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Three types of prokaryotes possess outer membranes with distinct
compositions and structures, suggesting they evolved independently
Milton H. Saier, Jr.
acterial cytoplasmic membranes consist primarily of amphipathic phospholipids with lesser amounts of
glycolipids, providing a degree of
protection from deleterious, toxic
substances that is lacking in organisms having a
single membrane. Almost all bacterial membranes are assembled in bilayers with embedded
integral and associated peripheral membrane
proteins.
In the case of gram-negative bacterial outer
membranes, phospholipids are present in the
inner leaflet of the bilayer, while lipopolysaccharides predominate in the outer leaflet. Carbohydrates in lipopolysaccharides and in glycolipids
and glycoproteins usually extend outwards.
Meanwhile, many gram-positive bacteria, particularly firmicutes and mollicutes with genomes
of low or moderate G⫹C content, lack an outer
B
Summary
• Prokaryotes show far greater diversity at the
molecular level than eukaryotes—particularly
in terms of the structures of their membranes.
• The protective outer membranes of many prokaryotes have very different compositions from
those of their inner membranes.
• With respect to lipid and protein constituents,
the outer membranes of gram-negative bacteria
differ drastically from those of high G⫹C grampositive bacteria and of archaea.
• The outer membranes, along with associated
components for transport and their assembly, of
gram negatives, gram positives, and archaea
appear to have evolved independently of one
another.
membrane. However, acid-fast, high G⫹C
gram-positive bacteria such as mycobacteria
and corynebacteria have mycolic acid-containing outer membranes that are structurally very
different from the outer membranes of gramnegative bacteria.
Some archaea contain membranes of very different protein and lipid constituents, typically
consisting of hydrophobic tails linked by ether
rather than ester bonds to the glycerol-containing lipid backbone. A few archaea have complex
envelopes which, like those in some bacteria,
consist of inner and outer membranes that differ
in their lipid and protein constituents. These
prokaryotic outer membranes are so different as
to suggest independent evolutionary origins.
Perspective
Structure and Evolution of
Prokaryotic Cell Envelopes
Bacterial Cytoplasmic Membranes
The cytoplasmic membrane (CM) of any
bacterium is about 70 Å (7 nm) thick, separating the interior of the cell from the external environment and preventing diffusion of
most substances in and out of the cytoplasm. Thus, the CM acts as a selective
barrier, concentrating metabolites and nutrients within the cell while secreting waste
products and toxins.
Bacterial CMs typically consist of nearly
equal amounts of phospholipid and protein,
accounting for about 70% of cellular phospholipids and 25% of cellular proteins. The
phospholipids are amphipathic, having hydrophobic tails and hydrophilic heads. They
contain a glycerol backbone to which is
attached two fatty acid molecules and a
phosphoryl head group. The membrane is
stabilized by hydrophobic interactions, hy-
Milton H. Saier, Jr.,
is Professor of
Molecular Biology
in the Division of
Biological Sciences,
University of California, San Diego.
Volume 3, Number 7, 2008 / Microbe Y 323
Bacterial Compartments and Organelles
Two-membrane prokaryotic cells are divided into at least five compartments: the cytoplasm, the inner membrane, the periplasm between the two
membranes, the outer membrane, and the extracellular milieu. Moreover,
the inner and outer leaflets of both membrane bilayers can be considered to
be distinct compartments. Numerous dissimilar and evolutionarily distinct
protein insertion complexes are responsible for integrating inner and outer
membrane proteins into the envelope.
In addition to these membranes, some bacteria have membrane-bound
organelles, including magnetosomes, which allow bacteria to orient in
the Earth’s magnetic field, chromatophores where photosynthesis occurs, gas vacuoles that provide for flotation, and sulfur granuoles that
house elemental sulfur. While magnetosome and chromatophore membranes are lipid bilayer based, gas vacuole and sulfur granuole membranes consist of two-dimensional arrays of proteins.
FIGURE 1
Integral membrane proteins in the CM are
also amphipathic, anchored into the membrane with one or more hydrophobic transmembrane segments. However, peripheral
membrane proteins are loosely bound and
interact transiently, often due to ionic attractive forces. The largest known functional
class of CM proteins includes transport proteins that account for about 10% of the cell
proteome. Cytoplasmic membrane proteins
also mediate transmembrane electron flow,
energy generation and interconversion, biosynthesis of lipids and cell envelope precursors, and translocation of these precursors to
an extracytoplasmic locale where they are
assembled. Owing to their hydrophobic
characters, membrane proteins are difficult to study, and consequently, they
account for fewer than 1% of the
known high-resolution protein structures.
