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Chapter 2
The Outer Membrane of Gram-negative
Bacteria and the Cytoplasmic Membrane
The Outer Membrane of Gram-Negative Bacteria
The major permeability barrier in any membrane is the lipid bilayer structure, and
its barrier property is inversely correlated with its fluidity. Bacteria cannot make
this membrane much less fluid or it will start to interfere with the normal functions
of the membrane proteins, so some bacteria have constructed an additional structure
that surrounds the cell outside the cytoplasmic membrane. An example of this are
Gram-negative bacteria, such as E. coli, which surround themselves with a second
outer membrane which functions as an effective barrier.
It was actually shown by electron microscopy that the Gram-negative bacteria
are covered by a membrane layer outside the peptidoglycan layer. This outer
membrane (OM) should not be confused with the cytoplasmic or inner membrane.
The two membranes differ by their buoyant densities, and OM can be isolated from
bacterial lysates by sucrose equilibrium density centrifugation.
The outer leaflet of the outer membrane bilayer is composed of an unusual lipid,
lipopolysaccharide (LPS), rather than the usual glycerophospholipid found in most
other biological membranes. LPS is composed of three parts: a proximal hydrophobic lipid A region, a core oligosaccharide region connecting a distal O-antigen
polysaccharide region to lipid A. This distal region protrudes in the medium.
All the fatty acid chains present in LPS are saturated which significantly reduces
the fluidity. Also, the LPS molecule contains six or seven covalently linked fatty
acid chains, in contrast to the glycerophospholipid that contains only two fatty acid
residues.
Hydrophobic probe molecules have been shown to partition poorly into the
hydrophobic portion of LPS and to permeate across the outer membrane bilayer
at about one-fiftieth to one-hundredth the rate through the usual lipid bilayers. The
vast majority of clinically important antibiotics and chemotherapeutic agents show
some hydrophobicity that allows them to diffuse across the membrane. The LPScontaining asymmetric bilayer of the bacterial outer membrane serves as an
G.N. Cohen, Microbial Biochemistry,
DOI 10.1007/978-90-481-9437-7_2, # Springer ScienceþBusiness Media B.V. 2011
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2 The Outer Membrane of Gram-negative Bacteria and the Cytoplasmic Membrane
efficient barrier against rapid penetration by these lipophilic antibiotics and chemotherapeutic agents.
Bacteria with this barrier must develop methods to bring in nutrients from their
surroundings, however. Apart from other systems which will be developed in
Chapter 4, the outer membrane contains for this purpose porins, a special class of
proteins, which produce non-specific aqueous diffusion channels across the membrane. The properties of the porin channels exclude antibiotics crossing them by
having a very small diameter (7 by 10 Å in their most constricted portion) which
slows down or completely stops antibiotic influx, and by lining the channel with
charged amino acid residues which orient the water molecules in a fixed direction.
These charged residues make the influx of lipophilic molecules difficult because the
energetically favorable orientation of the water will be disturbed.
Gram-positive bacteria, mycobacteria, have also been found to have developed
an outer leaflet to protect themselves from drugs. The mycobacterial barrier also
consists of an unusual lipid, mycolic acid. Mycolic acid contains more than
70 carbon atoms with only a few double bonds. Also, where LPS had six or seven
fatty acids joined to a single head group, here, hundreds of mycolic acid residues are
covalently linked to a common head group, an arabinogalactan polysaccharide,
which in turn is covalently linked to the underlying peptidoglycan structure.
Nutrients diffuse into the cells through mycobacterial porin, which is present in
very small amounts and allows only very slow diffusion of small molecules through
its channel. Antibiotics are severely retarded in entering the bacteria by the low
permeability combination of the porin channels and the lipid matrix, which allows
some resistance.
The Cytoplasmic Membrane
The cytoplasmic membrane, also called cell membrane or plasma membrane, is
about 7 nm thick. It lies internal to the cell wall and encloses the cytoplasm of the
bacterium.
Like all biological membranes in nature, the bacterial cytoplasmic membrane is
composed of phospholipid and protein molecules. In electron micrographs, it
appears as two dark bands separated by a light band and is actually a fluid
phospholipid bilayer imbedded with proteins. With the exception of the mycoplasmas (the only bacteria that lack a cell wall), prokaryotic membranes lack sterols.
