352 BACTERIALTRANSPORT [28]"

the absence of added Na *, at pH 9.0, respiring cells generate a pH alka-

line inside. Upon addition of Na ÷ the polarity of ApH is reverted. 66
In conclusion, the evidence supporting a role for the K + transport
systems, the K+/H + antiporter and the Na+/H + antiporter is mounting.
The details of the overall mechanisms and the possible interactions be-
tween them are yet to be elucidated. Thus, it is not clear whether the two
antiporters function in a concerted way or whether they are alternative
mechanisms functioning at different physiological conditions. Are there
other systems (other ions or leaks of Na ÷ and K ÷) part of the regulatory
systems as well? After failure of the homeostatic response, is there need
for a signal to activate the regulatory mechanism (pHo, pHi, Ark) or is the
new steady state reached by a mere rearrangement of the new rates of
transport of the various ions? It is clear that nothing is known about the
structure and regulation of the synthesis of the antiporters. Finally, the
coupling between pHi and cell division is very striking: relatively small
changes in pHi bring about an immediate cessation of cell division. From
the results published it would seem that there is a specific and sensitive
sensor of the hydrogen ion concentration that somehow controls cell
division. Elucidation of the nature of this control mechanism may shed
light on other processes in which cytoplasmic pH has been postulated as a
signal.

[28] R e g u l a t i o n o f I n t e r n a l p H in A c i d o p h i l i c a n d
Alkalophilic Bacteria
By T. A. KRULWICH and A. A. GUFFANTI

As noted in the preceding chapter of this volume 1 and in other recent

reviews,2,3 many if not all bacteria can maintain an astonishingly constant
cytoplasmic pH (pHin) when incubated or grown at very different external
pH values and even after being subjected to rather dramatic shifts in
external pH. This ability to regulate phi. is a crucial physiological func-
tion that is made all the more important--even when the vicissitudes in
external pH are not that great--by the likelihood that at least some criti-

1 E. Padan and S. Schuldiner, this volume [27].

2 E. Padan, D. Zilberstein, and S. Schuldiner, Biochim. Biophys. Acta 650, 151 (1981).
3 I. Booth and R. G. Kroll, Biochem. Soc. Trans. 11, 70 (1983).

Copyright © 1986 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 125 All fights of reproduction in any form reserved.
[28] REGULATION OF INTERNAL pH 353

cal cell process(es) is tightly controlled by pHin. 4 In neutralophilic bacteria

a large array of primary ion pumps and secondary antiport-, symport-,
and uniport-mediated ion movements have been proposed to play a role in
pH homeostasis. ~,3,5-9 Presumably, different mechanisms are required to
raise pHtn from those that serve to lower pHin. Moreover, a single organ-
ism may possess dominant and auxiliary mechanisms whose relative im-
portance could depend upon the specific pH range and/or special circum-
stances such as light. There are a few mutants whose phenotype supports
a role for a particular pump or porter in pH homeostasis in a neutralo-
phile; ultimately, clarification of cytoplasmic pH regulation may depend
upon the availability of larger numbers of well-characterized mutants. At
present, the precise roles of potassium and sodium in facilitating acidifica-
tion of the interior during alkali stress of a neutralophile remain somewhat
controversial. Similarly, in neutralophiles, the relative roles of proton-
pumping ATPases, respiration, and other possible means of raising pHin
during acid stress are still very incompletely determined. Examination of
acidophilic and alkalophilic bacteria offers an opportunity to study the
problems of pH homeostasis in extremis. Bacteria which grow well at
extremes of pH may have evolved special mechanisms for coping with
their most obvious biological problem; such mechanisms may or may not
be qualitatively similar to those employed in less demanding growth con-
ditions. Even if they are not, studies in the extreme ranges should indicate
directions for experimentation in the more subtle region of neutrality.
Moreover, extreme acidophiles and alkalophiles are of intrinsic interest
because of their broader bioenergetic problems and characteristics, the
properties of their surfaces, and those processes that are exposed to the
external milieu, their industrial potential, and their relationships to the
ecosystem. 10-13
Our own studies have focused upon species of Bacillus that grow
obligately at very low (pH 2-3.5) or very high (pH 10-11) pH values. In
the acid range, we have studied the thermoacidophile Bacillus acido-

