Vol. 131, No. 1
OF BACTEIUOLOGY, July 1977, p. 34-41 Copyright © 1977 American Society for Microbiology
JOURNAL
Printed in U.S.A.
Phototaxis and Membrane Potential in the Photosynthetic Bacterium Rhodospirillum rubrum SHIGEAKI HARAYAMA* AND TETSUO IINO Genetics, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan Laboratory of
Received for publication 23 February 1977
Cells of the photosynthetic bacterium Rhodospirillum rubrum cultivated anaerobically in light show phototaxis. The behavior of individual cells in response to the phenomenon is reversal(s) of the swimming direction when the intensity of the light available to them abruptly decreases. The tactic response was inhibited by antimycin, an inhibitor of the photosynthetic electron transfer system. The inhibitory effect of antimycin was overcome by phenazine methosulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic response; however, valinomycin or potassium alone had no effect. A change in membrane potential of the cells was measured as an absorbance change of carotenoid. Changes in the membrane potential caused by "on-off' light were prevented by antimycin and by valinomycin plus potassium, but not by antimycin plus phenazine methosulfate nor valinomycin or potassium alone. The results indicated that the phototactic response of R. rubrum is mediated by a sudden change in electron flow in the photosynthetic electron transfer system, and that the membrane potential plays an important role in manifestation of the response.
systems. The chemotactic behavior of individual cells of E. coli and S. typhimurium has also been examined (1-3, 16, 26, 30). The bacteria modulate the frequency of change in direction when they swim up or down a spatial gradient of a chemotactic stimulus; thus, their attraction to or migration from it is attained. This modulation is also observed when they are exposed to a temporal change in concentration of the stim-
Bacterial phototaxis has been observed in photosynthetic bacteria (6, 22) and in Halobacterium halobium (12). The photoreceptor pigments responsible for positive phototaxis (phototaxis towards light) have been identified as bacteriochlorophyll and carotenoids in photosynthetic bacteria (5, 19) and as bacteriorhodopsin in H. halobium (12). The phototactic behavior of individual cells of photosynthetic bacteria has been extensively examined (10): the bacteria show abrupt changes in direction when they swim from a light to a dark field, and some of them return to the light field; however, they fail to show such a response when they swim from a dark to a light field. As a consequence of these responses, the number of organisms in a light spot projected onto a suspension of bacteria increases with time. The change in swimming direction can be induced by temporal decrease of an actinic light intensity impinging on them. This observation suggested that the phototactic response is the result of sensing temporal, not spatial, differences in light intensity. In chemotaxis of Escherichia coli and of Salmonella typhimurium, chemoreceptor molecules of several attractants (chemicals that induce positive chemotaxis) have been identified (1, 11, 18). The sugar chemoreceptors are proteins that are constituents of active transport
ulating chemicals. Whereas cellular receptors of the tactic stimuli and swimming behaviors induced by the stimuli have been well documented in bacterial phototaxis, as well as in bacterial chemotaxis, how the stimuli are converted to a tactic signal that modulates the frequency of change in their swimming direction is unclear. In this paper we show evidence that reversals of the swimming direction of the photosynthetic bacterium Rhodospirillum rubrum induced by a sudden decrease in the actinic light intensity are mediated by a sudden decrease of electron flow in the photosynthetic electron transfer system and that membrane potential plays an important role in the response. MATERIALS AND METHODS Bacterial strains and growth of cells. Wild-type R. rubrum (S-1) was obtained from G. Soe, and the carotenoid-less blue-green mutant (G-9) was from Y. 34
PHOTOTAXIS IN R. RUBRUM
VOL. 131, 1977 Kobayashi, to whom we are indebted. The bacteria were cultivated in light at 28°C in a 120-ml screwcapped bottle containing a synthetic medium whose composition was as described previously (8), except that Casamino Acids was omitted from the medium. Illumination was provided by one 300-W flood lamp (Toshiba) placed 80 cm from the culture bottle. The bacteria at the late exponential to stationary growth phase were harvested, centrifuged, washed at least two times with 10 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.6), suspended in the appropriate solution, and then used for experiments. In the experiments reported in Table 3, the bacteria were washed in 10 mM sodium phosphate buffer (pH 6.8). Measurement of phototactic response. To examine quantitatively the phototactic response of the bacteria, an apparatus for giving successive phototactic stimuli to the bacteria was devised (Fig. 1). Two light beams, one for an actinic light and another for an observing light, were projected onto a phase-contrast microscope condenser through a half mirror. The actinic light beam from a tungstenhalogen lamp (Ushio, JC-24V-150W) was passed through a 4-cm cuvette containing water, chopped by a rotary sector for 0.5 s every 2 s, and then projected on a bacterial suspension on a glass slide through a phase-contrast condenser. The intensity of the actinic light, measured with a YSI-Kettering model 65 radiometer, was 500 W/m2. The observing light beam from a 30-W tungsten lamp (Nikon) was filtered by a thermo-cut-off filter (Hoya, HA-50) and a green interference filter (Toshiba, KL-56; peak transmission at 558 nm, with a half band width of 14 nm) and then continuously projected on the bacterial suspension. The intensity of the observing light was 3 W/m2 or below. The tactic response of R. rubrum induced by a sudden decrease in actinic light intensity is simple: the bacteria reverse their swimming direction after being stimulated (6, 10). Therefore, as an index of their phototactic activity, we measured the fraction of bacteria that showed reversal(s) of swimming direction within 0.5 s after the actinic light was turned off in repeated "on" (1.5 s) and "off" (0.5 s) circles, by direct microscopic observations made at an ambient temperature of 300C. When more than 50 bacteria were counted, the standard deviation of the measured values from the same sample did not exceed 20% of the mean value. Measurement of an absorbance change in bacterial suspension. Measurement of light-induced absorbance (A) change was carried out with a Hitachi 356 double-beam spectrophotometer. The wavelength used for measuring the absorbance change of carotenoid in wild-type cells (S-1) was 525 nm, with 510 nm as a reference. Those used for measuring that of cytochrome c2 and reaction center bacteriochlorophyll in cells of the carotenoid-less mutant (G9) were 552 and 600 nm, with 540 and 585 nm, respectively, as references. Actinic light from a tungsten-halogen lamp (Ushio, JC-24V-150W) filtered through an R-72 filter (Hoya), allowing transmission of light only above 700 nm, was projected onto a cuvette (light path, 1 cm) at a right angle to
35
Li II IF
c
.' *
HM
I
Ch
S
I
I
IHo 12 L2
RD
c7 FIG. 1. Apparatus for observation of bacterial phototactic response. Abbreviations: Li, tungsten lamp (Nikon, 30 W); L2, tungsten-halogen lamp (Ushio, JC-24V-150W); IF, interference filter (Toshiba, KL-56) plus thermo-cut-off filter (Hoya, HA50); 11 and 12, collector lens; H20, 4 cm of water; HM, half-reflecting mirror; Ch, rotary chopper cutting actinic light for 0.5 s every 2 s; RD, phase ring diaphragms; C, condenser lens; S, specimen; and 0, 40 x phase objective (Nikon, long-working distance).