Many bacteria, however, do contain sterol-like molecules called hopanoids. Like
the sterols found in eukaryotic cell membranes, the hopanoids most likely stabilize
the bacterial cytoplasmic membrane.
The phospholipid bilayer is arranged so that its polar ends (the phosphate and
glycerol portion of the phospholipids) form the outermost and innermost surface of
the membrane while its hydrophobic ends (the fatty acid portions of the phospholipids) are insoluble in water.
Energy Generation
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The cytoplasmic membrane is a selectively permeable membrane that determines what goes in and out of the organism. All cells must take in and retain all the
various chemicals needed for metabolism. Water, carbon dioxide and oxygen, and
lipid-soluble molecules simply diffuse across the phospholipid bilayer, diffusion
being powered by the potential energy of a concentration gradient and does not
require the expenditure of metabolic energy. All other molecules require carrier
molecules to transport them through the membrane. Mechanisms by which materials move across the cytoplasmic membrane will be examined in Chapter 4.
A number of other functions are associated with the cytoplasmic membrane: in
addition of being the site of peptidoglycan synthesis, both in the growing cell wall
and in the transverse septum that divides the bacterium during bacterial division
and the site of phospholipid and some protein synthesis required for the production
of more cytoplasmic membrane, it is the site of energy production through the
electron transport system for bacteria with aerobic and anaerobic respiration and
photosynthesis for bacteria converting light energy into chemical energy.
Energy Generation
Many cells use respiratory processes to obtain their energy. During respiration,
organic or inorganic compounds that contain high energy electrons are broken
down, releasing those electrons to do work. These electrons find their way to the
membrane where they are passed down a series of electron carriers. During this
operation, protons are transported outside the cell. The outside of the membrane
becomes positively charged; the inside becomes negatively charged.
This proton gradient energizes the membrane, much like a battery is charged.
The energy can then be used to do work directly, a process known as the proton
motive force, or can be channeled into a special protein known as ATP synthase.
ATP synthase can convert ADP to ATP, the ATP doing the work.
ATP Synthase
The ATP synthase enzymes have been remarkably conserved through evolution.
The bacterial enzymes are essentially the same in structure and function as those
from mitochondria of animals, plants and fungi, and the chloroplasts of plants. The
early ancestry of the enzyme is seen in the fact that the Archaea have an enzyme
which is clearly closely related, but has significant differences from the Eubacterial
branch. The H+-ATP-ase found in vacuoles of the eukaryote cell cytoplasm is
similar to the archaeal enzyme, and is thought to reflect the origin from an archaeal
ancestor.
In most systems, the ATP synthase sits in the membrane (the “coupling”
membrane), and catalyses the synthesis of ATP from ADP and phosphate driven
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2 The Outer Membrane of Gram-negative Bacteria and the Cytoplasmic Membrane
by a flux of protons across the membrane down the proton gradient generated by
electron transfer. The flux goes from the protochemically positive (P) side (high
proton electrochemical potential) to the protochemically negative (N) side. The
reaction catalyzed by ATP synthase is fully reversible, so ATP hydrolysis generates
a proton gradient by a reversal of this flux. In some bacteria, the main function is to
operate in the ATP hydrolysis direction, using ATP generated by fermentative
metabolism to provide a proton gradient to drive substrate accumulation, and
maintain ionic balance.
ADP þ Pi þ nHþ P , ATP þ nHþ N
In bacteria, the P side is the outside (the periplasm in gram negative bacteria), the
N side the cytoplasm in chloroplasts.
Subunit Composition of the ATP Synthase
The simplest system is that from E. coli. The ATP synthase can be dissociated into
two fractions by relatively mild salt treatments.
A soluble portion, the F1 ATPase, contains five subunits, in a stoichiometry of
3a:3b:1g:1d:12. Three substrate binding sites are in the b-subunits. Additional
adenine nucleotide binding site in the a-subunits are regulatory. The F1 portion
catalyzes ATP hydrolysis, but not ATP synthesis.