4 D. Zilberstein, V. Agmon, S. Schuldiner, and E. Padan, J. Bacteriol. 158, 246 (1984).

5 T. A. Krulwich, Biochim. Biophys. Acta 726, 245 (1983).
6 H. Kobayashi and T. Unemoto, J. Bacteriol. 143, i187 (1980).
7 j. Lanyi, Biochim. Biophys. Acta 559, 377 (1984).
8 S. Kan-Dron, R. Shnaiderman, and Y. Avi-Dor, Arch. Biochem. Biophys. 229, 640 (1984).
9 H. Tokuda and T. Unemoto, J. Biol. Chem. 257, 10007 (1982).
l0 T. A. Krulwich and A. A. Guffanti, Adv. Microb. Physiol. 24, 173 (1983).
ii K. Horikoshi and T. Akiba, "Alkalophilic Microorganism." Springer-Verlag, Berlin and
New York, 1982.
12 j. G. Cobley and J. C. Cox, Microbiol. Rev. 47, 579 (1983).
~s T. A. Langworthy, in "Microbial Life in Extreme Environment" (D. J. Kushner, ed.).
Academic Press, New York, 1978.
354 BACTERIAL TRANSPORT [28]

caldarius, first isolated and described by Darland and Brock.14 The obli-
gate alkalophiles have included Bacillus alcalophilus 15and Bacillus firmus
RAB. j6 Such obligately alkalophilic organisms can be isolated with sur-
prising ease from various soil samples, perhaps reflecting the high alkaline
clay particle content of many soils. In this review of methodology, we will
outline the protocols used to characterize features and mechanisms of pH
homeostasis in the obligately acidophilic and alkalophilic bacilli; informa-
tion derived from studies by other investigators of gram-negative acido-
philes and of alkaline-tolerant organisms (those with a range of growth pH
values that is lower than that of the obligate alkalophiles and includes
near-neutral values) will be introduced only peripherally to indicate where
important differences in experimental detail may obtain. Also, we will
confine this review to those methods that have employed whole cells. It
should be noted, however, that studies of pH regulation in bacteria have
been conducted both in vivo and in vitro. The in vitro studies, using
isolated membrane vesicles of the type first prepared by Kaback, 17 have
made some major contributions to this area. In both the acidophile B.
acidocaldarius and in alkalophilic B. alcalophilus and B. firmus RAB,
work with right-side-out membrane vesicles has provided unequivocal
evidence of outward, respiration-dependent proton translocation, ~8,~9and
has been part of the battery of approaches to the characterization of
antiport activities relevant to pH regulation, t8-2°

Acidophilic Bacteria
It has been clearly established that any one of several acidophilic
bacteria will maintain an almost constant cytoplasmic pH, at some value
between 5.8 and 7.1, over a range of external pH values including pH 2-
3.12.21 There is general agreement that respiration-dependent proton trans-
location is required for the generation of the enormous ApH. 12 It has also
been generally found that acidophiles, in the very acid range of pH, pos-

t4 G. Darland and T. Brock, J. Gen. Microbiol. 67, 9 (1971).

t5 R. E. Buchanan and N. E. Gibbons (eds.), "Bergeys' Manual of Determinative Bacteriol-
ogy." Williams & Wilkins, Baltimore.
t6 A. A. Guffanti, R. Blanco, R. A. Benenson, and T. A. Krulwich, J. Gen. Microbiol. 119,
79 (1980).
t7 H. R. Kaback, this series, Vol. 22, p. 99.
t8 T. A. Krulwich, A. A. Guffanti, R. F. Bornstein, and J. Hoffstein, J. Biol. Chem. 257,
1885 (1982).
19 A. A. Guffanti, M. Mann, T. L. Sherman, and T. A. Krulwich, J. Bacteriol. 159, 448
(1984).
20 K. G. Mandel, A. A. Guffanti, and T. A. Krulwich, J. Biol. Chem. 255, 7391 (1980).
21 M. Michels and E. P. Bakker, J. Bacteriol. 161, 231 (1985).
[28] REGULATION OF INTERNAL p H 355

sess a "reversed A0," a transmembrane electrical potential that is posi-

tive in. ~0,~2Most likely, the reversed Ark has a role in the maintenance and,
perhaps, the generation of the ApH. ~°,~2,22 The nature and origin of the
reversed A0 are as yet unknown, and pH shift experiments, that might
clarify aspects of ApH and A0 generation, have yet to be reported for
acidophiles. Thus, we will present protocols for measurement of the ApH,
from which calculations of pHin are made, and look forward to the appli-
cation of these methods to physiological experiments in which cells equili-
brated at neutral pH are shifted to highly acidic media. Because of sugges-
tions that cytoplasmic buffering capacity and a special impermeability of
the acidophile membrane to cations may be involved in pH homeostasis in
at least some gram-negative acidophiles, 12,22-25we will also consider the
methodological approaches to determinations of these parameters.

