Bacterial Membrane Advanced article

Transport: Organization of . Introduction

Article Contents

Membrane Activities . Permeability of the Matrix of the Outer or Inner

Membranes

. Passage of Metabolites across the Outer Membrane of

Etana Padan, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel Gram-Negative Cells
. Passage of Metabolites through the Inner Membrane
Based in part on the previous version of this Encyclopedia of Life Sciences
(ELS) article, Bacterial Membrane Transport: Organization of . Primary and Secondary Transport Systems

Membrane Activities by Peter C Maloney. . Chemiosmotic Circuits

. Integrating Primary and Secondary Transporters

Bacteria have transport systems enabling them to accumulate needed nutrients, . Channels
extrude unwanted by-products and modify their cytoplasmic content of protons and . Virtual Proton Pumps: A Proton-motive Force Emerges
salts so as to maintain a composition conducive to growth and development. Most from Combining Transport and Metabolism

bacterial transport systems resemble their counterparts in eukaryotic cells, and similar . Is a Proton(ion)-motive Necessary?

principles operate in both cell types. Because bacterial transporters are often easier to . pH Homeostasis in Bacteria

deal with experimentally, they have become important models for their eukaryotic . Conclusion

brethren. The recent determination of the crystal structure of several bacterial Online posting date: 15th September 2009
transporters including ones that share homology with human transporters that are
important in health and disease has been a major breakthrough. It opened the way both
to the understanding of the mechanism of active transport as well as to rational drug
design.

Introduction more), Ca2+ (a free ionic concentration of 0.1 mmol L21 or

Most bacteria share in common two barrier structures: an
external cell wall, of varying composition in different cell
types, and an inner phospholipid bilayer, the plasma mem-
brane. The cell wall resists cell swelling, the unavoidable
consequence of enclosing a cytoplasm rich in impermeable
molecules within a semipermeable and flexible plasma
membrane. This membrane itself functions as a partial
barrier to the diffusion of water-soluble materials, allowing
a temporary functional isolation of the cytoplasm from the
external world. It is the job of transport proteins embedded
in the plasma membrane bilayer to ensure that over the
long run the cytoplasm sustains a composition hospitable
to growth, development and cell division. This is done by
the transport of nutrients inward and waste products out-
ward, and by the restoration of ionic gradients dissipated
by diffusive events. Indeed, transport systems comprise the
essential homeostatic mechanism for regulating cellular
ionic balance of protons (usually approximately pH 7.5),
Na+ (usually 10 mmol L21 or less), K+ (200 mmol L21 or
ELS subject area: Microbiology
How to cite:
Padan, Etana (September 2009) Bacterial Membrane Transport:
Organization of Membrane Activities. In: Encyclopedia of Life Sciences
(ELS). John Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0001418.pub2
less) and so on. In most cases, these transporters draw on an
external source of energy (e.g. adenosine triphosphate
(ATP) hydrolysis, or an electrochemical ion gradient), but
in some settings the transporters themselves act to establish
these gradients. See also: Bacterial Cytoplasmic Mem-
brane; Bacterial Membrane Transport: Superfamilies of
Transport Proteins; Intracellular Transport; Ion Trans-
port Across Nonexcitable Membranes
Beyond the cell wall, different bacteria organize their
surroundings in different ways. All bacteria shape their
environment, most commonly by secretion of enzymes,
toxins or pheromones, and by decoration of their external
surfaces with an ensemble of macromolecules. By far the
most extravagant scenario occurs in Gram-negative cells,
which enclose their cell wall with a second bilayer, the outer
membrane, whose outer leaflet is enriched for complex
phospholipids and polysaccharides. In these cells, the space
between the inner and outer bilayers becomes yet another
functional compartment, the periplasmic space. Indeed,
the volume of the periplasm can in some cases rival that of
the cytoplasm, attesting to its potential, if not its actual
function. See also: Bacterial Cell Wall
Although, cell walls and membranes may differ greatly in
structure and composition among the main bacterial
groups (the archaea, the Gram-positive and Gram-nega-
tive eubacteria), the various transport proteins of the
plasma membrane are strikingly homologous with each
other and often equally homologous to their eukaryotic
counterparts. As a result, despite their variable settings, we
believe such proteins exploit the same fundamental

ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net 1
 Bacterial Membrane Transport: Organization of Membrane Activities
principles relating structure and function. It seems likely
this commonality of design (structural, biochemical and
physiological) reflects an evolutionary chain that extends
back to times well before the diversification of cells into the
now-recognized three kingdoms: the archaea, the eubacte-
ria and the eukaryotes. Indeed, one may argue that the
origin of a cellular basis of life was feasible only with
discovery of how to construct such transport systems.
See also: Archaeal Cell Walls
Permeability of the Matrix of the Outer
or Inner Membranes
Membranes as barriers
The lipid nature of cell membranes presents a barrier to the
rapid and free diffusion of water-soluble molecules. Prac-
tically speaking, such membranes are absolute barriers to
macromolecules, but only partial barriers to passage of
small to moderately sized molecules. Inward diffusion of,
say, glucose (a nonelectrolyte) can take tens of minutes or
several hours. Now, this might not seem such a long time to
wait: but if competitors are waiting as well, it may be to
your advantage to avoid even this partial barrier to entry
(or exit). Thus, when there is (or has been) selective ad-
vantage, one finds membrane proteins accelerating the
movement of molecules as small and permeant as glycerol
(GlpF in Escherichia coli), urea (UreI in Helicobacter
pylori) and even water itself (AqpZ in E. coli and else-
where). Much of the story of membrane transport, then, in
bacteria as everywhere, is a recounting of how cells cir-
cumvent this partial diffusion barrier to increase the input
or output of compounds so as to favourably modify the
internal or external environment. See also: Bacterial
Cytoplasmic Membrane; Bacterial Intracellular Mem-
branes; Protein Translocation Across Membranes; Water
Channels; Water Channels: Aquaporins; Water Transport
across Cell Membranes
Passage of Metabolites across the
Outer Membrane of Gram-Negative
Cells
Porins
The Gram-negative cell outer membrane allows nearly un-
restricted passage of small to moderate size molecules (up
to approximately 600 Da) into the periplasm. This is due to
the presence of large channels known as porins, present in
hundreds to perhaps tens of thousands of copies per cell,
depending on the example. The outer membrane is there-
fore a selective sieve that admits most common nutrients
while still excluding macromolecules. Porin structure has
been revealed by crystallography. The most numerous
2 ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net
porins are highly stable trimers whose subunit is a
16–18stranded b barrel (but see later discussion). The lu-
men of the individual barrels is penetrated by loops of var-
ying size and charge. In some cases, this confers a degree of
selectivity that varies from weak to absolute.
Substrate-selective porins
Some porins are specifically designed to aid the flux of their
substrates. This seems most often to reflect cases in which
the substrate has a mass, shape or charge that would oth-
erwise restrict its passage. A well-understood example is
maltoporin (LamB), which facilitates the influx of glucose
polymers (maltotriose and oligomers of up to 7–8 units),
because the shape and composition of loops lining the
maltoporin b-barrel allow favourable interaction with the
pyranose ring. Similarly, the PhoE porin provides a selec-
tivity enhancing flux of anionic phosphate and organic
phosphates, due to the presence of a positive charge on the
barrel lining. In the typical laboratory experiment, the need
for such substrate-selective porins is not always obvious, as
it is common to supply the target compounds in high
amounts or to provide alternative materials of smaller size.
On the other hand, these selectivities confer distinct ad-
vantage when substrate is present at a concentration low
enough to limit growth, a condition that might easily be
encountered in the wild (Bishop, 2008). See also: Mem-
brane Proteins
Porins, as one might imagine, serve not only to perm-
eabilize selectively the outer membrane. They can also serve
as receptors for external elements such as phage, toxins or
micronutrients and their chelates. For example, a porin is
used by E. coli as the receptor for iron-siderophore com-
plexes (FepA) or ferric hydroxylates (FhuA). The recent
crystallization of FepA and FhuA suggests that these porins
may actually act as selective transporters, something not
anticipated by early studies. As 22-stranded b-barrels, FepA
and FhuA are the largest known porins, yet they appear
completely plugged at their periplasmic end by a globular N-
terminal domain and at their external face by extracellular
loops. Evidently, opening and closing at both ends is coor-
dinated with ligand binding at the outer surface, so the side-
rophore can be trapped in the periplasm (Postle, 1999). This
method of transport across the outer membrane is orches-
trated by a protein known as TonB (the name reflects that
TonB mutants are resistant to phage T1). In a way not yet
understood, TonB associates with both its target porin/s and
several inner membrane proteins to provide the energy
needed for substrate accumulation within the periplasm.
Subsequently, substrate is taken into the cell by an adenosine
triphosphate (ATP)-dependent transporter located in the
inner membrane. Importantly, porins contribute to drug
resistance in bacteria (Pages et al., 2008)
Donnan effects
The periplasmic space of Gram-negative cells, although
devoid of deoxyribonucleic acid (DNA) and ribonucleic
 Bacterial Membrane Transport: Organization of Membrane Activities
acid (RNA), does contain proteins and oligosaccharides
that are too big to escape through porins. The concentra-
tion of these impermeant solutes, relative to the solute
content of the cytoplasm and the external medium, deter-
mines the balance of water flux into and out of the peri-
plasm, and hence periplasmic volume. Since the solute
content of the cytoplasm is typically well regulated, move-
ment of water into and out of periplasm is largely deter-
mined by the concentration of impermeant species in the
external medium. In dilute solutions, the periplasm will
swell; if external (impermeable) solute content rises, the
periplasm will shrink.
The periplasmic proteins and oligosaccharides have nega-
tive charge at neutral pH, and the presence of so-called
‘fixed’ negative charges actually leads to development of an
electrical potential, negative within the periplasm, across the
outer membrane. All mobile ions are in equilibrium with this
Donnan potential (Kutchai, 1998) in accordance with the
Nernst relationship. Thus, with respect to the external
world, permeant cations (e.g. Na+, K+, H+, Ca2+) accu-
mulate, whereas anions (e.g. Cl2, H2 PO
2