the measuring beam of the spectrophotometer. The intensity of the actinic light was 1.3 kW/m2. Between the cuvette and a photomultiplier tube, two filters (Corning 9782 and HA-50) were placed to protect the photomultiplier tube from the cross-illuminating actinic light. Intermittent illumination of the actinic light (1.5 s on and 0.5 s off) was attained by insertion or removal of a sector connected to a rotary solenoid operated by an autotimer. Estimation of cellular content of bacteriochlorophyll. Bacteriochlorophyll was extracted with acetone-methanol (7:2, vol/vol), and then the cellular content of the pigment was determined spectrophotometrically by using the absorption coefficient given by Clayton (5). Chemicals. Valinomycin and antimycin were purchased from Boehringer Manheim. Other reagents used were of analytical grade.
36
HARAYAMA AND IINO
J. BACTERIOL.
its oxidation (decrease in A552-540) by light was inhibited, and its reduction after the light was turned off was prevented by the inhibitor. Redox changes of reaction center bacteriochlorophyll, measured as A605585, were hardly affected by the inhibitor. Changes in carotenoid absorbance, which reflects changes in membrane potential (14, 15), were affected by the inhibitor: an increase in membrane potential (hyperpolarization) was observed after the light was first turned on, but a decrease in membrane potential (depolarization) after the light was turned off was prevented by the inhibitor. That the observed absorbance change at 525 to 510 nm corresponded to that of carotenoid, reflecting changes in membrane potential, was confirmed by the fact that, in a cell suspension of the carotenoid-less mutant (G-9), no lightinduced absorbance change between the two wavelengths was observed with or without antimycin and PMS (Fig. 2). Effect of valinomycin on phototactic response and light-induced absorbance change of carotenoid. Valinomycin is an ionophore that increases conductivity of K+, Rb+, and Cs+ in biological membranes; in the presence of valinomycin, these cations move down the electrochemical potential across the membrane and discharge it (9, 28). We used the ionophore to examine the role of membrane potential in the bacterial phototactic response because its effect on biological membranes was well characterized. When valinomycin was added to the cell susantimycin. of R. rubrum with or without KCl, it pension Antimycin affected light-induced redox on the phototactic response or on effect had no as measured A552-540: changes of cytochrome c2 the light-induced absorbance change of carotenoid. Since it is known that the outer memTABLE 1. Effect of antimycin and PMS on brane of gram-negative bacteria acts as a penephototactic response tration barrier for hydrophobic antibiotics having a molecular weight of 1,100 (20), it is possiPhototaxis Nb Additiona ble that the insensitivity of intact R. rubrum cells to the ionophore is due to the existence of 60d 224 Ethanol (0.2%, vol/vol) an intact outer membrane. It was reported that 5d 129 Antimycin (20 ,uM) 84e ethylenediaminetetraacetate (EDTA) removed Ethanol (0.2%, vol/vol) + PMS 180 (0.2 mM) the penetration barrier (17), and thus E. coli, a 67e Antimycin (20 gM) + PMS (0.2 163 gram-negative bacterium, became sensitive to mM) valinomycin (27). Upon EDTA treatment, cells a Cells ofR. rubrum S-1 were suspended in 10-2 M of R. rubrum also became sensitive to valinoTris-hydrochloride buffer (pH 7.6) containing the mycin: the phototactic activity of the EDTAaddition. treated cells was strongly inhibited by valinobNumber of the bacteria examined. mycin. Spontaneous reversal frequency of the ' Fraction of the bacteria that showed reversal(s) cells, under steady light illumination, was also of swimming direction 0.5 s after actinic light was diminished by valinomycin. The concentration turned off. d Phototactic response was observed 1 to 1.5 h of EDTA required to render the cells sensitive to valinomycin differed in different cell culafter the addition of ethanol or antimycin. tures: cells in the stationary growth phase were e PMS was added 1.5 h after the addition of ethanol or antimycin, and then the phototactic re- more resistant to EDTA than those of the exponential growth phase. Cumulative results on sponse was observed 10 to 20 min afterwards.
