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Received 2 August 1903
Multiple mechanisms, roles and controls of K transport in Escherichia coli
+
Wolfgang Epstein,* Ed Buurman, Debbie McLaggant and Josef Naprstekt
*Department of Molecular Genetics and Cell Biology, 920 E. 58th Street, University of Chicago, Chicago, IL 60637,
U.S.A.,+Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen, U.K., and $Institute of
Microbiology,Czech Academy of Sciences, Prague, Czech Republic
T h e relatively high concentration of K + found in
most bacteria is a major contributor to cytoplasmic
osmotic pressure and thereby to the maintenance of
turgor pressure, the difference between internal and
external osmolarity [ 11. T h e other important roles
of K + , the activation of some cytoplasmic enzymes
and the contribution to ionic and to p H homeostasis, seem to require only relatively low concentrations of this ion and therefore do not give rise to
the quantitative requirement for K + [2].In view of
the role of K + in determining turgor, it makes sense
that turgor appears to control many systems that
transport K + in to or out of the cell.
K+-influx systems
Escherichia coli has three major K -uptake systems,
+
each with distinct kinetic properties and encoded by
different sets of genes (Table 1). Trk, formerly
called TrkA or TrkG plus TrkH, is the most active
uptake system and the one whose activity predominates under most conditions [Z-41. T r k can take up
Volume 21
K+ at a very high rate in K+-depleted cells, has a
moderate affinity for K C and has a dual energy
requirement in that both the protonmotive force
and A T P are required. T h e product of the trkA
gene is absolutely required, as is the product of
either the trkG or the trM genes that encode homologous proteins with 12 predicted membranespanning regions similar to those of other
transporters [5, 61. T h e trkE gene product is
required for normal activity, with only partial
activity in its absence. T h e basis of the dual energy
requirement is not understood, but it has been
shown that the TrM protein has two NAD-binding
sites and that it binds NAD in nitro [7]. T h e trkE
gene is in an operon that is similar in structure to
Opp, the oligopeptide-transport system (W. Epstein,
E. Ruurman, D. McLaggan and J. Naprstek, unpublished work). TrkE is a homologue of oppD and
similar proteins in other systems and has the
sequence that, in other members of this family, is
associated with binding of ATP.
Bacterial Membrane Proteins
Table I
K+-uptake systems in E. coli
Kinetics
Kn
I007
"ma,
(,umol.g-' min-I) Other properties
Name
Genetics
(mM)
Kdp
6 genes, 2 operons:
kdpFABC encodes
structural proteins;
kdpDE encodes
the regulatory
proteins
0.002
100- I 50
KdpB is the site of acylphosphorylation
KdpA is the site of K+ binding
Expression of Kdp is inducible by growth at low K + ,
and is believed to be controlled by turgor pressure
Expression is mediated by KdpD, a sensor-kinase and
KdpE, a response regulator
Trk
4 unlinked genes:
trkA, trkE, trkG and
trkH
I .5
300-500
Constitutively expressed
Requires both ATP and protonmotive force in vivo
TrkA is a cytoplasmic, peripheral membrane protein
that binds NAD
TrkG and TrkH are membrane-spanning
transporters; either one suffices
TrkE has ATP-binding site
Kup
I gene, kup
0.5
0-50
Transports Cs+ as well as K +
-
Kdp is a K+-transport ATPase that is homologous to the P-type transport ATPases found in all
phyla [9]. Kdp is not expressed under most conditions in wild-type cells. It is only expressed when
the cells cannot accumulate sufficient K+ by other
paths. The high affinity of Kdp for K + makes it
ideal for its role of scavenging K + when the ion is
present at very low concentrations. Kdp differs
from most other P-type ATPases in being made up
of three large protein subunits. The largest, the 72
kDa KdpR subunit, is homologous to the large subunit of other P-type ATPases and forms the acylphosphate intermediate characteristic of these
transport ATPases. The 59 kDa KdpA subunit is
unique and is not found in other P-type ATPses. It
is predicted to span the membrane 12 times and
there is genetic evidence that it has a binding site for
K'. The 20.5 kDa KdpC subunit seems to have a
role in the assembly of the Kdp complex, while the
function of the 29-residue hydrophobic KdpF peptide is not known.