4 and SO4 ) are
expelled. Yet, under acidic extracellular pH, the periplasmic
pH has recently been shown to equal the external pH (Wilks
and Slonczewski, 2007). See also: Cell Membranes:
Intracellular pH and Electrochemical Potential
It is important to appreciate that the origins of a Donnan
potential at the outer membrane differ from those of the
electrical potential usually present across the inner membrane
(later). A Donnan potential reflects the equilibrium distribu-
tion of small mobile ions, whereas the electrical component of
an ion-motive force reflects the fact that such small ions are
not at equilibrium. Work (e.g. accumulation of something in
the periplasm) cannot be accomplished by linking an event to
ion movement across the outer membrane, because the ion is
at equilibrium on both sides of the membrane. By contrast,
work is readily accomplished by a coupling of an event to
ionic movement at the cytoplasmic membrane, where ions
can flow down their electrochemical gradients.
Passage of Metabolites through the
Inner Membrane
Passive and facilitated diffusion
As noted earlier, small water-soluble molecules (water,
urea, glycerol) can show significant passive diffusion
through the lipid bilayer. Even so, one sometimes finds
proteins that further accelerate these movements, presum-
ably reflecting a past history of selective pressure. In the
three examples just cited, the mechanism of facilitation
appears to be that of a channel; in two of these cases, the
structural basis of the channel is known to be a ring of a
helices, quite different from the b barrels forming porin
channels. But while such channels do ‘facilitate’ diffusion,
the term ‘facilitated diffusion’ is typically reserved for a
different context; the term was originally coined to describe
ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net
the catalysed (facilitated) movements of sugars and amino
acids into animal cells, and it retains something of this
character even today.
Most lipid-soluble molecules pass readily through phos-
pholipid membrane(s), so that access to the bacterial cyto-
plasm (or escape from it) is relatively easy for gases,
detergents, hydrophobic peptides, the neutral species of
organic weak acids or bases and so on. In some of these
cases, evolution faced the problem of extruding such ma-
terials after their initial inward diffusion. As a result, one
often finds transporters devoted to export, such as the
eponymous ‘drug’ transporters. One presumes that the au-
thentic substrates of such transporters are not the synthetic
drugs against which they now act. Instead, their true sub-
strates are bacteriostatic or bacteriocidal materials made
by plants or other bacteria (or possibly by themselves).
Gram-negative cells have an especially interesting class of
exporters that manage to extrude substrate directly to the
outside world. In these instances, substrates are placed in
the lumen of a long and complex tubular molecule that
extends from the surface of the inner membrane all the way
through the outer membrane. AcrAB-TolC of E. coli which
pumps out a very wide range of drugs and is responsible for
many drug resistant bacteria has been most extensively
studied with respect to structure and function (Murakami,
2008; Nikaido and Takatsuka, 2009).
Primary and Secondary Transport
Systems
It is common practice to classify various transport systems
as either primary or secondary, according to their use of
energy. An external source of energy – ATP hydrolysis,
say, or the absorption of light – drives reactions of primary
transport. The list of mechanistically distinct primary
transporters is quite small (although there are a large
number of separate examples). In bacteria, a list of com-
mon examples includes: (1) the F0F1 ATPases (F, V or A
types) that couple ATP hydrolysis or synthesis to the
movement of H+ or Na+; (2) the redox pumps linked to H+
or Na+ movement and (3) the P type (or E1E2) ATPases
that move H+, Na+, K+ or Mg2+, Ca2+ and other metals.
Less frequently encountered would be (4) an Na+-translo-
cating decarboxylase; (5) the light-driven bacterio- and
halorhodopsins that move H+ or Cl2 and (6) cases in which
methyl transfer reactions are coupled to cation extrusion in
the archaea. By transporting ions (usually cations), these
systems generate transmembrane ionic gradients, and this
is significant to the later discussion of chemiosmotic cir-
cuits (later). (7) The ABC solute ATPases move a wide
variety of substrates, inward or outward, at the expense of
ATP hydrolysis and in eukaryotes cause resistance of can-
cer cells to chemotherapy. They are discussed elsewhere in
this encyclopedia. See also: ATPases: Ion-motive; Bacterial
Membrane Transport: Superfamilies of Transport Pro-
teins; Ion Motive ATPases: V- and P-type ATPases
3
 Bacterial Membrane Transport: Organization of Membrane Activities