RESULTS Effect of antimycin and PMS on phototactic response and photosynthetic electron transfer in the bacteria. Cells of R. rubrum reverse their swimming direction when the actinic light intensity impinging onto them is abruptly decreased. Since the phototactic phenomenon, i.e., accumulation of the bacteria in a light spot projected onto a bacterial suspension, is a result of this response (6, 10), we estimated the phototactic activity of the bacteria by counting the fraction that reversed swimming direction in a 0.5-s dark period after the actinic light was turned off. The value of the fraction depended on decreased intensity of the actinic light as well as intensity of the observing light. Furthermore, sensitivity of the cells to the phototactic stimulus differed in different cultures. However, under the conditions described in Materials and Methods, 60 to 95% of the cells responded to the stimulus in control experiments. Antimycin, a specific inhibitor of photosynthetic electron transfer (13, 33), inhibited the phototactic response of the bacteria (Table 1). Motility of the bacteria was not affected by the inhibitor under aerobic conditions. The inhibitory effect of antimycin on the phototactic response was abolished when phenazine methosulfate (PMS) was subsequently added to the bacterial suspension. PMS had a stimulative effect on phototactic response when it was added to the bacterial suspension containing no
VOL. 131, 1977
PHOTOTAXIS IN R. R UBRUM Ethanol
I (GA6004585 9)
Antimycin
Ethanol PMS
37
Antimycin PMS
1444444444
Anti,rnycI
fff tftffff 2
7A552540 Iftt fIftit A52s-5
it ltfttlIft ftf itftftt f tftff t tftt tftftt
o
t0.01
fl fttft
ft tftttft
ftftffitt
ttt ftttti tt 208
FIG. 2. Effect of antimycin on the light-induced absorbance change in bacterial suspension. Cells of R. rubrum strains S-i and G-9 suspended in 10 mM Tris-hydrochloride buffer (pH 7.6) were incubated aerobically on a reciprocating shaker (120 strokes per min) at 28°C for 1 h. Then, 20 pM antimycin dissolved in ethanol (final concentration, 0.2% [vol/vol]) or 0.2% (vollvol) ethanol was added to the bacterial suspensions. They were further incubated aerobically for 1 to 1.5 h, and then light-induced absorbance changes were measured as described in the text. Freshly prepared PMS (0.2 mM) was added 1.5 h after the addition of ethanol or antimycin, and the light-induced absorbance changes were measured within 10 min. Arrows (.I t) indicate light on and light off, respectively. Absorbance changes of bacteriochlorophyll (A,5858 and cytochrome c2 (A552--W) were measured with the carotenoid-less mutant G-9, at a bacteriochrolophyll concentration of 18W. The mutant strain was used for the measurements because, in wild-type cells, these absorbance changes were obscured by associated carotenoid changes at these wavelengths (21). The absorbance change of carotenoid was measured with wild-type strain S-l, at a bacteriochlorophyll concentration of 23 pM, at A525 510, where no absorbance change was observed in carotenoid-less mutant G-9. Phototactic activities in the bacteria used in the experiment were 85% (+ethanol), 10% (+antimycin), 98% (+PMS), and 70% (+antimycin and PMS) in G-9 cells; and 73% (+ethanol), 9% (+antimycin), 86% (+PMS), and 73% (+antimycin and PMS) in S-i cells. The motility of G-9 cells has been observed to be impaired under high light intensity in aerobic conditions. This may be a result of photodynamic action (7). However, under the present experimental conditions, no impairment of motility was observed.
phototactic response are presented in Table 2. In these experiments, S-1 cells at a growth phase indicated in the table were suspended in 10 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mM KCI and were then incubated aerobically with shaking (120 strokes per min) at 280C. The indicated concentrations of EDTA and ethanol (1% vol/vol) or valinomycin (10 ,uM) dissolved in ethanol (final concentration, 1%, vol/vol) were added to the cell suspensions, which were further incubated aerobically for the indicated times. Since the bacteria treated with EDTA had a tendency to adhere to cover slips and glass slides, the phototactic response of the EDTA-treated cells was observed by using a bacteria counting chamber (20-gm depth) in which their motility was fair.
Light-induced absorbance changes of carotenoid were measured with the samples listed in Table 2; some of the results are presented in Fig. 3. In the cells used in experiment 2 ofTable 2, absorbance changes of carotenoid were normal when ethanol was added to the cell suspension, but in the presence of valinomycin they were prevented. In cells used in experiment 5 of Table 2, even without the addition of valinomycin, phototactic activity was strongly reduced by EDTA. The cells showed only the slight absorbance change of carotenoid. Why EDTA inhibited the reaction was unclear. These results showed that inhibition of the light-induced absorbance change of carotenoid, i.e., the light-induced change in membrane potential, by valinomycin or by EDTA resulted in a con-
38
J. BACTERIOL.
HARAYAMA AND IINO TABLE 2. Effect of valinomycin on phototactic response Expt
Growth phase
EDTA concn (M)
time in Incubation EDTA (min)
Additiona
Nb
Phototaxis (%
1
Stationary
10-3
42-52
E V
121 55
53 7
2
Stationary
10-3
280-285
E V
33
67
44
2
3
Stationary
10-3
240-245
E V
64 52
73 6
4
Late exponential
10-4
35-50
E V
51 35
49 6
5
Late exponential
10-4
80-83
E V
61 44
10 0
6
Stationary
10-3
100-105
E V
73 46
62 2
10-4
200-205
E V
92 64
78 78
a V, 10-5 M valinomycin; E, 1% (vol/vol) ethanol. b Number of bacteria examined.
* 4 +$* +4+4+ 4 +t
t
+ +
t+ +
9* + t+ + + + +* +
J0.001
A
r*,,,,,444
B T~lOs
FIG. 3. Light-induced absorbance changes of carotenoid in EDTA-treated cells with or without the addition of valinomycin. Light-induced absorbance changes at 525 nm, with 510 nm as a reference of the bacterial suspensions used in experiments of Table 2, were measured immediately after the measurements ofphototactic response. (A) Bacterial suspension used in experiment 2 of Table 2 (bacteriochrolophyll concentration, 23 pM). (B) Bacterial suspension used in experiment 5 of Table 2 (bacteriochrolophyll concentration, 13 M). (E) Ethanol (19%, vollvol) added. (V) valinomycin (10 pM) added.