Gram-Negative Bacterial
Outer Membranes
The outer membranes (OMs) of typical
gram-negative bacteria are asymmetric
lipid bilayers where the inner leaflet
consists of phospholipids and the outer
leaflet contains a preponderance of lipopolysaccharide (Fig. 1). Gram-negative bacteria lacking lipopolysaccharide
may instead have sphingolipids and/or
various glycolipids.
Because these bilayers show low permeabilities to many solutes, nutrients of
less than 600 Da in size cross the outer
membrane by diffusion through porin
channels. These channel proteins form
Schematic view of the E. coli cell envelope, typical of those of most gram-negative
␤-barrel structures with transmembacteria. Lipopolysaccharide (LPS), embedded within and extending from the outer
brane-spanning segments consisting of
surface of the OM, consists of three moieties: lipid A, core oligosaccharide, and the
O-antigen repeat polysaccharide side chains as indicated. A trimeric porin in the OM, and
amphipathic antiparallel ␤-strands.
integral membrane proteins in the CM, are depicted schematically. The peptidoglycan
␣-Helical proteins in the OMs of these
cell wall in the periplasm separates the two membranes. Modified from a figure on the
organisms are rare, just as ␤-structured
website http://www.microbialcellfactories.com/content/figures/1475–2859-5–13-1.jpg.
proteins in the CMs are rare. Other
compounds such as vitamin B12 and
drogen bonds, and divalent cations such as
iron siderophore complexes cross the OM via
Mg2⫹ and Ca2⫹. The asymmetric bilayer, with
substrate-specific, high-affinity, active transdifferent lipid and protein compositions for the
porters. These OMs also contain structural litwo apposed monolayers, is thus a stable strucpoproteins, membrane-anchored enzymes, and
ture that serves as an encapsulating “bubble”
multicomponent surface structures such as fimwith the cell cytoplasm inside.
briae (organelles of adhesion), pili (organelles of
324 Y Microbe / Volume 3, Number 7, 2008
conjugation), and flagella (organelles of
motility).
Lipopolysaccharides (Fig. 1) consist
of three parts: the proximal, hydrophobic lipid A region that is embedded in
the outer leaflet of the OM; the distal,
hydrophilic O-antigen polysaccharide
region that protrudes into the medium;
and the core oligosaccharide region
that connects lipid A to the O-antigen
repeat units. Lipid A is a polar lipid of
unusual structure in which a backbone
of glucosaminyl-␤-(136)-glucosamine
is esterified with six or seven saturated
fatty acids.
FIGURE 2
Outer Membranes of Acid-Fast,
Gram-Positive Bacteria
Acid-fast bacteria belong to a distinctive actinomycete taxon that includes
mycobacteria, corynebacteria, nocarThe mycobacterial cell envelope showing the most important structural components.
Passage of small hydrophilic molecules through the OM requires the involvement of
dia, rhodococci, and several other genporins (top). The figure illustrates the covalent linkages between cell wall peptidoglycan,
era. All these organisms have an unarabinogalactan and mycolic acids. The width of the CM is about 7 nm (bottom), but that
usual cell envelope composition and
of the outer membrane is about 10 nm.
architecture (Fig. 2). The envelope layers consist of typical CMs of phospholipid and protein, a characteristic wall
The OMs of these bacteria may be the thickest
of unusual structure, and a complex outer layer.
of
all biological membranes yet identified. Their
Although more detail is available for mycobaccell
walls are formed by thick peptidoglycan
teria, the envelopes of these related bacteria are
layers,
covalently linked to arabinogalactan via
similar, especially in terms of ultrastructure and
phosphodiester
linkages. The arabinogalactan,
cell wall composition. External to the mycobacin
turn,
is
esterified
with mycolic acids (Fig. 3).
terial CM is the cell wall, the arabinogalactan/
They
possess
very
long
chains (C60 –90) that may
arabinomannan polysaccharide layer, the OM,
contain
various
branches,
unsaturations, and
and sometimes an external proteinaceous suroxygen
functions
such
as
hydroxyl,
methylated
face (S)-layer. However, the permeability barrihydroxyl,
and
keto
groups.