Dissociation of the F1 ATPase from the membranes of bacteria or organelles
leaves behind a membrane embedded portion called Fo. This consists (in E. coli) of
three subunits a, b and c, with relative stoichiometries of 1:2:9–12. The c-subunit is
very hydrophobic, and forms a helix turn helix structure which spans the membrane
twice, with a hydrophilic loop on the side of attachment of F1. There is a conserved
acidic residue half-way across the membrane in the C-terminal helix.
After dissociation, the membranes are permeable to protons. The proton leak can
be stopped by addition of inhibitors, which are also inhibitors of ATP synthesis in
the functional complex. Two “classical” inhibtors are commonly used. Oligomycin
binds at the interface between Fo and F1; dicyclohexylcarbodiimide (DCCD) binds
covalently to the conserved acidic residue in the c-subunit of Fo. One DCCD per
ATPase is sufficient to block turn-over, suggesting a cooperative mechanism. The
action of these inhibitors indicates that the proton permeability of the Fo is a part of
its functional mechanism.
The proton leak can be plugged, and a functional ATP synthase can be reconstituted, by adding back the F1 portion to membranes containing the Fo portion.
A model of ATP synthase, derived from image averaging and cryo-electron
microscopy, showing a second stalk is presented in Figs. 1 and 2.
The structure of the soluble (F1) portion of the ATP synthase from beef heart
mitochondria, as well as the structure of the complete F1–Fo ATP synthase from
Subunit Composition of the ATP Synthase
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Fig. 1 Model of ATP
synthase. Fo is embedded in
the membrane (From
W. Junge, H. Lill and
S. Engelbrecht; TIBS, 22,
420–423 (1997))
δ
F1
α
β
γ
b
ε
b
c
a
F0
β β
Fig. 2 Model of ATPsynthase. As in Fig. 1, seen
from another angle
δ
α
α
γ
ε
b2
c12
a
yeast mitochondria have been solved by X-ray crystallography by John Walker and
his associates.
The ATP synthase operates through a mechanism in which the three active sites
undergo a change in binding affinity for the reactants of the ATP-ase reaction, ATP,
ADP and phosphate, as originally predicted by Paul Boyer. The change in affinity
accompanies a change in the position of the g-subunit relative to the a, b-ring,
which involves a rotation of the one relative to the other. In the direction of ATP
synthesis, the rotation is driven by a flux of H+ down the proton gradient, through a
coupling between the g-subunit, and the c-subunit of Fo. This rotation has now been
demonstrated experimentally.
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ATP Synthesis in Archaea
Archaea are a heterogenous group of microorganisms that often thrive under harsh
environmental conditions such as high temperatures, extreme pHs and high salinity.
As other living cells, they use chemiosmotic mechanisms along with substrate level
phosphorylation to conserve energy in form of ATP. Because some archaea are
rooted close to the origin in the tree of life, these unusual mechanisms are considered to have developed very early in the history of life and, therefore, may represent
first energy-conserving mechanisms. A key component in cellular bioenergetics is
the ATP synthase. The enzyme from archaea represents a new class of ATPases, the
A1A0 ATP synthases. They are composed of two domains that function as a pair of
rotary motors connected by a central and peripheral stalk(s). The structure of the
chemically-driven motor (A1) was solved by small-angle X-ray scattering in
solution, and the structure of the first A1A0 ATP synthases was obtained recently
by single particle analyses. These studies revealed novel structural features such as
a second peripheral stalk and a collar-like structure. In addition, the membraneembedded electrically-driven motor (A0) is very different in archaea with sometimes novel, exceptional subunit composition and coupling stoichiometries that
may reflect the differences in energy-conserving mechanisms as well as adaptation
to temperatures at or above 100 C.
Photosynthetic cells also have a membrane system. Here light excites electrons
and the electrons are again passed down through a series of electron carriers, a
proton motive force is generated and ATP is synthesized. All the photosynthetic
machinery is situated in the membrane.
The cytoplasmic membrane contains the bases of flagella used in motility.
Selected References
ATP Synthase
J. P. Abrahams, A. G. Leslie, R. Lutter and J. E. Walker, Nature 370, 621–628 (1994).
H. Noji, Y. Ryohei, Y. Masasuke and K. Kinosita Jr., Nature 386, 299–302 (1997).
W. Junge, H. Lill and S. Engelbrecht, TIBS, 22, 420–423 (1997).