comitant inhibition of bacterial phototaxis. The effect of incubation time of the cells in EDTA solution on their phototactic response in the presence or absence of valinomycin was examined (Fig. 4). In the experiment, 10 mM CaCl2 was added to the cell suspension to stop the action of EDTA at the indicated time. It was shown that sensitivity ofthe cells to valinomycin increased with increased incubation time in EDTA solution; however, prolonged incubation resulted in inhibition of phototactic response even without addition of valinomycin. Valinomycin discharges membrane potential
only when appropriate monovalent cations such as K+ and Rb+ are present. To examine whether the inhibitory effects of valinomycin are dependent on the presence of these cations, the effects of valinomycin on phototactic response and light-induced absorbance change of carotenoid in the EDTA-treated cells suspended in buffer free from these cations were examined. For this purpose, cells of R. rubrum S-1 at the late exponential growth phase were harvested, suspended in 10 mM sodium phosphate buffer (pH 6.8), and incubated aerobically with shaking (120 strokes per min) at 28°C for 1 h; 0.3 mM
VOL. 131, 1977
PHOTOTAXIS IN R. RUBRUM
39
TABLE 3. Requirement of K+ or Rb+ for the inhibitory effect of valinomycin on both phototactic response and light-induced absorbance change of carotenoid
Additiona
R t\
E V E V E V
I~~~~9I'Jr
0
Monovalent cation added water (1%, vol/vol) water (1%, vol/vol)
10-2 M KCl 10-2 M KCl 10-2 M RbCl 10-2 M RbCl
b
41 36 44 100 41 70
Photo- AAB/M
(%)xi BChlc5 73 86 73 15
80 16
3.0 2.6 2.3 0.8 2.6 1.0
E, Ethanol (1%, vol/vol); V, valinomycin (10-5 M). Number of bacteria examined. Mean of 10 absorbance changes of carotenoid (A525510) per micromolar bacteriochrolophyll (BChl) 0.5 s after the actinic light was turned off. a '
c
Val 2
15 45 85 INCUBATION TIME IN EDTA(MIN)
FIG. 4. Effect of incubation time in EDTA solution on phototactic response. S-i cells harvested in the late exponential growth phase were suspended in 10-2 M Tris-hydrochloride buffer containing 10 mM KCl and were incubated aerobically at 280C for 1 h. EDTA (0.1 mM) and ethanol ( %, vollvol) or valinomycin (10 pM) were added to the bacterial suspensions, which were further incubated aerobically at 28°C. CaCl2 (1 mM) was added to stop the action of EDTA at a time, indicated on the figure, after the addition of EDTA, and then the phototactic response was observed 15 min afterwards.
EDTA and ethanol (1%, vol/vol) or valinomycin (10 AM) were added to the cell suspension and further incubated aerobically for 30 min. CaCl2 (2 mM) was added to stop the action of EDTA on the cells. Thirty minutes after the addition of CaCl2, 10 mM KCl, 10 mM RbCl, or water (final concentration, 1% [vol/vol]) was added to the cell suspensions, and phototactic activities of the cells in these suspensions were measured 20 to 35 min and 40 to 55 min afterwards. The mean of two measurements is presented in Table 3. The absorbance changes of carotenoid within 0.5 s after the actinic light was turned off were measured 60 to 65 min after the addition of KCl, RbCl, or water (Table 3). When the bacteria were suspended in the 10 mM sodium phosphate buffer containing no KCI, addition of valinomycin after EDTA treatment of the cells did not affect significantly their phototactic response or the light-induced absorbance change of carotenoid. Further addition of 10 mM KCl or RbCl to the bacterial suspension inhibited the phototactic response as well as the light-induced absorbance change of carotenoid (Table 3). The results clearly indicated that the inhibi-
tory effects of valinomycin on the phototactic response and light-induced absorbance change of carotenoid were dependent on the existence of appropriate cationic species such as K+ and Rb+, but not Na+.
DISCUSSION Pigmented cells of R. rubrum reverse their swimming direction when the intensity of light impinging on them is abruptly decreased. Since the reversal of swimming direction is a key movement in attaining phototactic accumulation (6, 10), we examined the fraction of bacteria that reverse their swimming direction after a sudden decrease in light intensity to estimate their phototactic activity. Under the conditions of the experiments, very few cells showed repeated reversals within 0.5 s after a sudden decrease in actinic light intensity; i.e., almost all of the cells showed one or no reversals after the stimulus. Photoreceptor pigments responsible for the phototactic response of the bacteria have been identified as bacteriochlorophyll and carotenoids (4, 19). These are pigments driving photosynthesis. Thus, the hypothesis that phototaxis is induced by a transient disturbance of photosynthetic metabolism was proposed and widely accepted (6). A requirement of photosynthetic electron transfer for the manifestation of phototaxis has been claimed by Throm (32). He observed that inhibitors of photosynthetic electron transfer affected the maximum level of accumulation of cells in a light spot. However, the rate and the maximum level of bacterial accumulation in a light spot may be affected complexly by changes in the speed of the bacteria or the fraction of motile bacteria, as well as their phototactic activity. Therefore, it is preferable to observe the behavior of individual
40
J. BACTERIOL.
HARAYAMA AND IINO
cells rather than the mass accumulation of bacteria in a light spot for determination of their phototactic response. The conclusion of Throm was confirmed by the present experiments in which antimycin, an inhibitor of photosynthetic electron transfer, prevented phototactic response. Motility of the bacteria was not affected by the inhibitor under aerobic conditions, indicating that the energy for motility is provided by respiratory metabolism, which is not affected by antimycin (data now shown), as well as by photosynthetic metabolism. The inhibitory effect of antimycin on both phototactic response and photosynthetic electron transfer was annulled by PMS (Table 1). The effect of PMS has been interpreted as the formation of a bypass around the blocking site of antimycin in the electron transfer pathway (29). From these observations, it is inferred that the reversal of swimming direction after a sudden decrease in actinic light intensity is a consequence of the decrease in photosynthetic electron flow. Accompanying the decrease of electron flow is a change in the redox state of components of the electron transfer system and a decrease (depolarization) of membrane potential measured as an absorbance change of the carotenoid (Fig. 3). Both the reduction of cytochrome c2 and the decrease in membrane potential after the light was turned off were inhibited by antimycin. Thus, by considering only the action of antimycin on phototactic response, one cannot determine whether a redox change of electron transfer components or another event, such as depolarization of membrane potential, is important in the induction of a phototactic response in bacteria. Valinomycin is an ionophore that increases the permeability of biological membranes to certain cations (K+, Rb+, and Cs+). In the presence of these cations, the electrochemical gradient across the membrane is affected. Intact cells ofR. rubrum were insensitive to the antibiotic, presumably because of their intact outer membrane. However, when they were "properly" treated with EDTA, valinomycin plus 10 mM KCl inhibited both the phototactic response and the light-induced absorbance change of carotenoid (Table 2, Fig. 4) but not the redox changes of components in the photosynthetic electron transfer system (data not shown). The inhibitory effect of valinomycin on the bacterial phototactic response is probably due to depolarization of the membrane, not to permeability of any particular cation, since the phototactic response was affected by the ionophore only when the membrane was depolarized by the presence of both valinomycin and an interacting cation (Table 3).