It is easier to conceptualize the reactions of secondary
transport. Here, substrates simply move from one side of
the membrane to the other. The reaction is thermodynam-
ically ‘downhill’, and there is no chemical transformation
of its participants. Moreover if more than one substrate is
moving, it is even possible that the downhill movement of
one substrate might actually drive uphill movement of an-
other, leading to what is sometimes called secondary
‘active’ transport. A good example of this latter reaction is
the coupled transport of H+ and lactose in E. coli. Because
the cell is electrically negative relative to the outside, and
because internal H+ concentration is low (alkaline), the elec-
trical and chemical forces promoting H+ entry can be used to
drive sugar accumulation as the two enter together. (This
specific example is treated more quantitatively later on.)
Chemiosmotic Circuits
Note that the primary pumps (classes 1–6, earlier) establish
ion gradients, whereas the secondary transporters might
use an ion as a substrate. If the two reactions occur in the
same membrane, a kind of cycle can develop as the ion is
pumped in one direction, ‘uphill’, and returns to the origi-
nal surface, ‘downhill’, in co- or countertransport with an-
other substrate (Figure 1). This arrangement is known as a
chemiosmotic circuit.
Intellectual and experimental origins
Our view of chemiosmotic circuits comes as the merger of
two lines of thought, both initiated in the early 1950s. The
earlier of the two concerned a new idea about how sec-
ondary transport might occur. The later idea, envisioned
by Peter Mitchell, dealt with primary transport events,
particularly proton pumps, and how they might integrate
with secondary processes in the form of a chemiosmotic
circuit. In 1952, W Widdas developed a new model, the
‘mobile carrier’, to describe the transport of glucose by the
sheep placenta. Before this, the transport of sugars or
amino acids was discussed using terms set out by physiolo-
gists interested in the distribution of Na+, Cl2 and K+
across muscle and nerve membranes or in the movement of
salt and water across epithelia. The field was rich in coeffi-
cients of permeability and diffusion and in arguments
based on thermodynamics, none of which really helped in
understanding glucose transport. So Widdas took a differ-
ent tack. He imagined that a catalytic element (the ‘carrier’)
associated with substrate (glucose) in a one-for-one com-
plex that could diffuse (or reorient) across the thickness of
the membrane. At the other surface, substrate could be
discharged by dissociation, allowing the unloaded carrier
to return (reorient) to the original side, where it would be
available to take part in yet another cycle. One full turn-
over, then, gave the net transport of a single substrate
molecule. (Note how this differs from transport through
channels. In the equivalent turnover time – that is, between
the opening and closing of a channel – many tens of thou-
sands of substrate ions might move, each with only a fleet-
ing glance at the protein.) See also: Mitchell, Peter Dennis;
Oxidative Phosphorylation
This new view accounted for the kinetics of glucose trans-
port by the placenta, Widdas’ experimental target. Indeed,

S2 H
+ H+ 2HG6P1-
S1 Na+ S3

3 4
2
1 5

Na+
H+ G6P2-
H+ ETC proton- 2H+ G6P2-
pumps
H+ ADP+Pi ATPi
pHin 7.5
−−
+ −
+
+ H+/ATPase

H+

Figure 1 Chemiosmotic organization at the bacterial plasma membrane. A chemiosmotic H+ circuit initiated by electron transport-linked proton pumps is found
in aerobes and in facultative organisms (e.g. E. coli ) that are growing aerobically. Secondary transporters that complete this circuit (nos 1–5) are described in the
text. The H+/ATPase is using the proton electrochemical gradient to synthesize ATP. In anaerobic bacteria similar circuit can be maintained by anaerobic electron
transport or by the H+/ATPase that can function in reverse, hydrolizing ATP to maintain a chemiosmotic circuit (see Harold and Maloney, 1996). ETC proton
pumps: electron transport-linked proton pumps.

