Journal of General Microbiology (19831, 129, 367-373.
Printed in Great Britain
367
The Interrelation of Phototaxis, Membrane Potential and K+/Na' Gradient
in Halobacterium halobium
By V L A D I M I R A. B A R Y S H E V , t A L E X E I N . G L A G O L E V * A N D
V L A D I M I R P. S K U L A C H E V
A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry,
Moscow State University, Moscow 117234, U.S.S.R.
(Received 2 June 1982; revised 28 July 1982)
The attractant effect of green light and the repellent effect of blue light on Halobacterium
halobium were studied. It was found that addition of CN- and dicyclohexylcarbodiimide,which
block the redox chain and H+-ATPase, respectively, increased both the amplitude of lightdependent changes of membrane potential (A$), monitored by the distribution of tetraphenylphosphonium, and the sensitivity of the green-light taxis. A direct proportionality between A$
and the green-light sensitivity was revealed. The sensitivity of the green-light taxis was
decreased by glucose, histidine and Na+/K+gradient, i.e. factors reducing the light-dependent
changes of protonic potential. These factors did not affect the blue-light taxis. The uncoupler
carbonyl cyanide m-chlorophenylhydrazone appears to repel H . halobium cells. These data are in
agreement with the assumption that the green-light taxis is governed by a sensing of protonic
potential, whereas the blue-light taxis is mediated by a specific photoreceptor.
INTRODUCTION
Halobacterium halobium cells show two phototactic reactions. They swim towards greenyellow light, absorbed by bacteriorhodopsin (&,-,, 565 nm), and away from blue light. The latter
taxis is governed by a retinal-dependent pigment, P370 (Hildebrand & Dencher, 1975; Dencher
& Hildebrand, 1979). Recently, we studied the role of the energy level in the sensitivity of both
photosystems and found that the green-light induced phototaxis was observed only if light
changes produced a decrease in motility rate, presumably reflecting a lowering of the
protonmotive force (AiH+)level. In the case of the blue-light taxis, such a correlation was absent
(Baryshev et al., 1981). These results have been extended in the present study to quantitative
measurements of the membrane potential and taxis. The interplay of the phototaxis and AEH+
buffering by Na+/K+ gradient was also investigated.
METHODS
Bacteria and growth conditions. Halobacterium halobium R,M I strain, lacking gas vacuoles and bacterioruberin,
was a kind gift of Professor D. Oesterhelt. The cells were grown for 96 h (to the stationary phase) under
illumination in complex peptone (Oxoid) medium (Oesterhelt & Stoeckenius, 1974) as described previously
(Arshavsky et al., 1981).
Manipulation of the cells. Bacteria were harvested by centrifugation, washed and resuspended to 2 x lo9
cells ml-' in a salts solution containing 4270 mM-NaC1,27 rnM-KC1,SO mM-MgSO,, 14 mM-sodium citrate, 2 mMMOPS, pH 7.0. Before use the cells were incubated in the basal salts medium for 24 h at 37 "C to increase the
fraction of motile cells. After incubation, cells were again washed and resuspended.
Media. The Na+ medium contained 4270 mM-NaC1, 27 mM-KCl, 81 mM-MgSO,, 25 mM-Tris/MOPS, pH 8.0;
K + medium contained 1570 mM-NaC1, 2700 mM-KCl, 81 mM-MgSO,, 25 mM-Tris/MOPS. pH 8.0.
~-
______-__-
I
_
_
Ahhrriiations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,N'-dicyclohexylcarbodiimide;
TPP+, tetraphenylphosphonium; &+, transmembrane electrochemical potential of H + ions; A$, transmembrane electrical potential difference ; MCP, methyl-accepting chemotaxis protein.
t Deceased I 1 December 1982.
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368
V . A . BARYSHEV, A. N . GLAGOLEV A N D V . P . SKULACHEV
Reagents. Chemicals were obtained fram the following sources: DCCD, Fluka; CCCP, Serva; NaCN, BDH;
Tris and MOPS, Sigma; peptone, Oxoid. All other chemicals used were of reagent grade.
