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LptDE 2
OM translocon
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O-Antigen
Glu Hep
OM
Glu D
Glu Gal
E
core P
Hep Hep
PEtN Hep
periplasm
Kdo Kdo
O
O A
P O O
HO– O
O O
O NH
HO
O O
LPS
LptBFGC
F/G F/G IM
B B cytoplasm
ATP ADP + Pi
ATP ADP + Pi
Figure 1. Transport of LPS across the cell envelope. After synthesis, lipid A-core oligosaccharide (LPS) molecules are flipped across the IM in an ATP-dependent manner by
MsbA. At the outer leaflet, the O antigen is ligated to the outer core to form full-length LPS (not shown). LPS is transported across the periplasm and OM, and assembled at
the cell surface by LptB2FGCADE. LptB2FG form an ABC transporter that uses ATP hydrolysis to extract LPS from the IM and push it along a periplasmic bridge built of
homologous domains in LptCAD. It is unclear whether LptF, LptG or both, interact with LptC. At the OM, the LptDE form a plug-and-barrel translocon that inserts LPS
into the outer leaflet. Structural composition of E. coli LPS is shown on the left. Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; Hep, L-glycero-D-manno-heptose; Etn,
ethanolamine; P, phosphate; Glu, D-glucose; Gal, D-galactose.
its chemical composition varies among species [2,3]. In fact, present in the outer leaflet, the asymmetry of the OM is
some bacteria synthesize a shorter version of LPS without O broken, effectively creating patches of phospholipid bilayer,
antigen known as lipooligosaccharide or LOS [2]. In addition which allow the diffusion of small hydrophobic compounds
to the lipid asymmetry conferred by the presence of LPS (or into the cell. Indeed, a decrease in the levels of LPS at the cell
LOS) in the outer leaflet, the OM is structurally different from surface leads to hypersensitivity to hydrophobic toxic mol-
the IM in that its integral OM proteins adopt a b-barrel confor- ecules such as bile salts and some antibiotics. Furthermore,
mation instead of the typical a-helical structure of integral IM these studies also revealed that some bacterial species such as
proteins. Both LPS and b-barrel proteins contribute to the E. coli, S. typhimurium and Pseudomonas aeruginosa cannot sur-
unique permeability properties of the OM. Molecules of LPS vive without LPS, while others such as Neisseria meningitidis,
pack tightly in the outer leaflet of the OM and it is thought Acinetobacter baumannii and Moraxella catarrhalis can [6–11].
that the hydrophilicity of its sugars and charges of its phosphates Therefore, it is crucial that, during growth at each cell cycle,
serve as a barrier to small hydrophobic compounds that nor- Gram-negative bacteria properly assemble millions of LPS
mally partition and diffuse through phospholipid bilayers like molecules at their cell surface at a speed estimated as high as
the IM. By contrast, many small hydrophilic molecules can 70 000 molecules per minute [3].
easily cross the OM by diffusing through the hydrophilic For more than 40 years, we have known that although LPS
lumen of porins—integral b-barrel proteins that function as must be present at the cell surface, its synthesis occurs at the IM
non-specific, ungated pores or sieves [4]. As the IM lacks ungated [12,13]. In the past two decades, the players involved in the
pores, entry of small hydrophilic molecules through the IM relies transport of LPS across the cell envelope have been identified.
on transporters that must recognize them to allow their transport This review and the accompanying paper [14] highlight the
into the cytoplasm. Therefore, many molecules that can diffuse research that has led to our current understanding of how, fol-
across the IM (small hydrophobic compounds) cannot easily lowing synthesis at the inner leaflet of the IM, LPS is flipped
penetrate through the OM because of LPS, and many molecules across the IM, extracted from the IM, transported across the
that can diffuse across the OM (small hydrophilic compounds) aqueous periplasm and translocated through the OM so that
cannot easily penetrate through the IM. The fact that these two it can finally assemble at the cell surface (figure 1). This paper
membranes have opposite permeability properties protects focuses on the early steps that take place at the IM in the
cells from many harmful compounds and is one of the major journey of LPS across the cell envelope. The accompanying
reasons why it is so difficult to develop antibiotics that are paper [14] will delve into the details of LPS transport across
effective against Gram-negative bacteria. the periplasm and insertion into the OM.