The involvement of membrane potential in bacterial tactic response has been proposed by several authors (1). Recently, Szmelcman and Adler (31) reported that a chemotactic stimulus induces changes in membrane potential of E. coli cells. They discussed the possible role of ion fluxes in regulating flagellar rotation. The important role of ions in chemotactic responses was suggested by Ordal and Goldman (23-25). They reported that uncouplers of oxidative phosphorylation, inhibitors of electron transport, and permeable anions induce transient "tumbling"; that A23187, an ionophore for divalent cations, plus EDTA induces incessant "tumbling"; and that permeable cations inhibit "tumbling" in Bacillus subtilis. Ordal (23) proposed that binding of certain cations to the switch of a hypothetical chemotactic machinery inhibits "tumbling." Two possible roles of the membrane potential in the phototactic response of R. rubrum are conceivable: (i) the tactic signal inducing reversal of swimming direction is the depolarization of membrane potential; (ii) a normal membrane potential is required for flagellar reversal to occur. Since we could not determine the role of membrane potential in the phototactic response, we present three alternative possibilities for the signal that induces the response: a change in the redox state of the components in the photosynthetic electron transfer system; uptake of H+ (decrease in ApH across the membrane); and depolarization of the membrane potential. Bacterial motility was not paralyzed by valinomycin plus potassium (or rubidium). The relationship between membrane potential and cell motility will be discussed elsewhere (S. Harayama and T. Iino, manuscript in preparation). ACKNOWLEDGMENTS This work was supported by research grants 048068 and 178097 from the Japanese Ministry of Education, Science and Culture. LITERATURE CITED 1. Adler, J. 1975. Chemotaxis in bacteria. Annu. Rev.
Biochem. 44:341-356. 2. Berg, H. C. 1975. Bacterial behaviour. Nature (London) 254:389-392. 3. Berg, H. C., and P. M. Tedesco. 1975. Transient response to chemotactic stimuli in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:3235-3239. 4. Clayton, R. K. 1953. Studies in the phototaxis of Rhodospirillum rubrum. I. Action spectrum, growth in
green light, and Weber law adherence. Arch. Mikrobiol. 19:107-124. 5. Clayton, R. K. 1963. Toward the isolation of a photochemical reaction center in Rhodopseudomonas spheroides. Biochim. Biophys. Acta 75:312-323.
VOL. 131, 1977 6. Clayton, R. K. 1964. Phototaxis in microorganisms, p. 51-77. In A. C. Giese (ed.), Photophysiology, vol. 2. Academic Press Inc., New York. 7. Griffiths, M., W. R. Sistrom, G. Cohen-Bazire, and R. Y. Stanier. 1955. Function of carotenoids in photosynthesis. Nature (London) 176:1211-1214. 8. Harayama, S., and T. Iino. 1976. Phototactic response of aerobically cultivated Rhodospirillum rubrum. J. Gen. Microbiol. 94:173-179. 9. Harold, F. M. 1972. Conservation and transformation of energy by bacterial membranes. Bacteriol. Rev. 36:172-230. 10. Haupt, W. 1966. Phototaxis in plants. Int. Rev. Cytol. 19:267-299. 11. Hazelbauer, G. L. 1975. Maltose chemoreceptor ofEscherichia coli. J. Bacteriol. 122:206-214. 12. Hildebrand, E., and N. Dencher. 1975. Two photosystems controlling behavioural responses of Halobacterium halobium. Nature (London) 257:46-48. 13. Izawa, S., and N. E. Good. 1972. Inhibition of photosynthetic electron transport and photophosphorylation. Methods Enzymol. 24:355-377. 14. Jackson, J. B., and A. R. Crofts. 1971. The high energy state in chromatophores from Rhodopseudomonas spheroides. FEBS Lett. 4:185-189. 15. Jackson, J. B., and P. L. Dutton. 1973. The kinetic and redox potentiometric resolution of the carotenoid shifts in Rhodopseudomonas spheroides chromatophores: their relationship to electric field alterations in electron transport and energy coupling. Biochim. Biophys. Acta 325:102-113. 16. Koshland, D. E., Jr. 1974. Chemotaxis as a model for sensory systems. FEBS Lett. 40:S3-S9. 17. Leive, L. 1965. Release of lipopolysaccharide by EDTA treatment of E. coli. Biochem. Biophys. Res. Commun. 21:290-296. 18. Lengeler, J. 1975. Mutations affecting transport of the hexitols r.-mannitol, D-glucitol, and galactitol in Escherichia coli K-12: isolation and mapping. J. Bacteriol. 124:26-38. 19. Milatz, J. M. W., and A. Manten. 1953. The quantitative determination of the spectral distribution of phototactic sensitivity in the purple bacterium Rhodospirillum rubrum. Biochim. Biophys. Acta 11:17-27. 20. Nikaido, H. 1976. Outer membrane of Salmonella typhimurium. Transmembrane diffusion of some hydro-
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21.
22.
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28. 29. 30.
31. 32. 33.