4 ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net
 Bacterial Membrane Transport: Organization of Membrane Activities
the model is as useful a simplification today as it was in
1952, and no better way of summarizing the kinetic prop-
erties of these proteins has appeared. More important,
this view changed the intellectual and experimental land-
scape of the field. In particular, the suggestion of a
stoichiometric binding between carrier and substrate legit-
imized use of biochemistry in the study of membrane
transport.
Widdas studied sugar transport mediated by what is
now known as GLUT1 (glucose uniporter), a glucose
transporter in mammalian systems. Proof of the pro-
teinaceous and catalytic nature of the carrier (and proof
that bacteria have a cell membrane) was offered a year
later by Peter Mitchell, who described the exchange of
inorganic phosphate in Staphylococcus aureus (then
called Micrococcus pyogenes), a reaction mediated by
the hexose 6-phosphate transporter, UhpT. A short time
later, in 1956, the lactose transporter (LacY) of E. coli
was described, setting the stage for a genetic analysis.
The final formative event in the field of secondary trans-
port was the insight of Crane (in 1959) – that a carrier
might combine with more than one substrate, as in the
cotransport of glucose and Na+ (by SGLT1, glucose/
Na+ symporter) in the mammalian gut. See also: Blood–
Brain Barrier
As it happens, these founding events also introduced the
main biochemical mechanisms we now associate with sec-
ondary transport. Thus (using Mitchell’s terminology),
GLUT1 mediates the reaction of uniport, in which a subst-
rate moves alone, along its electrochemical gradient. UhpT
exemplifies antiport, in which substrates (here, phosphate
and sugar phosphate) exchange one with the other, while
LacY (or SGLT1) engages in symport or cotransport, as its
two substrates (H+ and lactose, Na+ and glucose) move
together in the same direction.
The coordination of secondary and primary transporters
in a chemiosmotic circuit was not appreciated until Mitc-
hell’s later work, begun in the 1960s, transformed our view
of how mitochondria and chloroplasts carry out oxidative
and photophosphorylation.
The contemporary view
In bacteria as elsewhere, secondary reactions integrate with
one or more primary reactions in a chemiosmotic circuit of
the sort shown in Figure 1. In that example, the circuit is
initiated by an electron-transport-linked proton pumps, as
might occur in aerobic bacteria or in facultative organisms
growing in aerobic conditions. Here, outward movement
of protons, driven by redox reactions, generate a proton-
motive force, Dp, comprised of both chemical and electrical
parts and commonly evaluated in electrical terms (mV) at
approximately 278C:
Dp ¼ Df
ð2:3RT=FÞDPH
½1
¼ Df
60DPH ðunits of mVÞ
where Dj gives the membrane potential (interior negative),
DpH indicates the pH gradient across the membrane
ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net
(external pH less internal pH) and R, T and F have their
usual values. (R, the universal gas constant, is 1.99 kcal -
mol21 K, T temperature in units of K and F Faraday’s
constant, equal to 23 kcal mol21 volt.) By convention, the
negative sign of Dp reflects the net tendency for protons to
move inward. In bacteria, maximal values for Dp are on the
order from 2150 to 2180 mV (Padan et al., 1976). See also:
Electron Carriers: Proteins and Cofactors in Oxidative
Phosphorylation
In anaerobic bacteria and in facultative organisms
growing under anaerobic conditions anaerobic electron
transport may operate or the H+-ATPase exports H+ on
the expense of ATP hydrolysis to drive the chemiosmotic
circuit (Figure 1).
A proton-motive force is the organizing element in
Figure 1, but this is not the only way such circuits are
arranged; other ion-motive forces are also established
under most conditions. For example, because bacteria
prefer K+ as the main internal cation, there are usually
mechanisms to extrude the Na+ that might otherwise
accumulate in response to a long-standing membrane
potential. Accordingly, internal Na+ is usually low, and
there is an inwardly directed Na+ chemical gradient
(DpNa). This together with the membrane potential es-
tablishes a sodium-motive force. The argument extends
to other cations (and anions), but H+ and Na+ merit
special attention, since most bacteria initiate a chemios-
motic circuit using either H+ or Na+ pumps (of a variety
of types).
Work performance by an ion-motive force
The proton (ion)-motive force underlies operation of an
extraordinary range of events. Quite literally, Dp is a res-
ervoir of potential energy to be used in performance of
chemical, mechanical and osmotic work. Chemical work is
typically discussed in the context of ATP synthesis during
oxidative or photophosphorylations; mechanical work is il-
lustrated by rotation of bacterial flagella as ions move in-
ward under the influence of electrical and chemical driving
forces. Osmotic work, the main concern of this article, relies
on the proton-motive force to provide the driving force for
much of the cell’s ongoing metabolite accumulation and
efflux.
To illustrate how the proton-motive force drives solute
transport, it is helpful to calculate the extent to which an
uncharged substrate (a nonelectrolyte) might accumulate
within a cell if its transport were mediated by a one-for-one
coupling with protons during a simple symport reaction. In
this case, the free energy (in mV) made available for sub-
strate (S) accumulation is that liberated as a single proton
(n=1) moves down its electrochemical potential gradient
of, say, 2180 mV:
pDp ¼ Df
60DPH ¼
180 mV ¼ energy available ½2
In principle, all this could be recaptured as the chemical
potential (in mV) of the transported molecule, reflecting its
5
 Bacterial Membrane Transport: Organization of Membrane Activities
accumulation within the cell:
chemical potential ¼
ð2:3 RT=FÞ log½Sin =½Sout 
½3
¼
60 log½Sin =½Sout 
Setting these relationships equal to each other leads one to
conclude that in a perfect world the perfect cell could attain
an internal substrate concentration 1000-fold higher than
in the outside medium. Higher proton/substrate stoic-
hiometry (n) would support higher accumulation ratios,
but such elevated ratios are unusual among bacterial me-
tabolite transporters.
Integrating Primary and Secondary
Transporters
The five secondary transporters shown in Figure 1 connect
in different ways to the overall chemiosmotic circuit. For
example, the uniporter shown (no. 1) would work inde-
pendently of ambient electrical and pH gradients, except
insofar as its substrate might carry a net charge or operate
as a weak acid or weak base. In the case of proton-linked
symport with a nonelectrolyte (no. 2), both electrical and
chemical components of the proton-motive force are rel-
evant as driving forces (eqn [1]). Example no. 3 shows an-
tiport, or exchange. This is of special interest for it
illustrates how one might broaden the selectivity of a
chemiosmotic circuit. Thus, by catalysing Na+ efflux, Na+/
H+ antiport supports a subsidiary circulation of Na+
within the dominant H+ circuit, allowing this cell to carry
out Na+-coupled symport (no. 4). In the same way, if the
dominant circulation were one of Na+, Na+/H+ antiport
would allow coexistence of subsidiary H+-linked reactions.
The final example shown (no. 5) illustrates one of the re-
actions underlying sugar phosphate transport. Here, note
that while the fundamental event is one of the neutral anion
exchange, the net reaction is equivalent to the cotransport
of 2H+ with G6P22, so that the exchange has the behav-
ioural phenotype of symport. The scheme of Figure 1 co-
ordinates the activity of secondary transporters of varying
chemical specificity and mechanistic type. It goes without
saying that other transporters are also present, some of
which, such as the ABC solute ATPases, can duplicate the
actions shown here without interacting with this circuitry.
See also: Ion Transport Across Nonexcitable Membranes;
Sodium Channels; Sodium, Calcium and Potassium
Channels
Channels
Several bacterial channels have been noted already (GlpF,
AqpZ, UreI). These are channels for nonelectrolytes,
whose movement does not perturb the components of Dp;
one can easily imagine these channels operating silently
6 ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net
within the larger chemiosmotic circuit (Martinac et al.,
2008). See also: Ion Channels; Ligand-Gated Ion Channels
When considering channels whose substrates are
charged, one should worry about the electrochemical
potentials across the membrane. In mammalian cells, the
activity of such channels determines the size and polarity of
the membrane potential, which normally rests at a rather
constant (interior negative) value. But in bacteria, fungi
and probably in plants and in most eukaryote organelles, it
is the activity of various ion pumps that sets the value of the
membrane potential (as in Figure 1). In fact, the membrane
potential of bacteria may assume any number of steady
state values, depending on the value of DpH that ensures a
near-neutral internal pH. This is because the value of Dp
(but not its composition) is set by the thermodynamics of
ion pumping. Therefore, at external pH 7–8, where the pH
gradient is rather small (E. coli regulates its internal pH at
approximately 7.5–7.8) (see later discussion), the electrical
gradient is the main element supporting a chemiosmotic
circuit (Figure 1; eqn [1]). By contrast, the electrical gradient
diminishes considerably as DpH expands to allow growth
at lower external pH. In addition, we should not forget that
bacteria are small (a radius of 0.5 mm gives an internal vol-
ume near 5  10216 L). This means their internal ionic pool
is limited and that opening a channel may have unexpected
consequences. For example, because there are only ap-
proximately 7  107 K+ ions inside E. coli (approximately
0.2 mol L21), the inappropriate opening of a single
K+ channel of the kind found in animal cells could deplete
cell K+ in a fraction of a second. In fact, this is exactly how
a number of colicins kill E. coli; opening of even a single
colicin channel is lethal. See also: Voltage-gated Potassium
Channels
Nevertheless, a spectacular recent accomplishment has
been the crystallization of a K+ channel from Streptomyces
lividans. In the inner membrane of E. coli there is an equally
well-characterized mechanosensitive channel that relieves
sudden osmotic stress (swelling) by allowing outward pas-
sage of small to moderately sized metabolites. Hence, it is
suggested that principles governing channel activity in
bacteria are probably very different from those used in
much larger eukaryotes. See also: Protein Translocation
Across Membranes
Virtual Proton Pumps: A Proton-
motive Force Emerges from
Combining Transport and Metabolism
Chemiosmotic circuits, largely built on the circulation of
H+ or Na+, dominate solute traffic at the bacterial mem-
brane (Harold and Maloney, 1996). Most often, such cir-
cuits are initiated by primary transport reactions, as
described earlier (Figure 1), but on more than one occasion
the same result appears from an unexpected association of
secondary transport and metabolism. In these cases, H+