Determinations o f ApH
The principles involved in measurements of ApH have been elucidated
and described in detail by many others, including a review by Rotten-
berg 26 in an earlier volume; the equations required for calculation can be
found there. In acidophilic as in conventional bacteria, the distribution of
weak acids is used to assay the pH gradient across the membrane; since
the pHout is known, the pHin c a n then be readily ascertained. 26The special
considerations for work with acidophiles include the choice of buffer, the
choice of weak acid, and the controls used to assess binding of the weak
acid (as opposed to its localization, free, within the cytoplasmic compo-
nent). In any measurements of the ApH, it should be recognized that the
amount of the accumulated probe that is truly free in the cytoplasm is not
easily evaluated.
We have used citrate buffer, 25 to 50 mM, to maintain the pHout at low
pH values in experiments with B. acidocaldarius. 27 Matin and his col-
leagues 23 have used 100 mM fl-alanine buffer in some of their determina-
tions of ApH in the gram-negative acidophile Thiobacillus acidophilus,
but it has recently been suggested that use of fl-alanine buffer may not be
optimal if protonophore effects are to be examined as part of the experi-
mental series. 21 The external pH of buffered cell suspensions should be

22 W. J. lngeldew, Biochim. Biophys. Acta 683, 89 (1982).

23 A. Matin, B. Wilson, E. Zychlinsky, and M. Matin, J. Bacteriol. 150, 582 (1982).
24 E. Zychlinsky and A. Matin, J. Bacteriol. 153, 371 (1983).
_,5 E. Zychlinsky and A. Matin, J. Bacteriol. 156, 1352 (1983).
26 H. Rottenberg, this series, Vol. 55, p. 547.
27 T. A. Krulwich, L. F. Davidson, S. J. Filip, Jr., R. S. Zuckerman, and A. A. Guffanti, J.
Biol. Chem. 253, 4599 (1978).
356 BACTERIAL TRANSPORT [28]

monitored to assess dirft and account for it in the calculations. With B.

acidocaldarius, suspensions are prepared from logarithmically growing
cells (100-200 Klett units with a red filter) that are harvested by centrifu-
gation and washed at least once with the buffer of choice. For determina-
tions using a flow dialysis assay, cells are resuspended to a final concen-
tration of 5 mg cell protein/ml; when the determination of ApH is to be
made using a filtration assay, the cells are resuspended to a final concen-
tration of approximately 0.5 mg cell protein/ml. It is necessary to know
the internal cell volume in order to calculate the distribution of weak acid
probe. We have used tritiated water to measure the total water space and
either radiolabeled inulin or dextran to measure the external water space
as described by Rottenberg26; the difference between the two values is the
internal water volume. In flow dialysis assays, it is important for this
volume to represent a significant part of the experimental chamber
volume.
The weak acid probes of ApH that are most commonly used for con-
ventional bacteria, e.g., 5,5-dimethyl-2,4-oxazolidinedione (DMO), are
inappropriate for use at very acid external pH v a l u e s . 21,28 Since the pKa of
DMO is 6.3, an accumulation ratio, inside to out, of only 3 to 5 would be
expected at typical values of pHout 2 and pHin 6.5. Such a small ratio is
difficult to measure accurately. Therefore, either benzoic acid (pKa 4.17)
or acetylsalicylic acid (pKa 3.48) are the probes of choice for work with
acidophiles. The latter weak acid is usually commercially available with a
radioactive label in the acetyl group; it may be important to show that
extramembranal cleavage of the probe does not occur. As in all determi-
nations of ApH from the distribution of weak acids, it is also essential that
the acid be nonmetabolizable and that the entry of the acid be entirely by
passive diffusion. The weak acids should be used at concentrations less
than 10/xM to avoid dissipation of the gradient that is being measured.
The accumulation of the weak acid can be measured by filtration
through GFC filters (Whatman) as described by Zilberstein et al. 4 With
the aerobic thermoacidophile, B. acidocaldarius, the cell suspensions are
incubated at the growth temperature with rapid mixing to ensure aeration.
Either [14C]acetylsalicylic acid (30 mCi/mmol) or [14C]benzoic acid (20
mCi/mmol) are added at concentrations of 1 to 10/zM. The ApH deter-
mined should be independent of the concentration of probe and of cell
protein used within the experimental range. Samples, usually 1 ml from a
total incubation mixture of 5 to 10 ml, are removed at various times after
the addition of the labeled acid. Samples must be taken until a steady-
state level of accumulation is reached, generally 5 to 10 min. The samples