41
phobic substances. Biochim. Biophys. Acta 433:118132. Nishimura, M., and B. Chance. 1963. Studies on the electron-transfer systems in photosynthetic bacteria. I. The light-induced absorption-spectrum changes and the effect of phenylmercuric acetate. Biochim. Biophys. Acta 66:1-16. Nultach, W. 1973. Phototaxis and photokinesis in bacteria and blue-green algae, p. 70-81. In A. Perez-Miravete (ed.), Behaviour of micro-organisms. Plenum Press, London. Ordal, G. W. 1976. Control of tumbling in bacterial chemotaxis by divalent cation. J. Bacteriol. 126:706711. Ordal, G. W., and D. J. Goldman. 1975. Chemotaxis away from uncouplers of oxidative phosphorylation in Bacillus subtilis. Science 189:802-805. Ordal, G. W., and D. J. Goldman. 1976. Chemotactic repellents of Bacillus subtilis. J. Mol. Biol. 100:103108. Parkinson, J. S. 1975. Genetics of chemotactic behavior in bacteria. Cell 4:183-188. Pavlasov&, E., and F. M. Harold. 1969. Energy coupling in the transport of ,3-galactosides by Escherichia coli: effect of proton conductors. J. Bacteriol. 98:198-204. Pressman, B. C. 1976. Biological applications of ionophores. Annu. Rev. Biochem. 45:501-530. Siebeama, C. 1970. Photosynthetic bacteria, p. 57-93. In P. Halldal (ed.), Photobiologie of microorganisms. Wiley-Interscience, London. Spudich, J. L., and D. E. Koshland, Jr. 1975. Quantitation of the sensory response in bacterial chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 72:710-713. Szmelcman, S., and J. Adler. 1976. Change in membrane potential during bacterial chemotaxis. Proc. Natl. Acad. Sci. U.S.A. 73:4387-4391. Throm, G. 1968. Untersuchungen zum Reaktionsmechanismus von Phototaxis und Kinesis an Rhodospirillum rubrum. Arch. Protistenkd. 110:313-371. van Grondelle, R., L. N. M. Duysens, and H. N. van der Wal. 1976. Function of three cytochromes in photosynthesis of whole cells of Rhodospirillum rubrum as studied by flash spectroscopy. Evidence for two types of reaction center. Biochim. Biophys. Acta 449:169187.
OF BACTEIUOLOGY, July 1977, p. 34-41 Copyright © 1977 American Society for Microbiology
JOURNAL
Printed in U.S.A.
Phototaxis and Membrane Potential in the Photosynthetic Bacterium Rhodospirillum rubrum SHIGEAKI HARAYAMA* AND TETSUO IINO Genetics, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan Laboratory of
Received for publication 23 February 1977
Cells of the photosynthetic bacterium Rhodospirillum rubrum cultivated anaerobically in light show phototaxis. The behavior of individual cells in response to the phenomenon is reversal(s) of the swimming direction when the intensity of the light available to them abruptly decreases. The tactic response was inhibited by antimycin, an inhibitor of the photosynthetic electron transfer system. The inhibitory effect of antimycin was overcome by phenazine methosulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic response; however, valinomycin or potassium alone had no effect. A change in membrane potential of the cells was measured as an absorbance change of carotenoid. Changes in the membrane potential caused by "on-off' light were prevented by antimycin and by valinomycin plus potassium, but not by antimycin plus phenazine methosulfate nor valinomycin or potassium alone. The results indicated that the phototactic response of R. rubrum is mediated by a sudden change in electron flow in the photosynthetic electron transfer system, and that the membrane potential plays an important role in manifestation of the response.
systems. The chemotactic behavior of individual cells of E. coli and S. typhimurium has also been examined (1-3, 16, 26, 30). The bacteria modulate the frequency of change in direction when they swim up or down a spatial gradient of a chemotactic stimulus; thus, their attraction to or migration from it is attained. This modulation is also observed when they are exposed to a temporal change in concentration of the stim-
Bacterial phototaxis has been observed in photosynthetic bacteria (6, 22) and in Halobacterium halobium (12). The photoreceptor pigments responsible for positive phototaxis (phototaxis towards light) have been identified as bacteriochlorophyll and carotenoids in photosynthetic bacteria (5, 19) and as bacteriorhodopsin in H. halobium (12). The phototactic behavior of individual cells of photosynthetic bacteria has been extensively examined (10): the bacteria show abrupt changes in direction when they swim from a light to a dark field, and some of them return to the light field; however, they fail to show such a response when they swim from a dark to a light field. As a consequence of these responses, the number of organisms in a light spot projected onto a suspension of bacteria increases with time. The change in swimming direction can be induced by temporal decrease of an actinic light intensity impinging on them. This observation suggested that the phototactic response is the result of sensing temporal, not spatial, differences in light intensity. In chemotaxis of Escherichia coli and of Salmonella typhimurium, chemoreceptor molecules of several attractants (chemicals that induce positive chemotaxis) have been identified (1, 11, 18). The sugar chemoreceptors are proteins that are constituents of active transport
ulating chemicals. Whereas cellular receptors of the tactic stimuli and swimming behaviors induced by the stimuli have been well documented in bacterial phototaxis, as well as in bacterial chemotaxis, how the stimuli are converted to a tactic signal that modulates the frequency of change in their swimming direction is unclear. In this paper we show evidence that reversals of the swimming direction of the photosynthetic bacterium Rhodospirillum rubrum induced by a sudden decrease in the actinic light intensity are mediated by a sudden decrease of electron flow in the photosynthetic electron transfer system and that membrane potential plays an important role in the response. MATERIALS AND METHODS Bacterial strains and growth of cells. Wild-type R. rubrum (S-1) was obtained from G. Soe, and the carotenoid-less blue-green mutant (G-9) was from Y. 34
PHOTOTAXIS IN R. RUBRUM
