewlyidentified componentsby X-raycrystal-
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 thehydrophobic environment of the membrane
are the driving forces, it may be possible toset up an in vitro system for OMP assembly
with purified components. For LPS, a majorissue remains how it is transported throughthe 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. Forlipoproteins, 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 thebeginning.
Importantly, much progress in the fieldhas
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 twomodel organisms, it is likely that studies in
other bacteria will uncover new, unanticipatedfeatures.
Further studies in this field will remainimportant, because they will uncover funda-
mental biological processes. In addition, theknowledge gained from these studies may be
useful for medical applications: The essentialnature 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 proteinOmp85, which is evolutionary conserved and found even in the OM of mitochondria.
3. Omp85 recognizes its substrate OMPs by virtue of their C-terminal signaturesequences.
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-attachedreceptor.
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 integralOMP 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 ofthis 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 149:1265–732. Ames GFL, Spudich EN, Nikaido H. 1974. Protein composition of the outer membrane
of Salmonella typhimurium: effect of lipopolysaccharide mutations. J. Bacteriol. 117:406–163. 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 periplasmicPPIase lacking its parvulin domains functions in vivo and has chaperone activity. EMBO
J. 20:285–946. Biemans-Oldehinkel E, Doeven MK, Poolman B. 2006. ABC transporter architecture
and regulatory roles of accessory domains. FEBS Lett. 580:1023–357. Bitter W. 2003. Secretins of Pseudomonas aeruginosa: large holes in the outer membrane
Arch. Microbiol. 179:307–148. 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 cellsurface.
9. Bos MP, Tefsen B, Geurtsen J, Tommassen J. 2004. Identification of an outermembrane 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. 45:1289–30212. Casjens S. 2000. Borrelia genomes in the year 2000. J. Mol. Microbiol. Biotechnol. 2:401–1013. 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. Structuralinsights into the secretin PulD and its trypsin-resistant core. J. Biol. Chem. 280:37732–4115. Charlson ES, Werner JN, Misra R. 2006. Differential effects of yfgL mutation on Es-
cherichia coli outer membrane proteins and lipopolysaccharide. J. Bacteriol. 188:7186–9416. Chen R, Henning U. 1996. A periplasmic protein (Skp) of Escherichia coli binds a class of
outer membrane proteins. Mol. Microbiol. 19:1287–9417. 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
8/11/2019 Om Bio Genesis
http://slidepdf.com/reader/full/om-bio-genesis 19/27
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 theperiplasmic chaperone Skp of Escherichia coli for phospholipids, lipopolysaccharides andnon-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 PhoEof Escherichia coli K-12. J. Mol. Biol. 269:473–78
22. De Cock H, Tommassen J. 1996. Lipopolysaccharides and divalent cations are involvedin 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. 60:2034–5224. 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 thetranslocon for E. coli capsular polysaccharides defines a new class of membrane protein.
Nature 444:226–2929. 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 membraneprotein 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. Lossof Hfq activates the σE-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
8/11/2019 Om Bio Genesis
http://slidepdf.com/reader/full/om-bio-genesis 20/27
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, whichencodes 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. 268:16551–5637. 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 occursat the cytoplasmic membrane. J. Biol. Chem. 276:18804–1138. 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 differentiatedmembrane domains from Escherichia coli and Salmonella typhimurium, including a fraction
containing attachment sites between the inner and outer membrane and the mureinskeleton 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 outermembrane protein PhoE of Escherichia coli . Eur. J. Biochem. 267:3792–800
43. Johansen J, Rasmussen AA, Overgaard M, Valentin-Hansen P. 2006. Conserved smalnoncoding RNAs that belong to the σE regulon: role in down-regulation of outer mem-
brane proteins. J. Mol. Biol. 364:1–844. 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 forpilus 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. 279:36470–8046. Kellenberger E. 1990. The ‘Bayer bridges’ confronted with results from improved elec-
tron microscopy methods. Mol. Microbiol. 4:697–70547. 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 deletionsinthegeneforan Escherichia coli outer membrane protein define an area possibly important
for recognition of the outer membrane by this polypeptide. J. Biol. Chem. 263:13291–9649. Koebnik R, Locher KP, Van Gelder P. 2000. Structure and function of bacterial outer
membrane proteins: barrels in a nutshell. Mol. Microbiol . 37:239–5350. 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. 278:24586–9351. 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
8/11/2019 Om Bio Genesis
http://slidepdf.com/reader/full/om-bio-genesis 21/27
52. Kornd ¨ orfer IP, Dommel MK, Skerra A. 2004. Structure of the periplasmic chaperone
Skp suggests functionalsimilarity with cytosolic chaperonesdespite different architecture.