In strains lacking Trk, Kup and Kdp, there is
a low, residual rate of K + uptake that does not discriminate between different monovalent cations, in
contrast to the known systems, all of which prefer
K + over any other ion. This residual activity has
not been defined further; it probably represents illicit uptake in which K + is acting as an analogue for
another ion and is being transported via systems
whose physiological roles are the transport of some
other ion.
K+-efflux systems
In the steady state there is a moderate rate of
exchange of cytoplasmic with external K + , the rate
of which seems to be determined by the properties
of the uptake process [lo]. Whether exchange represents movement in both directions through the
influx system or is the result of uptake through an
influx system and exit through a separate efflux
system is not known. The only defined systems that
can mediate the efflux of K' are KetB and KefC,
but neither of these appear to be important in maintaining cell K' [ll]. These systems are normally
inactive, but can be activated by mutation or by
inhibitors that interact with glutathione [ 121.
Control of K + transport activity by
turgor
The dependence of cell K + concentration upon the
osmolarity of the medium [13, 141 seems to be
determined by the effects of turgor on transport.
Upshock, a sudden increase in external osmolarity
that reduces or abolishes turgor, results in an
immediate stimulation of influx but no change in
the rate of efflux, to produce a net uptake of K+
I993
Biochemical Society Transactions
I008
[lo]. Eflux, by contrast, is stimulated by an
increase in turgor. When turgor is increased at a
rate that is not too fast, either by diluting the
external medium [ 151 or by massive accumulation
of another solute [ 111, there is a rapid efflux of K + .
Very little is known about the paths for such turgorstimulated efflux. It is not dependent on any known
uptake system, nor on either KefB or KefC.
The mechanism(s) whereby changes in turgor
alter transport are not understood. The effects are
very rapid; in the case of uptake there is no measurable lag ( < 5 s) between upshock and the onset of
uptake. The rapid response suggests the effect is
very direct, possibly a direct effect of turgor on the
conformation and thereby activity of the transport
systems. The effect is analogous to feedback inhibition of an enzyme. Here the end product of transport, turgor, appears to be controlling the systems
responsible for its maintenance. During steady-state
growth where cells must maintain a slow rate of K +
uptake, it is assumed that turgor pressure is slightly
below the optimum level so that the rate of influx is
slightly greater than efflux. When growth stops, as,
for example, due to the limitation of some essential
nutrient, turgor would rise slightly to reduce influx
and/or stimulate efflux and thereby achieve a
steady state for K + .
A very different mechanism is presumed to
operate in downshock, a term that describes a
sudden and large increase in turgor due to a large
reduction in external osmolarity. The result is a
very rapid and relatively nonspecific efflux of many
small solutes and even some small proteins [13, 16,
171. Efflux of solutes in downshock is probably via
pressure-activated channels that appear to be found
both in the inner and in the outer membranes of E.
coli, as well as in other bacteria [ 18, 191.
Control of expression of Kdp by
turgor
In wild-type strains, Kdp is expressed only in
medium of very low K + concentration; full expression requires growth under conditions of K + starvation. In strains defective in K + transport,
expression occurs during growth at much higher
K + concentrations. Studies of Kdp expression
using a transcriptional k d p - l a d fusion have
strongly implicated turgor pressure as the signal for
expression [20]. Conditions where turgor is
expected to be low are associated with expression.
There is no consistent correlation of expression
either with external or with internal K + or with the
presence of any particular K +-transport system;
there is always expression when the growth rate is
Volume 21
limited by the availability of K +. Since turgor cannot be measured readily, many aspects of the model
remain speculative and other models have been
suggested [21].