itself is not moved. Instead, the end result of the metabolic
cycle is as if a proton were actually moved.
Virtual pumps
This emergent feature of bacterial cell biology was first
noted in studies of the anaerobe, Oxalobacter formigenes,
which uses oxalate decarboxylation to generate a proton-
motive force (Anantharam et al., 1989). In Klebsiella
aerogenes a membrane-bound decarboxylase functions to
initiate a circuit by extruding Na+, but in O. formigenes the
decarboxylation reaction [I] is catalysed by a soluble, cyto-
solic enzyme.


OOC
COO
þ Hþ ! HCOO
þ CO2 þ energy ½I
The energy released by this decarboxylation is dissipated
as heat and cannot be used to drive ion transport. Instead, a
new kind of proton pump appears when decarboxylation is
considered together with the ways in which precursor (oxa-
late22) is made available and product (formate2) removed
(Figure 2). In this case, OxlT, a secondary transporter, ca-
talyses the one-for-one antiport of divalent oxalate and
monovalent formate, leaving a single negative charge be-
hind in the cytoplasm. At the same time, there is consump-
tion of a single proton during the decarboxylation reaction.
Notice that the ion-motive gradients arising from this
overall cycle (an interior negative membrane potential, an
interior alkaline cytoplasm) are identical to those that arise
Oxalate
–OOC COO–
OxIT
Decarboxylase*
Formate
HCOO–
+ –
Acid
+ –
Alkaline
+ –
+ –
ADP
F0F1
4H+
*10% soluble protein
ATP
Figure 2 A virtual proton pump. In Oxalobacter formigenes, the
thermodynamic equivalent of a proton pump emerges from the functional
association of OxlT, an oxalate22:formate12 antiporter, and an intracellular
decarboxylation system. Modified from Anantharam et al. (1989).
ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net
Bacterial Membrane Transport: Organization of Membrane Activities
when a traditional proton pump operates. In this way, the
combined action of OxlT and the decarboxylase comprise a
metabolic cycle formally identical to a proton pump whose
stoichiometry is 1H+ pumped per cycle. Continuing oper-
ation of this cycle ensures constant outward flux of pro-
tons, and this is what supports all proton-linked activities
in O. formigenes, including ATP synthesis, flagellar rota-
tion, solute transport and so on (Figure 2).
Similar other virtual proton pumps linked with dec-
arboxylations are known (oxalate!formate, histi-
dine!histamine, aspartate!alanine, lysine!cadaverine
and so on). Any number of intermediate steps might sep-
arate precursor and product. A good example of this more
complex construction occurs in Leuconostoc spp., where
the antiport of divalent precursor (citrate) and monovalent
product (lactate) is feasible only after a number of inter-
mediate steps.
There is no need for the coupled antiport of precursor and
product. These might instead move by entirely separate
transporters, so long as the net import of negative charge (or
its equivalent, the export of a positive charge) is in one-
for-one stoichiometry with cytosolic proton/s that disap-
pear. Here, the citrate/lactate couple provides a model. In
the same way, one can dispense with anions as charge car-
riers. In at least two cells that contain urease (Bacillus paste-
urii and Ureaplasma urealyticum), a virtual pump based on
urea (precursor) entry and ammonium (product) exit sus-
tains the proton-motive force. (Urease catalyses hydration
of urea: NH2 –CO–NH2+H2O!2NH3+CO2. In these
cases, it is likely that urea moves inward in exchange for
the NHþ 4 that arises when water combines with ammonia.)
Finally, if the scalar metabolic reaction generates a gas or
H+
other liquid-soluble material, one can avoid the step of car-
rier-mediated efflux of product. For this last example, one
could speculate about bacterial production of CH4 and H2,
since a membrane potential could arise from the entry of a
monovalent precursor (acetate, formate or bicarbonate)
whose subsequent metabolism would consume a single pro-
ton for each CH4 or H2 that appears.
CO2
Is a Proton(ion)-motive Necessary?
Harold and Van Brunt (1977) showed that Enterococcus
faecalis, whose chemiosmotic circuitry resembles that of
Figure 1, grows indefinitely when the proton (and so-
dium)-motive force is collapsed by the channel-forming
ionophore, gramicidin. Survival without Dp requires a
medium at pH 7.5, with high K+, very low Ca2+ and
Na+, and millimolar levels of nutrients. This permissive