Measurement of the membrane potential (A$). The A$ across the H . halobium cytoplasmic membrane was
determined by a method using lipophilic ions (Liberman & Skulachev, 1970). TPP+ concentration was monitored
with a selective electrode (Grinius et al., 1980), which was a kind gift of Dr L. Grinius. The electrode was inserted
into a 2 ml transparent plastic chamber. A 200 pl sample of the cell suspension was added to the incubation
mixture to obtain a final turbidity of 2-3 at 578 nm. The A$ was calculated from the Nernst equation:
The relationship between the turbidity at 578 nm and the protein content in the cells used in our study was the
same as reported by Michel & Oesterhelt (1980).Therefore, we used the figures of the above authors to estimate the
content of intracellular water, i.e. a turbidity of 1.O was assumed to correspond to 0.8 mg intracellular water per ml
of suspension.
TPP+ is absorbed by the cells in an energy-independent manner. To measure this absorption, the cells were
incubated for 24 h in K + medium in the dark with 2.5% (v/v) formaldehyde; a given amount of TPP+ was then
added and the fraction that bound to the cells was measured. Formaldehyde treatment abolished energydependent TPP+ accumulation. All measurements were corrected for the energy-independent TPP+ binding. The
plots shown in this paper are typical of a series of four to six experiments.
Illumination. The cells were illuminated by a 900 W Osram lamp as described previously (Arshavsky et al.,
198 l), by varying the intensity by neutral density filters. Light intensity was measured by a thermoelement (RTNlOC, USSR) connected to a millivoltmeter (VK2-16, USSR).
Photosensitivity measurements. Sensitivity was determined as the fraction of cells that reversed within 3 s after
the onset of the light stimulus. The stimuli were a decrease in green-yellow light or turning on of blue light. In the
latter case, a green-yellow background illumination was applied. Green light was delivered through the condenser
and blue light was transmitted through the incident light illuminator of a Univar (Reichert) photomicroscope. For
each stimulus, the behaviour of 40 cells was recorded. The fraction of cells that reversed was measured and
corrected for spontaneous reversals. Observations were made under phase contrast at 1000 x magnification. The
microscope slide was kept at 37 "C by a Biotherm thermostated microscope stage (Reichert).
Determination ofuncoupler taxis. Adler's capillary method was used to estimate the uncoupler taxis (Adler, 1973).
Capillaries, closed at one end, with an internal diameter of 0.35 mm, were filled with Na+ medium, pH 7.0, to a
length of 3 cm and placed in a transparent chamber filled with 1 ml of the same medium containing 2 x lo9
cells ml-' and a certain concentration of CCCP. The chamber was then placed into a luminostat at 37 "C for 120
min. The capillaries were rinsed and their contents were diluted three- to sixfold and placed in a cell-counting
chamber. The cells were counted under phase contrast. For each measurement, the average number of cells from
three or four capillaries is given.
RESULTS
Comparison of green- and blue-light taxis with light-induced changes in A$
It is known that A i H + may be composed of two constituents, A$ and ApH. To simplify the
experimental system, we took advantage of the fact that at pH 8.0 APH is absent in H . halobium,
so that Aj&+ is equal to A$ (Michel & Oesterhelt, 1980). All the experiments were performed at
pH 8.0, so that monitoring A$ by the TPP+ distribution actually gave the values of &+.
Blue light at an intensity inducing a 100% phobic response of H . halobium cells did not affect
the level of A$. This indicates that repellent taxis governed by P370 is not mediated by any
significant and steady changes in A$. On the other hand, variations in green-yellow light,
absorbed by bacteriorhodopsin, resulted in measurable A$ changes (Fig. 1a, b).
In our previous study, we found that inhibitors of the light-independent proton pumps,
DCCD and CN-, strongly increased the sensitivity of green-light taxis (Baryshev et al., 1981).