z8 j. C. Cox, D. G. Nicholls, and W. J. lngledew, Biochem. J. 178, 195 (1979).

[(28] REGULATION OF INTERNAL pH 357

should filter rapidly (around 5 sec). We have had best results with these
filters in experiments in which the filtered samples were not washed,
thereby avoiding efflux of the accumulated acid. Controls for nonspecific
binding are critical. Organic solvents, especially 5% (v/v) n-butanol, 29,3°
can be used to permeabilize the cells and give a background for nonspe-
cific binding. It should also be possible to use cells treated with
ionophores such as gramicidin as a control, as long as the treatment
reliably abolishes the ApH. We have used 2,4-dinitrophenol treatment of
B. acidocaldarius, and found the results to be the same with pro-
tonophore (50 /xM, 2,4-dinitrophenol) alone as with protonophore to-
gether with either NaSCN (5 mM) or valinomycin (2/zM). 27 In a different
protocol, Michels and Bakker 21 found that 2,4-dinitrophenol decreases
the ApH by greater than 80% in B. acidocaldarius, leaving a small residual
pH gradient. If uncouplers are to be used as a binding control in experi-
ments with acidophiles, however, it is important to consider that pro-
tonophores may be less effective at very low pH values than in the neutral
range, 12 requiring higher than usual concentrations and substantial incu-
bation times for effective use with at least some acidophiles. Some gram-
negative and archaebacterial acidophiles, especially, may retain a sub-
stantial pH even after treatment with protonophores ~2 unless a high
protonophore concentration is used and the specific buffering conditions
allow counterion movement to offset the effect of proton influx on the Ark.
For example, abolition of at least 85% of the ApH of Thermoplasma
acidophilum has been observed by Michels and Bakker 2~ using high con-
centrations of 2,4-dinitrophenol and citrate buffer, but lower pro-
tonophore concentrations or use of fl-alanine buffer resulted in a much
more substantial residual ApH. Their experimental findings may provide
the basis for explaining a number of earlier observations of a much larger
residual ApH in some gram-negative acidophiles upon protonophore
treatment or extensive starvation in/3-alanine buffer or water. 23,24 How-
ever, such observations are still suggestive of the ability of at least certain
gram-negative acidophiles to maintain an appreciable ApH by passive
means under some conditions. It will be of interest to try to examine the
relative importance of passive mechanisms to both viability and pH ho-
meostasis under ionic conditions that approximate those of growth media.
Determinations of viability are critical, since a passive mechanism for
maintaining a constant pHi. which does not facilitate the preservation of
viability could hardly represent a true adaptation of the organism, and is
more likely to be a laboratory phenomenon.

29 E. R. Kashket, A. G. Blanchard, and W. C. Metzger, J. Bacteriol. 143, 128 (1980).

30 E. P. Bakker, Biochim. Biophys. Acta 681, 474 (1982).
358 BACTERIALTRANSPORT [28]
Sample filters from the filtration assays of ApH should be dried, and
the radioactivity counted. We use scintillation spectrometry, and include
a control for quenching by the buffer in each experiment, i.e., an aliquot
of the radioactive probe suspended in the buffer used is placed on a dry
GFC filter and the filter is counted. The cpm recorded for a known
amount of acid probe in the presence of the appropriate buffer is then
used to calculate the concentration of accumulated acid.
For assays of ApH employing a flow dialysis method, the procedures
developed by Ramos et al. 31 for use with membrane vesicles are applied.
As already noted, we have used protonophore-treated cells of B. acido-
caldarius as a control; killed cells should give comparable results. Vigor-
ous aeration of the thick suspensions of aerobic cells is important, and can
be achieved by blowing a stream of water-saturated oxygen over a rapidly
mixing suspension. Nonetheless, it has been suggested that this proce-
dure results in consistent but somewhat artificially low values for pHi,
because of suboptimal aeration relative to that achieved in the filtration
procedurefl ~A falsely low value of pHi, would lead to an underestimate of
the capacity to exclude pumped protons and might also result in falsely
low calculated values of the total magnitude of the protonmotive force
(unless the experimental conditions reduced the magnitude of the re-
versed A~b to the same extent).