medium effectively mimics the main ionic conditions re-
quired for cytoplasmic health, and at the same time
provide nutrients at high enough concentration to initi-
ate metabolism. H+- and Na+-coupled solute transport-
ers continue to work, but a toxic build-up of internal H+
or Na+ is avoided as they return to the medium via the
ionophore. Understandably, growth ceases outside this
narrow window, showing that chemiosmotic circuits and
7
 Bacterial Membrane Transport: Organization of Membrane Activities
the genes that encode their constituent proteins are
essential in dealing with a variable and unpredictable
environment, the natural bacterial environment. There-
fore, in addition to energy transduction and a continu-
ous supply of nutrients the chemiosmotic circuit is
needed for homeostatic mechanisms such as maintaining
a constant intracellular pH of the cytoplasm.
pH Homeostasis in Bacteria
In line with being the most common ions in nature, H+
and Na+ are the main ions of chemiosmotic circuits.
Yet, when the concentration of these ions becomes too
high or too low they turn into most potent stressors to
all cells. Therefore, all living cells are crucially dependent
on processes that regulate their intracellular pH, Na+
and volume.
By applying various approaches to measure intracellular
pH it was demonstrated that bacteria have most potent pH
homeostatic mechanisms. Nonextermophilic bacteria
grow over a broad range of external pH values from 5.5
to 9.0 and maintain a cytoplasmic pH 7.5–7.6 (Padan et al.,
2005). The alkaliphilic bacteria grow up to extracellular pH
of 11–12 and their intracellular pH does not exceed pH 8.3
under the most extreme conditions. The acidophilic bac-
teria grow down to pH 2 and their intracellular pH is ap-
proximately 6.5. See also: Acidophiles; Alkaliphiles;
Intracellular pH Measurement
A large number of adaptive strategies are deployed for
pH homeostasis in the various bacteria. Here, we will focus
on the chemiosmotic circuit-related system, the Na+/H+
antiporters (Figure 1) that both in bacteria and eukaryotic
cells play primary role in pH and Na+ homeostasis. The
Na+/H+ antiporter activity was discovered by P Mitchell in
bacteria (West and Mitchell, 1974) and since then has been
found in the cytoplasmic membrane of all cells and in many
organellar membranes. The genome project has yielded a
multiplicity of genes that encode putative Na+/H+ anti-
porters, classified as members of the monovalent cation-
proton (CPA) superfamily (http://www.tcdb.org/). Two
subfamilies NHE (Na+/H+ exchangers) and NHA (Na+/
H+ antiporters) have orthologues from bacteria to humans
and are suggested to share a similar structural fold. The
family prototype E. coli NhaA has been studied most ex-
tensively studied (Padan, 2008). NhaA is indespensible for
pH and Na+ homeostasis in E. coli and many other en-
terobacteria. The NhaA protein has been over-expressed,
purified and reconstituted into proteoliposomes in a func-
tional form revealing the characteristics underpinning the
primary role of NhaA in pH homeostasis; NhaA activity is
pH dependent, a property it shares with many prokaryotic
and eukaryotic antiporters. NhaA has a high turnover rate
with a stoichiometru of 2H+:1Na+. Mutants deficient in
any of these properties cannot survive at alkaline pH in the
presence of Na+.
The recently determined crystal structure of NhaA has
provided insights into the mechanisms of NhaA Na+/
8 ENCYCLOPEDIA OF LIFE SCIENCES & 2009, John Wiley & Sons, Ltd. www.els.net
H+ antiporter activity and its unique regulation by pH.
It also paved the way to model the human antiporter,
NHE1 which plays an important role in human path-
ological conditions and therefore is a target for drug
design.
Conclusion
Even now, examination of DNA sequence shows that there
are several thousands of distinct bacterial membrane trans-
porters; and with completion of the bacterial genomes in
the current pipeline, this total could increase 10-fold. These
primary and secondary transporters display an enormous
range of chemical specificity, yet because their ongoing ac-
tivity creates a chemiosmotic circuit, they find themselves
working as a coordinated whole and regulated by Na+/H+
antiporter activity. This common organizational principle
directs microbial lifestyles in niches as disparate as the hu-
man intestine and deep-sea vents.
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