VOL. 131, 1977 Kobayashi, to whom we are indebted. The bacteria were cultivated in light at 28°C in a 120-ml screwcapped bottle containing a synthetic medium whose composition was as described previously (8), except that Casamino Acids was omitted from the medium. Illumination was provided by one 300-W flood lamp (Toshiba) placed 80 cm from the culture bottle. The bacteria at the late exponential to stationary growth phase were harvested, centrifuged, washed at least two times with 10 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.6), suspended in the appropriate solution, and then used for experiments. In the experiments reported in Table 3, the bacteria were washed in 10 mM sodium phosphate buffer (pH 6.8). Measurement of phototactic response. To examine quantitatively the phototactic response of the bacteria, an apparatus for giving successive phototactic stimuli to the bacteria was devised (Fig. 1). Two light beams, one for an actinic light and another for an observing light, were projected onto a phase-contrast microscope condenser through a half mirror. The actinic light beam from a tungstenhalogen lamp (Ushio, JC-24V-150W) was passed through a 4-cm cuvette containing water, chopped by a rotary sector for 0.5 s every 2 s, and then projected on a bacterial suspension on a glass slide through a phase-contrast condenser. The intensity of the actinic light, measured with a YSI-Kettering model 65 radiometer, was 500 W/m2. The observing light beam from a 30-W tungsten lamp (Nikon) was filtered by a thermo-cut-off filter (Hoya, HA-50) and a green interference filter (Toshiba, KL-56; peak transmission at 558 nm, with a half band width of 14 nm) and then continuously projected on the bacterial suspension. The intensity of the observing light was 3 W/m2 or below. The tactic response of R. rubrum induced by a sudden decrease in actinic light intensity is simple: the bacteria reverse their swimming direction after being stimulated (6, 10). Therefore, as an index of their phototactic activity, we measured the fraction of bacteria that showed reversal(s) of swimming direction within 0.5 s after the actinic light was turned off in repeated "on" (1.5 s) and "off" (0.5 s) circles, by direct microscopic observations made at an ambient temperature of 300C. When more than 50 bacteria were counted, the standard deviation of the measured values from the same sample did not exceed 20% of the mean value. Measurement of an absorbance change in bacterial suspension. Measurement of light-induced absorbance (A) change was carried out with a Hitachi 356 double-beam spectrophotometer. The wavelength used for measuring the absorbance change of carotenoid in wild-type cells (S-1) was 525 nm, with 510 nm as a reference. Those used for measuring that of cytochrome c2 and reaction center bacteriochlorophyll in cells of the carotenoid-less mutant (G9) were 552 and 600 nm, with 540 and 585 nm, respectively, as references. Actinic light from a tungsten-halogen lamp (Ushio, JC-24V-150W) filtered through an R-72 filter (Hoya), allowing transmission of light only above 700 nm, was projected onto a cuvette (light path, 1 cm) at a right angle to
35
Li II IF
c
.' *
HM
I
Ch
S
I
I
IHo 12 L2
RD
c7 FIG. 1. Apparatus for observation of bacterial phototactic response. Abbreviations: Li, tungsten lamp (Nikon, 30 W); L2, tungsten-halogen lamp (Ushio, JC-24V-150W); IF, interference filter (Toshiba, KL-56) plus thermo-cut-off filter (Hoya, HA50); 11 and 12, collector lens; H20, 4 cm of water; HM, half-reflecting mirror; Ch, rotary chopper cutting actinic light for 0.5 s every 2 s; RD, phase ring diaphragms; C, condenser lens; S, specimen; and 0, 40 x phase objective (Nikon, long-working distance).
the measuring beam of the spectrophotometer. The intensity of the actinic light was 1.3 kW/m2. Between the cuvette and a photomultiplier tube, two filters (Corning 9782 and HA-50) were placed to protect the photomultiplier tube from the cross-illuminating actinic light. Intermittent illumination of the actinic light (1.5 s on and 0.5 s off) was attained by insertion or removal of a sector connected to a rotary solenoid operated by an autotimer. Estimation of cellular content of bacteriochlorophyll. Bacteriochlorophyll was extracted with acetone-methanol (7:2, vol/vol), and then the cellular content of the pigment was determined spectrophotometrically by using the absorption coefficient given by Clayton (5). Chemicals. Valinomycin and antimycin were purchased from Boehringer Manheim. Other reagents used were of analytical grade.
36
HARAYAMA AND IINO
J. BACTERIOL.
its oxidation (decrease in A552-540) by light was inhibited, and its reduction after the light was turned off was prevented by the inhibitor. Redox changes of reaction center bacteriochlorophyll, measured as A605585, were hardly affected by the inhibitor. Changes in carotenoid absorbance, which reflects changes in membrane potential (14, 15), were affected by the inhibitor: an increase in membrane potential (hyperpolarization) was observed after the light was first turned on, but a decrease in membrane potential (depolarization) after the light was turned off was prevented by the inhibitor. That the observed absorbance change at 525 to 510 nm corresponded to that of carotenoid, reflecting changes in membrane potential, was confirmed by the fact that, in a cell suspension of the carotenoid-less mutant (G-9), no lightinduced absorbance change between the two wavelengths was observed with or without antimycin and PMS (Fig. 2). Effect of valinomycin on phototactic response and light-induced absorbance change of carotenoid. Valinomycin is an ionophore that increases conductivity of K+, Rb+, and Cs+ in biological membranes; in the presence of valinomycin, these cations move down the electrochemical potential across the membrane and discharge it (9, 28). We used the ionophore to examine the role of membrane potential in the bacterial phototactic response because its effect on biological membranes was well characterized. When valinomycin was added to the cell susantimycin. of R. rubrum with or without KCl, it pension Antimycin affected light-induced redox on the phototactic response or on effect had no as measured A552-540: changes of cytochrome c2 the light-induced absorbance change of carotenoid. Since it is known that the outer memTABLE 1. Effect of antimycin and PMS on brane of gram-negative bacteria acts as a penephototactic response tration barrier for hydrophobic antibiotics having a molecular weight of 1,100 (20), it is possiPhototaxis Nb Additiona ble that the insensitivity of intact R. rubrum cells to the ionophore is due to the existence of 60d 224 Ethanol (0.2%, vol/vol) an intact outer membrane. It was reported that 5d 129 Antimycin (20 ,uM) 84e ethylenediaminetetraacetate (EDTA) removed Ethanol (0.2%, vol/vol) + PMS 180 (0.2 mM) the penetration barrier (17), and thus E. coli, a 67e Antimycin (20 gM) + PMS (0.2 163 gram-negative bacterium, became sensitive to mM) valinomycin (27). Upon EDTA treatment, cells a Cells ofR. rubrum S-1 were suspended in 10-2 M of R. rubrum also became sensitive to valinoTris-hydrochloride buffer (pH 7.6) containing the mycin: the phototactic activity of the EDTAaddition. treated cells was strongly inhibited by valinobNumber of the bacteria examined. mycin. Spontaneous reversal frequency of the ' Fraction of the bacteria that showed reversal(s) cells, under steady light illumination, was also of swimming direction 0.5 s after actinic light was diminished by valinomycin. The concentration turned off. d Phototactic response was observed 1 to 1.5 h of EDTA required to render the cells sensitive to valinomycin differed in different cell culafter the addition of ethanol or antimycin. tures: cells in the stationary growth phase were e PMS was added 1.5 h after the addition of ethanol or antimycin, and then the phototactic re- more resistant to EDTA than those of the exponential growth phase. Cumulative results on sponse was observed 10 to 20 min afterwards.