Determinations of Cytoplasmic Buffering Capacity and Passive

Proton Permeability
The observations suggesting that some acidophiles can maintain a
substantial ApH by passive mechanisms led to consideration of the possi-
bility that these species possess a particularly cation-impermeable mem-
brane and/or are able to resist changes in cytoplasmic pH in part because
of a high cytoplasmic buffering capacity.~°,~5 Mitchell and c o l l e a g u e s 32-34
have described, in detail, the conceptual and mathematical considerations
in determinations of cytoplasmic buffering capacity; as part of those stud-
ies, they reported experimental results using methods in which bacterial
cells are permeabilized to ascertain the total buffering capacity (Bt) and
methods in which the capacity is determined from the pattern of pH
change after addition of a small acid pulse to a suspension of intact cells in
KC1. Briefly, the internal buffering capacity (Bi) is indirectly determined

3t S. Ramos, S. Schuldiner, and H. R. Kaback, this series, Vol. 55, p. 680.

32 p. Mitchell and J. Moyle, Biochern. J. 104, 588 (1967).
33 p. Mitchell and J. Moyle, Biochem. J. 105, 1147 (1967).
34 p. Scholes and P. Mitchell, Bioenergetics 1, 61 (1970).
[28] REGULATION OF INTERNAL pH 359

from the total buffering capacity (Bt) and the buffering capacity of the
outside surface(s) of the cell (Bo).
In a recent study, 35 we have employed both experimental approaches
to measurements in bacilli that grow in various ranges of pH and in
Escherichia coli. In methods using permeabilized cells, the Bo is first
determined on an untreated cell suspension of bacteria at about 5 mg cell
protein/ml of water or 200 mM KCI. Bo is calculated from the initial, rapid
change in the pH of the medium upon addition of successive, small ali-
quots of HCI (usually 10 to 20/xl of 0.05 M HCI). For determinations of B~,
the cell suspensions are then treated with 5-10% Triton X-100, 25'35 5% n-
butanol,3,35 o r with a combination of agents that abolishes the ability of the
cells to maintain a protonmotive force. 36 The changes in pH caused by
aliquots of acid are then recorded for the treated suspension. In our
hands, the presence or absence or carbonate dehydratase in these suspen-
sions made no difference. However, it is important that additions of KOH
(titrating up) give the same results as HCI (titrating down) over the same
range and that several cell concentrations are examined. It is also desir-
able to employ several different methods for permeabilizing the cells,
since at least some bacteria seem to open up more fully, for example, with
n-butanol than with Triton (see Table I). For comparisons between differ-
ent bacteria, care should be taken to calculate from measurements taken
at the same initial and final pH values since Bo and Bi are highly pH
dependent. In connection with the data shown in Table I, it is notable that
alkalophiles, at alkaline pH, exhibit among the highest cytoplasmic buff-
ering capacities found in our survey, values that are comparable to the
relatively high Bi of the acidophile in our study at acid pH. Nonetheless,
as described in the next section, a failure of the active mechanisms of pH
homeostasis results in an immediate rise in the pH~n of B. firmus RAB so
that it equals the pHout, usually 10.5. Thus while a high Bi (and the sub-
stantial Bo found in all the species we have examined) might play a role in
buffering minor perturbations in pHin or in protecting some specific organ-
elle or molecule, it is unlikely to be of importance in resisting large perma-
nent shifts in pH.
The second approach to measurements of cytoplasmic buffering ca-
pacity employs intact cells suspended in 200 mM KCI, and involves fol-
lowing the decay of the pH perturbation caused by a small acid pulse.
Scholes and MitchelP4 and Maloney37 have described this method and the

35 T. A. Krulwich, R. A. Agus, M. Schneier, and A. A. Guffanti, J. Bacterh~l. 162, 768

(1985).
36 S. H. Collins and W. A. Hamilton, J. Bacteriol. 126, 1224 (1976).
37 p. C. Maloney, J. Bacteriol. 140, 197 (1979).
 360 BACTERIAL TRANSPORT [28]

0
0

r~
o

<
<
r,..)