Nat. Struct. Mol. Biol. 11:1015–20
53. Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C. 2000. Crystal structure of thebacterial membrane protein TolC central to multidrug efflux and protein export. Nature
405:914–1954. 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 outermembrane. J. Biol. Chem. 278:48520–2355. Laird MW, Kloser AW, Misra R. 1994. Assembly of LamB and OmpF in deep rough
lipopolysaccharide mutants of Escherichia coli K-12. J. Bacteriol. 176:2259–6456. Lazar SW, Kolter R. 1996. SurA assists the folding of Escherichia coli outer membrane
proteins. J. Bacteriol. 178:1770–7357. Lee PA, Tullman-Ercek D, Georgiou G. 2006. The bacterial twin-arginine translocation
pathway. Annu. Rev. Microbiol. 60:373–9558. 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 sortingand transport of Escherichia coli lipoproteins destined for the outer membrane. EMBO J.
14:3365–7261. 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 outermembrane 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.
21:871–8463. 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. 182:1–665. Narita S, Tokuda H. 2006. An ABC transporter mediating the membrane detachment of
bacterial lipoproteins depending on their sorting signals. FEBS Lett . 580:1164–7066. Nikaido H. 2003. Molecular basis of bacterial outer membrane permeability revisited.
Microbiol. Mol. Biol. Rev. 67:593–65667. Onufryk C, Crouch ML, Fang FC, Gross CA. 2005. Characterization of six lipoproteins
in the σE regulon. J. Bacteriol. 187:4552–6168. Papenfort K, Pfeiffer V, Mika F, Lucchini S, Hinton JCD, et al. 2006. σE-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 426:862–6670. 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
8/11/2019 Om Bio Genesis
http://slidepdf.com/reader/full/om-bio-genesis 22/27
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. 183:49–5372. Pugsley AP. 1993. The complete general secretory pathway in gram-negative bacteria
Microbiol. Rev. 57:50–10873. Raetz CRH, Reynolds CM, Trent MS, Bishop RE. 2007. Lipid A modification systems
in gram-negative bacteria. Annu. Rev. Biochem. 76:295–32974. Raetz CRH, Whitfield C. 2002. Lipopolysaccharide endotoxins. Annu. Rev. Biochem
71:635–70075. 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. Demonstratesthat the C-terminalsignature sequenceof OMPs functionsas a targeting factor, recognizedby 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. 4:1984–9577. 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. 10:3170–8778. Ruiz N, Falcone B, Kahne D, Silhavy TJ.2005.Chemicalconditionality: a genetic strategy
to probe organelle assembly. Cell 121:307–1779. 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 andcharacterization of a newgeneof 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 proteinsTrends 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. 269:19701–683. Sen K, Nikaido H. 1990. In vivo trimerization of OmpF porin secreted by spheroplasts
of Escherichia coli . Proc. Natl. Acad. Sci. USA 87:743–4784. 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 aperiplasmic protein as new factorsrequired for thetransport of LPS tothe 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. 189:244–5386. 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. 157:547–5887. Spies C, Beil A, Ehrmann M. 1999. A temperature-dependent switch from chaperone to
protease in a widely conserved heat shock protein. Cell 97:339–4788. 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. 4:599–61189. Steeghs L, de Cock H, Evers E, Zomer B, Tommassen J, et al. 2001. Outer membranecomposition of a lipopolysaccharide-deficient Neisseria meningitidis mutant. EMBO J
20:6937–45
90. First demonstration that
N. meningitidis is viable without LPS,making thisorganism suitablefor further studieson 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 392:449–5091. Stegmeier JF, Andersen C. 2006. Characterization of pores formed by YaeT (Omp85)
from Escherichia coli . J. Biochem. 140:275–83
212 Bos · Robert · Tommassen
8/11/2019 Om Bio Genesis
http://slidepdf.com/reader/full/om-bio-genesis 23/27
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. 388:37–4693. 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. 280:34409–1994. 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. 218:141–4895. Surana NK, Grass S, Hardy GG, Li H, Thanassi DG, et al. 2004. Evidence for conserva-
tionof architecture andphysical properties of Omp85-like proteins throughout evolution.
Proc. Natl. Acad. Sci. USA 101:14497–503
96. Provideshigh-resolution structures of LolA and LolB,providing insightsinto the lipoproteintransport 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.
22:3199–20997. Tam C, Missiakas D. 2005. Changes in lipopolysaccharide structure induce the σE-
dependent response of Escherichia coli . Mol. Microbiol. 55:1403–1298. Tamm LK, Arora A, Kleinschmidt JH. 2001. Structure and assembly of β-barrel mem-
brane proteins. J. Biol. Chem. 276:32399–40299. 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. 280:35961–66100. Tefsen B, Geurtsen J, Beckers F, Tommassen J, de Cock H. 2005. Lipopolysaccharide
transport to the bacterial outer membrane in spheroplasts. J. Biol. Chem. 280:4504–9101. 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. 276:47690–94102. Tormo A, Almir ´ on M, Kolter R. 1990. surA, an Escherichia coli gene essential for survival
in stationary phase. J. Bacteriol. 172:4339–47103. Trent MS, Stead CM, Tran AX, Hankins JV. 2006. Diversity of endotoxin and its impact
on pathogenesis. J. Endotoxin Res. 12:205–23104. Van Ulsen P, Tommassen J. 2006. Protein secretion and secreted proteins in pathogenic
Neisseriaceae. FEMS Microbiol. Rev. 30:292–319
105. First
demonstration thatan integral OMP,Omp85, is requiredfor the assembly ofOMPs.