This effect was attributed to an increase in the amplitude of AZH+ changes following light stimuli,
which would be likely to happen if bacteriorhodopsin were the only operating proton pump. We
directly tested this assumption by registering the light-dependent changes of A@ in the presence
of DCCD and CN- (Fig. 1 b). Indeed, the amplitude of changes in the membrane potential level
appeared to be much larger than without inhibitors (Fig. la).
Since blue light had no effect on A$, further analyses were confined to the action of greenyellow light. The dependence of the amplitude of A$ changes in response to various decreases in
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Phototaxis in H . halobium
369
Fig. 1 . Effect of blue and green-yellow light on the membrane potential of H . halobium cells. Cells were
suspended in Na+ medium supplemented with 5 x 1 0 - 6 ~ - T P P + .The TPP+ concentration was
monitored by a TPP+ selective electrode. (a) Turning on and off green-yellow light ( A > 540 nm,
90 W rn-l). (6) Turning on and off green-yellow light (as in a ) in the presence of 5 mM-NaCN and
0.1 mM-DCCD. The scales in (a) and (6) are the same.
light intensity is shown in Fig. 2(a). Here again, the amplitude of changes in A$ was greater in
the cells with inhibitors at all the tested light intensity changes. The same changes in
illumination were used to study the sensitivity of taxis in the microscopic assay. The sensitivity
was found to be higher in the cells with inhibitors as compared to the control (Fig. 26). Taxis
sensitivity plotted against A(A$) (Fig. 2c) revealed that the taxis response paralleled A$ changes.
The plot was linear, regardless of the presence or absence of inhibitors, which indicates that cells
can perceive changes in A$ by a ‘voltmeter’ operating on a linear scale. Thus 20% taxis was
caused by a 85 mW cm-* decrease in green light intensity in the control, while only a
25 m W cmV2change was required to elicit the same response in the cells with inhibitors.
Therefore, there is no input of light changes per se (apart from its effect on &+) in the
photoresponses, in agreement with our previous findings (Baryshev et al., 198 1).
In a study of H . halobium photoresponses, Dencher (1978) found that Weber’s law, although
valid for the blue-light taxis, was inapplicable to the green-light taxis. Weber’s law holds that the
response is constant if the product of a stimulus and its duration are kept constant. The
inapplicability of Weber’s law to the green-light system seemed rather strange, if we take into
account the above finding of a very simple relationship, i.e. the direct proportionality between
the extent of taxis and the magnitude of A$ changes (see Fig. 2c). In an attempt to solve this
problem, we monitored the kinetics of the light-induced A$ changes during several minutes after
changes in light intensity. Turning off the light produced a decrease in A$, which then eventually
regained its original level. Subsequent illumination increased A$, but again the A+ level returned
to the initial value within a few minutes (Fig. 3). These observations clearly indicate that
constancy of the product of light decrease and stimulus duration will not provide a constant
product of A(A$) and stimulus duration, since both the light- and dark-induced A$ changes tend
to reverse in time. This is why, apparently, the cells seem not to obey Weber’s law.
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370
v.
A . BARYSHEV, A . N . GLAGOLEV AND V . P. SKULACHEV
100
80
n
>
E
3
3
60
.-E
.-
3
40
W
a
W
(c1
20
20
60
40
20
40
60
80
100
Decrease in light intensity (5%)
100
80
Decrease in light intensity (%)
100
80
-8
60
v
.-mX
40
20
2
4
6
Decrease in Aty (mV)
8
Fig. 2. Dependence of the green-light taxis sensitivity and of A+ on the light intensity. Cells were
prepared as in Fig. 1. 0,
control; a, in the presence of 5 mM-NaCN and 0.1 mM-DCCD. Initial
intensity of the green-yellow light was 90 W m-?. The membrane potential in the light was 172 mV for
the control cells and 202mV in the presence of inhibitors. (a) Dependence of changes in A$ on
illumination decrease. (b) Dependence of taxis sensitivity on illumination decrease. The taxis was
determined by following the fraction of cells that reversed in response to the indicated light decreases,
as described in Methods. (c) Dependence of taxis sensitivity on changes in membrane potential.