z
m
EL
EL

0
¢,q
)"
r..)
0

m
[.., oo
.,..2
0 q)
m

-1
< L~
>
m

c¢
<
%
Z

• ~. o o
m

¢o

.~ O ..~ e'~

~ N
[28] REGULATIONOF INTERNALpH 361

relevant calculations in detail. Bo is calculated from a value for the initial

pH deflection that is obtained by extrapolation back from the decay
c u r v e . Bt is determined from the final equilibration pH. Maloney37 has
indicated the importance of controls to show that equilibration has indeed
occurred. In work with aerobes, it may be important to add an inhibitor of
the respiratory chain in addition to the KSCN, valinomycin, and carbon-
ate dehydratase included by Maloney. At least some bacilli that we have
examined fail to achieve a fiat baseline value even after prolonged incuba-
tion of the cell suspension under N2 in the presence of such an inhibitor.
If the cells are more rigorously starved, to eliminate the residual proton
pumping, care must be taken to determine that the membrane is uncom-
promised. These considerations notwithstanding, there is an advantage to
the acid pulse method. That is, a value for the passive membrane conduc-
tance to protons (CmH+) can be obtained. CmH+ is calculated using a tj/2 for
the time required for equilibrium to be reached, as described by Mitchell
and Moyle. 3z
In initial determinations in our laboratory, the Cmn+ of B. acidocalda-
rius has been found to be in the same range as values reported for other
bacteria. These values are preliminary, however, and no attempts have
yet been made to systematically examine the passive permeability to
other cations. Such determinations will be of interest and, as noted ear-
lier, should include measurements made under various conditions of ex-
ternal pH and composition of the suspending medium.

Alkalophilic Bacteria
Obligately alkalophilic bacteria maintain a cytoplasmic pH of no
higher than 9.6 when suspended in Na+-containing alkaline buffers at pH
values as high as 11 (Table If). 16'38 Under growth conditions, or if a sub-
strate such as L-malate is added to the buffer, an even lower pHi, of about
8.5 is maintained. 39 Cells of B. firmus RAB that have been equilibrated at
pH 8.5 also exhibit impressive homeostasis after a rapid shift in pHout to
10.5 as long as Na ÷ is present in the medium. 4° Evidence from in vitro
studies 18,2° and in vivo experiments with both wild type and mutant
strains 39-41 supports the conclusion that (1) Na ÷ is required for pH homeo-
stasis in obligate alkalophiles; (2) Na~+. is exchanged electrogenically for
Ho+t ( H + translocated in > Na ÷ translocated out) by a Na+/H + antiporter

38 A. A. Guffanti, P. Susman, R. Blanco, and T. A. Krulwich, J. Biol. Chem. 253, 708 (t978).
39 M. Kitada, A. A. Guffanti, and T. A. Krulwich, J. Bacteriol. 152, 1096 (1982).
40 T. A. Krulwich, J. G. Federbush, and A. A. Guffanti, J. Biol. Chem. 260, 4055 (19851.
41 M. L. Garcia, A. A. Guffanti, and T. A. Krulwich, J. Bacteriol. 156, 1151 (1983).
362 BACTERIAL TRANSPORT [28]

T A B L E 11
CYTOPLASMIC pH OF BUFFERED CELL SUSPENSIONS OF
ALKALOPHILIC BACTERIA

pHin

pHout BacHlusalcalophilus" BacillusfirmusRAB b

9.0 9.0 9.0

10.0 9.5 9.3
10.5 9.5 9.3
I1.0 9.6 9.5

" Guffanti et a l ) 8
b Guffanti et al. ~6

that is active only above a cytoplasmic pH of about 8; and (3) Na ÷-

coupled solute uptake systems play a physiologically significant role in
pH homeostasis by providing an entry route for Na ÷, albeit as yet unclear
whether other entry routes exist. Although some obligate alkalophiles do
not exhibit marked growth dependence upon added Na* in the medium,
they probably utilize the same kind of Na ÷ cycle (perhaps augmented by
incompletely defined auxiliary mechanisms) except that the affinity for
Na ÷ is so high that it is difficult to achieve an operational "Na÷-free ''
condition. 18,40,41Work by other investigators with several other alkalophi-
lic and alkaline-tolerant organisms indicates that a Na+/H + antiporter may
have a widespread role, and usually a dominant one, in pH homeostasis in
the highly alkaline range. 42-47 We will outline the special considerations in
measuring ApH in alkalophiles and will describe several protocols
whereby the Na + requirement for pH homeostasis can be demonstrated
and characterized.