While the signal for Kdp expression remains
to be established, the path for expression is now
known to be mediated by the KdpD and KdpE proteins, members of the sensor-kinase/response activator family of regulators [22-241. KdpD, predicted
to have a middle section that crosses the membrane
four times, is an inner-membrane-bound autokinase
that can transfer phosphate to soluble KdpE, which
in turn binds to the promoter of the kdpFABC
operon to stimulate its transcription.
Osmotic activity of cytoplasmic solutes
Turgor pressure is not easily measured in cells as
small as bacteria, so that most studies infer or
estimate the magnitude of this parameter. One
method simply adds up the concentration of cytoplasmic solutes, assuming that the activity coefficients are similar to those in free solutions of similar
composition. However, a fraction of any cation
pools in the cell are involved in balancing the
charge on macromolecular anions. Ions that balance
such charge are often referred to as ‘bound
(although no specific binding is involved) because
they appear to have rather low ionic and osmotic
activity. W e have estimated the magnitude of
‘bound’ K + by downshock (D. McLaggan, J.
Naprstek, E. T. Huurman and W. Epstein, unpublished work). When cells are subjected to downshock with distilled water, ions associated with
macromolecules will remain with the cell. If a low
concentration of some salt is added to the shock
solution, all monovalent cations should be lost
through exchange. Using this assay we find that at
low osmolarity (0.17 osM) about 56% of cell K +
serves to balance macromolecular anions, while at
higher osmolarity this fraction falls to 38% in a
medium of 1.3 osM.
The major low-molecular-mass anion is
glutamate, whose concentration is also dependent
on medium osmolarity. Glutamate accumulation
balances the charge of about 80% of ‘free’ K + , that
is, the fraction of K + that is not involved in balancing macromolecular anions (D. McI,aggan, J.
Naprstek, E. T. Ruurman and W. Epstein, unpublished work) [25]. Most of the other major cytoplasmic osmotic solutes are neutral compounds
such as trehalose, proline and glycine betaine that
are accumulated to significant levels only in a
medium of elevated osmolarity [26]. When accumulation of these neutral molecules is prevented,
Bacterial Membrane Proteins
K' and glutamate alone must adjust cytoplasmic
osmolarity. The high ionic strength results in the
inhibition of many cellular processes and ultimately
in growth inhibition. When neutral molecules are
accumulated they displace a high proportion of K'
and glutamate [ l l ] and therefore allow growth at
considerably higher medium osmolarities since
turgor pressure can be established without reaching
inhibitory levels of ionic strength in the cytoplasm.
K' is a second messenger
Studies of upshock indicate that there is both a temporal order of events and a sequential dependence
of the responses, and that K + plays a pivotal role
[ 271. Significant degrees of upshock increases cell
K' in two ways: (1) in a few ms water leaves the
cell to reduce cell volume, resulting in plasmolysis,
and (2) there is a slower increase due to net uptake
of K + , which increases cell K' concentration and
increases cell volume to reverse plasmolysis. When
net uptake of K + is prevented, upshock results in a
modest reduction of internal pH (D. McLaggan, J.
Naprstek, E. T. Huurman and W. Epstein, unpublished work), but few other effects.
It is the uptake of K + rather than the upshock
itself that is responsible for the transient alkalinization of the cytoplasm (D. McLaggan, J. Naprstek,
E. T. Huurman and W. Epstein, unpublished work)
[ 281, the accumulation of glutamate, the stimulation
of phosphate uptake (1). McLaggan and W . hpstein,
unpublished work) and the stimulation of the expression of the proU gene that enhances the accumulation of proline and glycine betaine [20]. Part of
these responses may be a direct effect of higher K +
concentrations, since the stimulation of one of the
enzymes for synthesis of trehalose 1301 and the
transcription of proU [31] by K + has been
observed in vzvo. It has also been suggested that
changes in DNA supercoiling, in which ionic
strength should have a role, may be involved in the
increased expression of some genes at high osmolarity 1321. Not all the effects appear to be due to
K', since activation of transport by the Prop system can be demonstrated in vesicles and does not
seem to involve changes in K + concentration [33].