The importance of LPS in providing resistance against
hydrophobic antibiotics was revealed from studies using
wild-type cells where LPS was removed by chemical treatment 2. Synthesis, transport and biogenesis of the LPS
and mutant cells defective in LPS biogenesis [4,5]. Collectively,
these studies demonstrated that decreasing the amount of LPS layer
at the cell surface leads to the appearance or an increase of Electron microscopy studies drove the discovery that the
phospholipids at the cell surface. When phospholipids are Gram-negative cell envelope is delimited by two membranes
[15]. Electron micrographs produced by Kellenberger & Ryter synthesized LPS to the OM [27,28]. The function as a flippase 3
[16] were the first to clearly distinguish that there were three was finally verified by Doerrler et al. [29] by demonstrating
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layers in the E. coli cell envelope. The chemical composition of that modification of LPS with aminoarabinose and phos-
these multilayers took some time to describe. Bladen & phoethanolamine, which occurs at the outer leaflet of the IM,
Mergenhagen [17] found that the outer layer of the Gram- depends on MsbA.
negative Veillonella bacterium could be extracted with The flipping mechanism of MsbA has become a model to
phenol –water and that the extracted material retained the study structural changes that ABC exporters undergo during
endotoxic effects associated with LPS. The intermediate transport. As in other ABC transporters, ATP hydrolysis pro-
layer could be broken down by lysozyme indicating that it vides the energy for MsbA function [27,30]. Structural studies
was peptidoglycan [17]. Thus, it was soon deduced that have demonstrated that MsbA forms a homodimer with
Gram-negative bacteria have an inner and outer lipid fused nucleotide binding and transmembrane domains
membrane with peptidoglycan in between them. The asym- (NBD and TMD, respectively) [31]. Furthermore, X-ray crys-
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Phil. Trans. R. Soc. B 370: 20150029
open closed
(b)
AMPPNP
inward outward
closed nucleotide bound
Figure 2. MsbA undergoes structural conformational changes proposed to mediate LPS flipping. (a) Crystal structures of MsbA (monomers in blue and cyan) suggest
that the NBDs readily separate and dimerize in its inward-facing state (PDB accession no. 3B5W, 3B5X) [31]. (b) MsbA bound to a non-hydrolysable ATP analogue
(AMPPNP in yellow spheres) shows an outward-facing state and associated twisting of the transmembrane helices (PDB accession no. 3B5X, 3B5Y) [31].
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tute the TMD subunits of the LptB2FG ABC transporter [48]. accumulation of newly synthesized LPS at the IM [59].
LptC has a single transmembrane anchor in the IM and a Noting these phenotypes were similar to those resulting
large periplasmic domain that is structurally homologous to from depletion of LptABDE, the Polissi and Silhavy labora-
both LptA and the periplasmic domain of LptD [49,50]. As tories joined forces to demonstrate that, indeed, depletion of
described in §7, in our current model, the LptB2FG ABC trans- each LptABDE and newly characterized LptC resulted in
porter harnesses energy from ATP hydrolysis to power the the same phenotypes [56,59 –61]. This suggested that Lpt fac-
extraction of LPS from the outer leaflet of the IM and load tors function together. Having identified an Lpt protein in
the glycolipid onto LptC. every compartment of the cell suggested a model where
Transit of LPS through the periplasm is mediated through LptB was a cytoplasmic ATPase that provided the energy
structurally homologous domains found in LptC, LptA and source for transport, LptA was a chaperone that shuttled
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Furthermore, X-ray crystal structures of LptB in ATP- and
ADP-bound states have suggested that conformational
changes associated with ATP hydrolysis occur in a groove in
the LptB dimer that lines the membrane interface that interacts
with LptFG (figure 3) [47]. We have proposed that these struc-
tural changes reveal the means of coupling ATP hydrolysis by
LptB to the extraction of LPS by the IM components [47].
The action of the LptB2FG ABC transporter is intimately
related to the transport of LPS along the periplasmic com-
ponents of Lpt. So far the energetics of LPS flow has been
A large body of research conducted in the past two decades has Authors’ contributions. All authors made substantial contributions to con-
finally made great progress in elucidating how the glycolipid ception and design of this review. B.W.S. and N.R. drafted the
manuscript and J.M.M., D.J.S and D.K. revised it critically for import-
LPS is transported from its site of synthesis, the IM, to its final
ant intellectual content. All authors gave final approval of the version
location, the cell surface. This progress was possible by the to be submitted.
use of many different in vivo, in vitro and in silico approaches Competing interests. We declare we have no competing interests.
and the efforts of many laboratories. The accompanying Funding. N.R. is supported by funds from the Ohio State University
paper [14] resumes the discussion about the major discoveries and the National Institute of General Medical Sciences of the
that have been made regarding the periplasmic and OM National Institutes of Health under award no. R01GM100951. D.K.
is supported by funds from the National Institute of Allergy and Disclaimer. The content is solely the responsibility of the authors and 7
Infectious Diseases of the National Institutes of Health under does not necessarily represent the official views of the National
award no. R01AI081059 and U19AI109764. Institutes of Health.
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