Determinations of ApH
The pHin of alkalophiles is calculated from measurements of the ApH
using the same principles and methods as described above except that the
probe, buffers, and controls require accommodation to the alkaline pH

42 D. M c L a g g a n , M. J. Selwyn, and A. P. D a w s o n , F E B S Lett. 165, 254 (1984).

43 A. G. Miller, D. H. Turpin, and D. T. Canvin, J. Bacteriol. 159, 100 (1984).
44 B. V. C h e r n y a k , P. A. Dibrov, A. N. Glagolev, M. Y u Sherman, and V. P. Skulachev,
F E B S Lett. 164, 38 (1983).
45 T. N a k a m u r a , H. Tokuda, and T. Unemoto, Biochim. Biophys. Acta 776, 330 (1984).
46 M. Kitada and K. Horikoshi, J. Biochern. 87, 1279 (1980).
47 A. A n d o , I. Kusawa, and S. Fukui, J. Gen. Microbiol. 128, 1057 (1982).
[28] REGULATION OF INTERNAL pH 363

range. We have employed carbonate buffers at pH 9.0 and above; it can

be shown, using less optimal Tris buffers, that carbonate per se is not
required for homeostasis or Na+/H + antiport. For assays of a PHacid~,,
weak bases rather than weak acids are employed. [14C]Methylamine, with
a pKa of 10.6, is generally used. The probe concentration is kept at 25/~M
or below (52 mCi/mmol) in order to avoid dissipating the ApH. Since some
bacteria may have transport systems for methylamine/ammonium, it is
important to verify for each organism that the weak base enters by diffu-
sion and that its accumulation ratio reflects the ApH. Towards that end,
methylamine uptake should be examined over a wide range of concentra-
tions of the base to assess saturability, and uptake of the base should be
examined in deenergized cells across which an artificial pH gradient is
imposed. As with the weak acids used, the quenching effects of the buffer
(which may vary with the pH of the buffer) must be determined. Binding
controls are also needed here; we have employed killed cells and gramici-
din-treated (1 tzM) cells. Compounds that catalyze electroneutral ex-
change of OH-/anion or H+/cation could also be used. Weak acid pro-
tonophores at high concentration may collapse a pH gradient at
moderately alkaline pH, but cannot be used reliably at very alkaline pH.
Many of our own studies and some of the protocols described below
involve suspension of alkalophile cells at neutral or near-neutral pH val-
ues. If no methylamine uptake is observed with such suspensions, it is
still premature to conclude that ApH = 0 and pHin = pHoot unless the
uptake of a weak acid (e.g., DMO) is also measured and found to be zero.
Indeed, there are experimental conditions in which alkalophile cells are
found to generate a ApH, acid out. 39

Demonstration of the Na + Dependence of pH Homeostasis in

Obligately AIkalophilic Bacteria
We have employed three experimental protocols to examine the re-
quirement for specific ions for pH homeostasis in the obligate alka-
lophiles. As noted, each of the approaches has indicated a major role for
Na +. In the first, logarithmically grown cells ofB. firmus RAB at pH 10.5
have been washed and resuspended in either 50 mM potassium carbonate
or 50 mM sodium carbonate buffer at pH 10.5.39.4° The cells resuspended
in potassium carbonate show an immediate rise in the pHi, to 10.5 and a
rapid loss of viability, whereas those in sodium carbonate (or control
combinations of sodium and potassium carbonates) maintain a cytoplas-
mic pH below 9.0 and retain viability to a much greater extent over the
first 30 to 60 min of incubation. The cells' ability to lower pHi, and retain
364 BACTERIAL TRANSPORT [28]