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 proteininvolved in outer-membrane-protein assembly. Res. Microbiol. 155:129–35
107. Waller PR, Sauer RT. 1996. Characterization of degQ and degS , Escherichia coli genesencoding homologs of the DegP protease. J. Bacteriol. 178:1146–53
108. Demonstratesthat the C-terminasignature sequenceof OMPs triggersthe σE-dependent periplasmic stress
response when unfolded OMPsaccumulate in theperiplasm.
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 113:61–71109. 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 tohemophores. Annu. Rev. Microbiol. 58:611–47111. 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 andlipid-independent outer membrane proteins of Escherichia coli . Mol. Microbiol. 57:1450–
59
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Annual Review of
Microbiology
Volume 61, 2007 Contents
Frontispiece
Margarita Salas
40 Years with Bacteriophage Ø29
Margarita Salas
The Last Word: Books as a Statistical Metaphor for Microbial Communities
Patrick D. Schloss and Jo Handelsman
The Mechanism of Isoniazid Killing: Clarity Through the Scope
of Genetics
Catherine Vilchèze and William R. Jacobs, Jr.
Development of a Combined Biological and Chemical Process for
Production of Industrial Aromatics from Renewable Resources
F. Sima Sariaslani
The RNA Degradosome of Escherichia coli : An mRNA-Degrading
Machine Assembled on RNase E Agamemnon J. Carpousis
Protein Secretion in Gram-Negative Bacteria via the Autotransporter
Pathway
Nathalie Dautin and Harris D. Bernstein
Chlorophyll Biosynthesis in Bacteria: The Origins of Structural and
Functional Diversity
Aline Gomez Maqueo Chew and Donald A. Bryant
Roles of Cyclic Diguanylate in the Regulation of Bacterial Pathogenesis
Rita Tamayo, Jason T. Pratt, and Andrew Camilli Aggresomes and Pericentriolar Sites of Virus Assembly:
Cellular Defense or Viral Design?
Thomas Wileman
As the Worm Turns: The Earthworm Gut as a Transient Habitat for
Soil Microbial Biomes
Harold L. Drake and Marcus A. Horn
vi
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Biogenesis of the Gram-Negative Bacterial Outer Membrane
Martine P. Bos, Viviane Robert, and Jan Tommassen 191
SigB-Dependent General Stress Response in Bacillus subtilis and
Related Gram-Positive Bacteria
Michael Hecker, Jan Pané-Farré, and Uwe Völker 215
Ecology and Biotechnology of the Genus Shewanella Heidi H. Hau and Jeffrey A. Gralnick 237
Nonhomologous End-Joining in Bacteria: A Microbial Perspective
Robert S. Pitcher, Nigel C. Brissett, and Aidan J. Doherty 259
Postgenomic Adventures with Rhodobacter sphaeroides Chris Mackenzie, Jesus M. Eraso, Madhusudan Choudhary, Jung Hyeob Roh,
Xiaohua Zeng, Patrice Bruscella, Ágnes Puskás, and Samuel Kaplan 283
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 309
Endolithic Microbial Ecosystems
Jeffrey J. Walker and Norman R. Pace 331
Nitrogen Regulation in Bacteria and Archaea
John A. Leigh and Jeremy A. Dodsworth 349
Microbial Metabolism of Reduced Phosphorus Compounds Andrea K. White and William W. Metcalf 379
Biofilm Formation by Plant-Associated Bacteria
Thomas Danhorn and Clay Fuqua 401
Heterotrimeric G Protein Signaling in Filamentous Fungi
Liande Li, Sara J. Wright, Svetlana Krystofova, Gyungsoon Park,and Katherine A. Borkovich 423
Comparative Genomics of Protists: New Insights into the Evolution
of Eukaryotic Signal Transduction and Gene Regulation
Vivek Anantharaman, Lakshminarayan M. Iyer, and L. Aravind 453
Lantibiotics: Peptides of Diverse Structure and Function
Joanne M. Willey and Wilfred A. van der Donk 477
The Impact of Genome Analyses on Our Understanding of
Ammonia-Oxidizing Bacteria
Daniel J. Arp, Patrick S.G. Chain, and Martin G. Klotz 503
Co nt en ts v ii
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Morphogenesis in Candida albicans Malcolm Whiteway and Catherine Bachewich
Structure, Assembly, and Function of the Spore Surface Layers
Adriano O. Henriques and Charles P. Moran, Jr.
Cytoskeletal Elements in Bacteria
Peter L. Graumann
Indexes
Cumulative Index of Contributing Authors, Volumes 57–61
Cumulative Index of Chapter Titles, Volumes 57–61
Errata
An online log of corrections to Annual Review of Microbiology articles may be founat http://micro.annualreviews.org/
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