Derived from data of Figs 2(a) and (b).
200
: : : : :
-
I
I
I
I
I
'
>
h
E
3.
Q
on
160
170
Fig. 3. Kinetics of A$ changes following variations in green-yellow light illumination. Cells were
suspended in the growth medium. Light intensity was 90 W m-*, Iz > 540 nm.
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371
Phototaxis in H . halobium
190
->
180
s
160
-a
E
170
150
140
0
5
10
15
20
25
30
Time (min)
Fig. 4. Stabilization of A$ by the Na+/K+ gradient. Cells were suspended in Na+ ( 0 )or K+ ( 0 )
medium in the presence of 5 m - N a C N . Initial illumination (as in Fig. 3) was turned off as indicated by
the arrow.
Natural factors that aflect phototaxis sensitivity
Since photoresponses to light absorbed by bacteriorhodopsin appear to be mediated by A i H +
sensing (Baryshev et al., 1981, and the present study), we studied the interplay of A i H + sensing
and A i H +buffering. For effective A i H + sensing, changes in A i H + should be rapid. On the other
hand, A i H + may be stabilized by a Na+/K+ gradient (Skulachev, 1978). In H . halobium,
electrogenic K+ transport buffers changes in A$ (Wagner et al., 1978) and the electrogenic
Na+/H+antiport (Lanyi & MacDonald, 1976) may stabilize the overall A i H + (Arshavsky et al.,
1981). Therefore A i H + buffering would seem to oppose AllH+ sensing. To look into this
possibility, the sensitivity of H . halobium phototaxis in a Na+ medium was compared to that in a
K + medium. In the Na+ medium, both the outward-directed K+ gradient and the inwarddirected Na+ gradient are large, providing optimal conditions of A i H + buffering. Indeed,
turning off the light has little effect on the A$ of H . halobium cells (Fig. 4). In the K+ medium,
containing high K+ and lower Na+, the K+ gradient is absent and the Na+ gradient is lowered.
Accordingly, the stabilization of A$ is less efficient (Fig. 4). The sensitivity of photoresponses
to a decrease in green-yellow light was only half as great in the Na+ medium as compared with
the K+ medium (results not shown). Sensitivity of the blue-light response, which is assumed to be
independent of AzH+sensing, was about the same in both Na+ and K+ media. This means that
the differences in taxis sensitivity of the green-light system between the Na+ and K+ media can
hardly be accounted for by a non-specific inhibition of the taxis systems in Na+ medium and are
rather due to the AFH+ buffering effect.
Another way of changing the sensitivity of the green-light photoresponses would be by
modifying the activity of the light-independent proton pumps. As it was found that CN- and
DCCD increased the phototaxis sensitivity, it would be reasonable to expect that oxidizable
substrates would have the opposite effect. Indeed, glucose plus histidine inhibited the greenlight taxis more than twofold at 10 MM, while producing only a small effect on blue light taxis
(results not shown). Glucose and histidine were found to be attractants in H . halobium by Schimz
& Hildebrand (1979).
Repellent efect of an uncoupler
The existence of AFH+ sensing presupposes that cells must respond to A z H + changes
irrespective of what causes them (Glagolev, 1980). A simple way to modify A i H + without
interfering with the light intensity is to add an uncoupler. The ability of H . halobium to be
repelled by an uncoupler, CCCP, was tested in a capillary assay. A capillary containing a basal
salt solution was placed in a chamber containing CCCP and a cell suspension. The uncoupler
diffused into the capillary, forming a spatial gradient. At 5 x 1 0 - 6 ~ - C C C Pa, significant
number of cells were repelled (Fig. 5). At higher concentrations of CCCP, the repelling activity
declined, eventually falling below the control level, probably due to a decrease in motility rate.