Mycolic
acids
present
ers in these bacteria depend on both covalently
in
other
actinomycetes
are
similar
in
structure
wall-linked long-chain ␣-alkyl, ␤-hydroxy fatty
but contain shorter chains.
acids, the mycolates, and noncovalently bound
In addition to OM lipoproteins and enzymes,
lipids. Extractable lipids include mannosylated
mycobacteria
possesses several porins, one
inositol and phenolic glycolipids, glycopeptidolipids, and trehalose-based lipooligosaccharides whose properties have intrigued lipid
chemists for decades. These lipids and mycoOuter Membrane Vesicle-Mediated Communication
lates account in large measure for the remarkable drug resistance of mycobacteria,
Gram-negative bacteria sometimes release OM blebs or vesicles of 0.5–
rendering treatment of mycobacterial dis1.0 ␮m in diameter into the culture medium. Typically, they contain
enzymes and signalling molecules that may be delivered to other bacteria
eases difficult. This difficulty is particularly
when the vesicles fuse to the OM of the recipient cell, providing a
important to human health because onemechanism for prokaryotic communication. In other circumstances,
third of the global human population is inthese vesicles deliver bacterial protein toxins to mammalian cells.
fected with mycobacteria, and millions die
from mycobacterial diseases every year.
Volume 3, Number 7, 2008 / Microbe Y 325
FIGURE 3
entirely different from the typical trimeric porins of gram-negative bacteria
that seem to be lacking in acid-fast bacteria. The approximately 50-fold-lower
numbers of porins in acid-fast bacteria
compared to gram-negative bacteria,
and the increased lengths of mycobacterial pores, are two primary determinants of the low permeabilities of outer
mycobacterial membranes to small hydrophilic solutes.
Archaeal Membranes
Although similar in many respects, the
lipid compositions of archaeal and bacterial membranes differ radically. In
particular, archaeal membranes contain
L-glycerol ether-linked lipids rather than
ester-linked D-glycerol lipids found in
bacteria and eukaryotes. Additionally,
bacterial-type peptidoglycan cell walls
are altogether lacking in archaea, which
instead contain cell wall surface layer
Structures of mycolic acids (mycolates) in M. tuberculosis. ␣-Mycolates: the meromyproteins. OMs are not found in the
colate chains contain two cis-cyclopropanes. Methoxymycolates: the meromycolate
better-characterized archaea, although
chains contain an ␣-methyl-ether moiety in the distal position and a cis-cyclopropane or
they have been identified in one class of
an ␣-methyl trans-cyclopropane in the proximal position. Ketomycolates: the meromycolate chains contain an ␣-methyl ketone moiety in the distal position and proximal
these organisms and are probably
functionalities as in the methoxy series. Unsaturations are present in some meromycopresent in others.
late chains (not shown).
Polar ether lipids (Fig. 4) account for
80 –90% of the total membrane lipids
in archaea. The remainder consists of
of which is the low-activity channel protein
neutral
squalenes
and other isoprenoids. While
OmpATb, which enables such cells to adapt to
some
archaeal
species
have only the standard
low pH and survive in macrophages. In contrast
diether
core
lipids,
certain
sulfur-dependent arto other acid-fast bacterial porins, it shows sechaea
also
contain
tetraether
lipids. Polar headquence similarity to outer membrane porin A
groups
in
glycosidic
or
phosphodiester
linkage
(OmpA) homologues of gram-negative bacteria.
to
glycerol
consist
of
polyols,
other
carbohyAnother mycobacterial porin, MspA, forms a
drates, and amino compounds. Ether-containcone-like tetrameric complex with a single cening lipids are more stable than the ester-containtral pore 10 nm in length. This structure is
ing lipids of bacteria and eukaryotes, allowing
archaea carrying them to live in extreme
environments such as strongly acidic lakes
and in hot springs and thermal vents at temProtein Secretion and Mycobacterial Disease
peratures exceeding 100°C.
Mycobacteria secrete proteins that contribute to the pathogenesis of
Ignicoccus is a hyperthermophilic archaeon
many human and animal diseases. Information about how the secreted
belonging to the Desulfurococcales subdiviproteins and the polysaccharides of the capsule cross the outer lipid
sion of the Crenarchaeota. This chemolithobarrier is fragmentary. In any case, the secretion and construction of the
OM depends on very different components from those that are found in
autotrophic organism obtains its energy by
gram-negative bacteria. It is possible that additional knowledge of
reducuing elemental sulfur with molecular
mycobacterial envelope assembly and permeability will lead to new
hydrogen. Cells of Ignicoccus have yet anapproaches to treating mycobacterial diseases.
other unusual cell envelope consisting of cytoplasmic and outer membranes separated
326 Y Microbe / Volume 3, Number 7, 2008
by a periplasmic space of variable
widths containing membrane-bound
vesicles. The OM, approximately 9 nm
wide, contains three types of particles:
numerous irregularly packed single particles, about 8 nm in diameter, putative
pores with a diameter of 24 nm, and
particles arranged in rings surrounding
the pores with a diameter of 130 nm.