viability are significantly enhanced when a nonmetabolizable solute

whose inward transiocation is coupled to Na + uptake is added in addition
to Na +. We have used 500/zM a-aminoisobutyric acid (AIB). The results
support a role for Na + coupled symporters as a route of Na + entry that is
relevant to pH homeostasis. It is not surprising, but should be noted, that
the rate at which cells shifted to Na+-free medium lose viability is highly
dependent on the concentration of cells in the suspension and the number
of times the cells are washed on harvesting. Use of less concentrated cell
suspensions (e.g., 5-10 × 107 cells/ml) and more washes (2-3) result in
much more rapid loss of viability than with thicker, less rigorously
washed cell suspensions. This can be understood in terms of removal of
bound and even, perhaps, some cytoplasmic Na ÷ and adhering solutes
which might facilitate Na + cycling.
The other two protocols involve pH shifts and are patterened after
experimental designs used by Kroll and B o o t h , 48 Zilberstein et al., 4 and
McLaggan et al. 42 We have again used these protocols to demonstrate
Na ÷ dependence and AIB enhancement of pH homeostasis in B. f i r m u s
RAB. In one type of experiment, cells are equilibrated at pH 8.5 in the
absence of Na + and then subjected to an alkaline shift in pHout either with
or without the simultaneous addition of Na ÷ and/or AIB. Logarithmically
grown cells are harvested and washed in 50 mM potassium carbonate
buffer, pH 8.5. The cells are allowed to equilibrate for one hour at pH 8.5
with shaking at 30°; assays at that point confirm that pHin = 8.5. In our use
of this protocol, we then rapidly dilute (usually 1:10) the cells into potas-
sium carbonate buffer at pH 10.5. The cell concentration after the pH shift
is approximately 0.5 mg cell protein/ml. Some samples are shifted (di-
luted) into buffer containing Na ÷ (in concentrations from 2 to 50 mM) and/
or 500/zM AIB. Controls include samples with similar amounts of added
K + and a sample that is not shifted. Aliquots (usually 1 ml) are removed
periodically to assay the pH. The first sampling is soon (15 to 30 sec) after
the shift so that the "overshoot" phenomenon 4,42,48 can be observed.
Using this kind of protocol, a rapid rise in pHin up to 10.5 is observed in
cells shifted to pH 10.5 with no Na ÷ or low concentrations of Na +. The
presence of 50 mM Na + allows pH homeostasis with an initial overshoot
acidification of the interior. Notably, in the presence of AIB, but not in its
absence, concentrations of Na ÷ as low as 2.5 mM become completely
effective in facilitating pH regulation. These observations reinforce the
caveat against ruling out the possible efficacy of low, contaminating Na +
levels. Suppose, for example, that phosphate were a Na+-coupled solute.

48 R. G. Kroll and I. R. Booth, Biochem. J. 216, 709 (1983).

[29] P E P T I D E T R A N S P O R T IN B A C T E R I A 365

Unless cells were kept phosphate free, the combined effects of residual
Na + and residual phosphate might well facilitate the regulation of pHin.
Finally, in a third experimental design, cells equilibrated at pH 8.5 as
above are again shifted to pH 10.5, but only in potassium carbonate
buffer. After 1 or 2 min, with pHin already at 10.5, the suspensions are
"treated" with various concentrations of Na +, with added K + as a con-
trol, in some instances also adding AIB. While the cells under these
conditions can still regulate pHin, viability has been compromised, indi-
cating that the most pH sensitive process(es) is other than an alkali-
induced loss of the capacity for pH regulation.
While the protocols described will indicate Na + (or perhaps, with
other species, different ion) involvement in pH homeostasis, they do not
distinguish between the involvement of a secondary Na+/H ÷ antiporter
and, for example, a primary Na + pump. Distinction between possible
mechanisms of Na + function is generally made using inhibitors. Thus
evidence for an electrogenic Na+/H ÷ antiporter in obligately alkalophilic
bacilli includes: requirement of a A~b per se for Na + fluxes--22Na + efflux
from cells at high pH 41 and 22Na+ uptake by energized, everted vesi-
cles2°,49--and associated proton fluxes38A9; and inhibition of the Na+-de -
pendent acidification of the interior by protonophores, at least at the
moderately alkaline pH that can be tested. 38'4°

Acknowledgment
The work in our laboratory was supported by Research Grant PCM 8121557 from the
National Science Foundation.

49 A. A. Guffanti, FEMS Microbiol. 17, 307 (1983).

[29] P e p t i d e T r a n s p o r t in B a c t e r i a
By CHRISTOPHER F. HIGGINS and MARGARETM. GIBSON

Introduction
Three genetically distinct transport systems with overlapping sub-
strate specificities serve to mediate the transport of small peptides across
the cytoplasmic membrane and into the cytoplasm of both E. coli and S.
typhimurium.l This multiplicity of systems makes measurement of trans-

C. F. Higgins, Microbiology 17 (1984).

Copyright © 1986 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 125 All rights of reproduction in any form reserved.