At the maximal CCCP concentration used,
M, the motility rate dropped from 3.9 pm s-l to
2.9 pm s-l.
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372
V. A . BARYSHEV, A. N. GLAGOLEV A N D V. P . SKULACHEV
Fig. 5. Migration of H . halobium cells as a function of an uncoupler gradient. Cells were suspended in
the basal salts solution in the presence of indicated amounts of CCCP, and capillaries containing the
same solution without CCCP placed into this medium. Cells that swam into the capillaries were
counted after 120 min. The 100%level represents the number of cells that swam into capillaries when
the basal solution contained no CCCP.
DISCUSSION
Bacterial A i H + sensing is at present a subject of controversy, being advocated by some
researchers and denied by others. Monitoring A$ changes by fluorescence of a cyanine dye,
Ordal & Villani (1980) found no correlation between the magnitude of A$ changes and the
extent of uncoupler taxis in Bacillus subtilis. On the other hand, we found a good correlation
between CCCP-induced changes in A$, monitored by TPP+ distfibution, and the repelling
action of CCCP on a cyanobacterium Phormidium uncinatum (Murvanidze et al., 1982). Besides,
the potency of uncouplers as repellents was found to correlate with their known protonophorous
activity on B. subtilis (Ordal & Goldman, 1975) and Escherichia coli (Sherman et al., 1981).
Repaske & Adler (1981) recently suggested that the sensing of A i H + was in fact a reflection of
specific sensing of the internal pH. However, we found that uncoupler taxis of E. coli did not
depend on the main methyl-accepting chemotaxis proteins (MCPs), namely, MCP-I and MCPI1 (Sherman et al., 1981), whereas pH taxis specifically requires MCP-I. Uncoupler taxis in E .
coli seems to closely resemble O2 taxis, which was also ascribed to A i H + sensing (Laszlo &
Taylor, 1981) and was found not to be dependent on either MCP-I or MCP-11. In B. subtilis,
uncoupler taxis was reported to be entirely independent of methylation, which means that this
phenomenon should be placed apart from the remaining system of chemotaxis (Ordal, 1976).
Recently MCP methylation was found to accompany chemotaxis and the green-light taxis in H .
halobium (Schimz, 1981). Thus, in H . halobium, in contrast to E . coli and B. subtilis, AFH+sensing
seems to share the information processing pattern with conventional chemoreception.
The data of the present study indicate, in agreement with our previous results (Baryshev et al.,
1981) that there is no bacteriorhodopsin-linkedgreen-light sensing other than that mediated by
AF.H+.
Phototaxis to green light was quite predictably modified by naturally existing factors, namely,
by the Na+/K+gradient and oxidizable substrates. If green-light taxis is considered as a AiH+linked process, it seems quite reasonable that the sensitivity of photoresponses should be
diminished under conditions when A i H + was effectively stabilized by the fluxes of Na+ and K+
ions. The fact that glucose and histidine also suppressed phototaxis sensitivity may be explained
by decrease in light-dependent A i H + changes in the presence of an actively operating redox
chain. The blue-light taxis remained unchanged in spite of variations in substrates or in Na+/K+
buffer. Moreover, blue light at intensities causing a 100% behavioural response had no effect on
the level of A+ as measured by TPP+ distribution. This result is in apparent disagreement with
the study of Wagner et al. (1980), who found that blue light caused a K+ efflux from cells of H .
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Phototaxis in H . halobium
373
halobium, and with the study of Litvin et al. (1981) who observed a slow passive H+ influx
following blue light illumination. The reason for the discrepancy is probably the difference in
illumination intensity, which was 10 times lower in our experiment. To date, P375 appears to be
a specialized photoreceptor, unlike bacteriorhodopsin.
Thanks are due to Dr D. Oesterhelt for providing the H . halobium strain.
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