New K+-transport systems created by
mutation
We have used the high K' requirement for growth
of strains lacking Kdp, Trk and Kup to look for
other ways that cells can accumulate K + , by selecting for mutants that can grow at lower K' concentrations. Mutations that increase K + uptake have
now been identified in at least three loci where what
appears to be a single mutation allows enhanced
K + uptake. The only locus characterized to date is a
missense mutation in the oppB gene that encodes
one of the membrane-spanning proteins of the oligopeptide-transport system. Mutations in another
locus create a much higher rate of K + uptake, so
high that the growth of the cells is inhibited in a
medium containing more than about 40 mM K+.
Uptake by this system appears to exhibit no specificity for K+ over Rb' or cs'.
These 'new' systems provide an opportunity
to examine how substrate specificity of transport
systems might have changed during evolution, but
should also give us a new handle with which to
examine systems for K' efflux. W e presume that
the inhibition of growth at higher K + concentrations exhibited by some mutants is due to a rate of
influx that is higher than that which can be balanced
by efflux. Enhanced expression or activity of efflux
systems should allow these mutants to grow in a
standard medium and this should occur among the
mutants obtained in selections for growth in media
of high K + concentrations.
Research reported here was supported in part by grants
from the National Institutes of Health and the National
Science Foundation, as well as a fellowship to E.H. from
the Netherlands Organization for Scientific Research
(NWO).
1. Epstein, W. (1986) FEMS Microbiol. Rev. 39, 73-78
2. Walderhaug, M. O., Llosch, L). C. and Epstein, W.
(1987) in Ion Transport in I'rokaryotes (Kosen, €3.
and Silver, S., eds.), pp. 85-130, Academic Press,
New York
3. Hossemeyer, D., Horchard, A., Dosch, D. C., Helmer,
G. C., Epstein, W., Booth, I. R. and Hakker, E. 1'.
(1989)J. Hiol. Chem. 264, 16403-16410
4. Llosch, I). C., Helmer. G. I,., Sutton, S. H., Salvacion,
I;. F. and Epstein, W. (1991) J. Hacteriol. 173,
687-696
5. Schlosser, A., Kluttig, S., Hamann, A. and Bakker,
E. 1'. (1991) J. Hacteriol. 173, 3170-3176
6. Schlosser, A., Hamann, A,, Schleyer, M. and Bakker,
E. 1'. (1992) in Molecular Mechanisms of Transport
(Quagliariello, E. and I'almiere, F.?eds.), pp. 5 1-58,
Elsevier, Amsterdam
7. Schlosser, A., Hamann, A., Bossemeyer, D.,
Schneider, E. and Rakker, E. P. (1993) Mol.
Microbiol. 7, 533-543
8. Keference deleted
9. Altendorf, K., Siebers, A. and Epstein, W. (1992)
Ann. N.Y. Acad. Sci. 671,228-243
10. Rhoads, D. H. and Epstein, W. (1978)J. Gen. I'hysiol.
72,283-295
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11. Hakker, E. P., Booth, I. K., Llinnbier, U., Epstein, W.
and Gajewska, A. (1987)J. Bacteriol. 169, 3743-3749
12. Elmore, J. J., Lamb, A. J.. Ritchie, G. Y.. Douglas,
R. M., Munro, A,, Gajewska, A. and Booth, I. R.
(1990) Mol. Microbiol. 4,405-412
13. Epstein, W. and Schultz, S.G. (1965) J. Gen. Physiol.
49,221-234
14. Richey, H., Caley, L). S.,Mossing, M. C., Kolka, C.,
Anderson, C. F., Farrar, T. C. and Kecord, M. T.