FIGURE 4
Unique Archaeal Symbioses
Ignicoccus lives in symbiosis with another archaeon, a very small, singlecelled organism called Nanoarchaeum
equitans (Fig. 5). This cell has one of the
smallest genomes yet sequenced (fewer
than 500,000 bp). In fact, too few genes
are present to code for all the biological
functions that are essential for life,
meaning it can live only by being together with Ignicoccus. Among the
missing functions are the enzymes that
catalyze lipid biosynthesis. If these enzymes are really absent from this organism, how does Nanoarchaeum construct its CM?
Ultrastructural analyses reveal not
only the two-membrane envelope of Ignicoccus, but also the presence of intraperiplasmic vesicles. Based on analyses
showing that the lipid and protein compositions of the inner and outer membranes are different, it appears that
these vesicles derive from the CM of
Ignicoccus. Moreover, the lipids in
the nanoarchaeal membrane resemble
those in the CM of Ignicoccus. These
observations suggest that one organism
makes the lipids for both members of
this pair, and that the vesicles mediate
passage from one to the other. Moreover, some transport proteins in the
nanoarchaeal membrane, as well as essential cytoplasmic enzymes, apparently also derive from its symbiotic
partner, Ignicoccus. Thus, these two
symbiotic archaea apparently developed unusual mechanisms for intercellular communication and molecular
transfers involving periplasmic vesicles.
Structure of a typical archaeal ether lipid, based on L-glycerol (top), compared to that of
a typical bacterial ester lipid, based on D-glycerol (bottom). Primary structural differences
are illustrated. (Modified from the website http://www.ucmp.berkeley.edu/archaea
/archaeamm.html.)
FIGURE 5
An electron microscopic depiction of an Ignicoccus cell (bottom), showing the inner and
outer membranes, in symbiotic association with two Nanoarchaeum cells (top). (Reproduced from H. Huber, M. Hohn, R. Rachel, T. Fuchs, V. Wimer, and K. Stetter, A new
phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature
417:63– 67, 2002).
Volume 3, Number 7, 2008 / Microbe Y 327
Conclusions
Prokaryotes possess cell envelopes of extremely
varied composition and structure. In both
prokaryotic domains, as in organelles of eukaryotes, the envelopes may have one or two
membranes, which always possess different
combinations of lipids and proteins.
We are now coming to appreciate the complexities of the assembly machineries that function to construct these envelopes. Many are
present in specific membranes while others span
the entire cell envelope structure. Moreover,
completely different transport components are
found in the inner and outer membranes of
organisms that have both.
The structural data available for the OMs of
gram-negative bacteria, high G⫹C gram-positive bacteria, and archaea suggest that outer
prokaryotic membranes probably evolved independently in these three organismal types. Further research should help to clarify other important but poorly understood issues dealing with
basic aspects of the functions, structures, biogenesis, and evolution of these membranes.
Moreover, because fewer than 1% of prokaryotic life forms have been characterized, it seems
likely that other envelope types will be discovered, making this feature a snapshot of progress
rather than a definitive report.
SUGGESTED READING
Brennan, P. J. 2003. Structure, function, and biogenesis of the cell wall of Mycobacterium tuberculosis. Tuberculosis
83:91–97.
Dowhan, W. 1997. Molecular basis for membrane phospholipids diversity: why are there so many lipids? Annu. Rev.
Biochem. 66:199 –232.
Kartmann, B., S. Stengler, and M. Niederweis. 1999. Porins in the cell wall of Mycobacterium tuberculosis. J. Bacteriol.
181:6543– 6546.
Koga, Y., and H. Morii. 2005. Recent advances in structural research on ether lipids from archaea including comparative and
physiological aspects. Biosci. Biotechnol. Biochem. 69:2019 –2034.
Rachel, R., I. Wyschkony, S. Riehl, and H. Huber. 2002. The ultrastructure of Ignicoccus: evidence for a novel outer
membrane and for intracellular vesicle budding in an archaeon. Archaea 1:9 –18.
Saier, M. H., Jr. 2006. Protein secretion and membrane insertion systems in gram-negative bacteria. Microbe 1:414 – 419.
328 Y Microbe / Volume 3, Number 7, 2008