(1987)J. Hiol. Chem. 262,7157-7164
15. Meury. J., Robin, A. and Monnier-Champeix, P.
(1985) Eur. J. Biochem. 119,613-619
16. Tsapis, A. and Kepes, A. (1977) Hiochim. Biophys.
Acta 469, 1-12
17. Kundig, W., Kundig, F. L)., Anderson, €3. and
Roseman, D. (1966)J. Biol. Chem. 241.3243-3246
18. Martinac, H., Huechner, M., Delcour, A. H., Adler, J.
and Kung, C. (1987) I'roc. Natl. Acad. Sci. U.S.A. 84,
2297-2301
19. Herrier, C., Coulombe, A,, Szabo, I., Zoratti, M. and
Ghazi, A. (1992) Eur. J. Hiochem. 206, 559-565
20. 1,aimins. I,. A,, Rhoads, D. H. and Epstein, W. (1981)
Proc. Natl. Acad. Sci. USA. 78, 464-468
21. Asha, A. and Gowrishankar, J. (1993) J. Bacteriol.
175,4528-4537
22. Walderhaug, M. O., Polarek, J. W., Voelkner, P.,
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23. Nakashima, K., Sugiura, A., Momoi, H. and Mizuno,
T. (1992) Mol. Microbiol. 6, 1777-1784
24. Nakashima, K., Sugiura, A., Kanamaru, K. and
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25. McLaggan, D., Logan. T. M., Lynn. L). G. and
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26. Csonka, I,. N. and Hanson, A. D. (1991) Annu. Kev.
Microbiol. 45,569-606
27. Booth, I. R. and Higgins, C. F. (1990) FEMS
Microbiol. Rev. 75, 239-246
28. Dinnbier, U., Limpinsel, E., Schmid, R. and Hakker,
E. P. (1988) Arch. Microbiol. 150. 348-357
29. Sutherland, I,., Cairney, J., Elmore, M. J., Booth, 1. K.
and Higgins, C. F. (1986) J. Bacteriol. 168.805-814
30. Giaever, H. M., Styrvold, 0. B., Kaasen, I. and Strom,
A. R. (1988) J. Bacteriol. 170,2841-2849
31. Prince, W. S. and Villarejo, M. K. (1990) J. Hiol.
Chem. 265. 17673-17679
32. Higgins, C. F.,Dorman, C. J., Stirling, D. A,, Waddell,
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Chem. 263, 14900- 14905
J.?
Received 27 July 1993
Proton-translocating transhydrogenase in bacteria
J. Baz Jackson,*t Nick P. J. Cotton,* Ross Williams,* Tania Bizouarn,* Mick N . Hutton, Leonid A. Sazanov*
and Christopher M. Thomasf
Schools of *Biochemistry and $Biological Sciences, The University of Birmingham, Edgbaston, Birmingham B I 5 2TT,
U.K.
Introduction
Proton-translocating
transhydrogenase
(H'THase) catalyses the transfer of reducing equivalents (H -equivalents) between NAD(I-1) and
NADP(H) coupled to the transfer of H' across a
membrane.
~
NADH + NADP'
NAD'
+ xH,:,,
+ NADPH + xH,:
[ 1-31,
G=+
(1)
It is found in the inner mitochondria1 membrane
and in the cytoplasmic membrane of many bacteria.
The enzyme has no known prosthetic groups, and
Abbreviations used: AI'AL)', acetyl pyridine adenine
nucleotide; L)CCI>. N,N'-dicyclohexylcarbodi-imide;
H -THase, proton-translocating transhydrogenase.
?To whom correspondence should be addressed.
+
Volume 21
is remarkable in that the standard free energies of
the donor and acceptor are very similar. Thus, at
equilibrium, the energy of the proton electrochemical gradient is equivalent to that of the mass-action
ratio of the nucleotide substrates and products.
Recent reviews on H+-THase can be found in
Structure of the enzyme
Figure 1 summarizes the relationship between the
polypeptide structures of H +-THase from beef
heart mitochondria, Escherichia coli and RhodospirilZum rubrum. There are clear similarities in the amino
acid sequences of the mitochondrial and E. coli
enzymes, although the latter consists of two polypeptides ( a , M, 54 000 and p, M, 49 000) and the
former only one polypeptide (Mr 109000) [4-h].
Hydropathic profiles [4, 51 and experiments with