RESULTS Effect of antimycin and PMS on phototactic response and photosynthetic electron transfer in the bacteria. Cells of R. rubrum reverse their swimming direction when the actinic light intensity impinging onto them is abruptly decreased. Since the phototactic phenomenon, i.e., accumulation of the bacteria in a light spot projected onto a bacterial suspension, is a result of this response (6, 10), we estimated the phototactic activity of the bacteria by counting the fraction that reversed swimming direction in a 0.5-s dark period after the actinic light was turned off. The value of the fraction depended on decreased intensity of the actinic light as well as intensity of the observing light. Furthermore, sensitivity of the cells to the phototactic stimulus differed in different cultures. However, under the conditions described in Materials and Methods, 60 to 95% of the cells responded to the stimulus in control experiments. Antimycin, a specific inhibitor of photosynthetic electron transfer (13, 33), inhibited the phototactic response of the bacteria (Table 1). Motility of the bacteria was not affected by the inhibitor under aerobic conditions. The inhibitory effect of antimycin on the phototactic response was abolished when phenazine methosulfate (PMS) was subsequently added to the bacterial suspension. PMS had a stimulative effect on phototactic response when it was added to the bacterial suspension containing no
VOL. 131, 1977
PHOTOTAXIS IN R. R UBRUM Ethanol
I (GA6004585 9)
Antimycin
Ethanol PMS
37
Antimycin PMS
1444444444
Anti,rnycI
fff tftffff 2
7A552540 Iftt fIftit A52s-5
it ltfttlIft ftf itftftt f tftff t tftt tftftt
o
t0.01
fl fttft
ft tftttft
ftftffitt
ttt ftttti tt 208
FIG. 2. Effect of antimycin on the light-induced absorbance change in bacterial suspension. Cells of R. rubrum strains S-i and G-9 suspended in 10 mM Tris-hydrochloride buffer (pH 7.6) were incubated aerobically on a reciprocating shaker (120 strokes per min) at 28°C for 1 h. Then, 20 pM antimycin dissolved in ethanol (final concentration, 0.2% [vol/vol]) or 0.2% (vollvol) ethanol was added to the bacterial suspensions. They were further incubated aerobically for 1 to 1.5 h, and then light-induced absorbance changes were measured as described in the text. Freshly prepared PMS (0.2 mM) was added 1.5 h after the addition of ethanol or antimycin, and the light-induced absorbance changes were measured within 10 min. Arrows (.I t) indicate light on and light off, respectively. Absorbance changes of bacteriochlorophyll (A,5858 and cytochrome c2 (A552--W) were measured with the carotenoid-less mutant G-9, at a bacteriochrolophyll concentration of 18W. The mutant strain was used for the measurements because, in wild-type cells, these absorbance changes were obscured by associated carotenoid changes at these wavelengths (21). The absorbance change of carotenoid was measured with wild-type strain S-l, at a bacteriochlorophyll concentration of 23 pM, at A525 510, where no absorbance change was observed in carotenoid-less mutant G-9. Phototactic activities in the bacteria used in the experiment were 85% (+ethanol), 10% (+antimycin), 98% (+PMS), and 70% (+antimycin and PMS) in G-9 cells; and 73% (+ethanol), 9% (+antimycin), 86% (+PMS), and 73% (+antimycin and PMS) in S-i cells. The motility of G-9 cells has been observed to be impaired under high light intensity in aerobic conditions. This may be a result of photodynamic action (7). However, under the present experimental conditions, no impairment of motility was observed.
phototactic response are presented in Table 2. In these experiments, S-1 cells at a growth phase indicated in the table were suspended in 10 mM Tris-hydrochloride buffer (pH 7.6) containing 10 mM KCI and were then incubated aerobically with shaking (120 strokes per min) at 280C. The indicated concentrations of EDTA and ethanol (1% vol/vol) or valinomycin (10 ,uM) dissolved in ethanol (final concentration, 1%, vol/vol) were added to the cell suspensions, which were further incubated aerobically for the indicated times. Since the bacteria treated with EDTA had a tendency to adhere to cover slips and glass slides, the phototactic response of the EDTA-treated cells was observed by using a bacteria counting chamber (20-gm depth) in which their motility was fair.
Light-induced absorbance changes of carotenoid were measured with the samples listed in Table 2; some of the results are presented in Fig. 3. In the cells used in experiment 2 ofTable 2, absorbance changes of carotenoid were normal when ethanol was added to the cell suspension, but in the presence of valinomycin they were prevented. In cells used in experiment 5 of Table 2, even without the addition of valinomycin, phototactic activity was strongly reduced by EDTA. The cells showed only the slight absorbance change of carotenoid. Why EDTA inhibited the reaction was unclear. These results showed that inhibition of the light-induced absorbance change of carotenoid, i.e., the light-induced change in membrane potential, by valinomycin or by EDTA resulted in a con-
38
J. BACTERIOL.
HARAYAMA AND IINO TABLE 2. Effect of valinomycin on phototactic response Expt
Growth phase
EDTA concn (M)
time in Incubation EDTA (min)
Additiona
Nb
Phototaxis (%
1
Stationary
10-3
42-52
E V
121 55
53 7
2
Stationary
10-3
280-285
E V
33
67
44
2
3
Stationary
10-3
240-245
E V
64 52
73 6
4
Late exponential
10-4
35-50
E V
51 35
49 6
5
Late exponential
10-4
80-83
E V
61 44
10 0
6
Stationary
10-3
100-105
E V
73 46
62 2
10-4
200-205
E V
92 64
78 78
a V, 10-5 M valinomycin; E, 1% (vol/vol) ethanol. b Number of bacteria examined.
* 4 +$* +4+4+ 4 +t
t
+ +
t+ +
9* + t+ + + + +* +
J0.001
A
r*,,,,,444
B T~lOs
FIG. 3. Light-induced absorbance changes of carotenoid in EDTA-treated cells with or without the addition of valinomycin. Light-induced absorbance changes at 525 nm, with 510 nm as a reference of the bacterial suspensions used in experiments of Table 2, were measured immediately after the measurements ofphototactic response. (A) Bacterial suspension used in experiment 2 of Table 2 (bacteriochrolophyll concentration, 23 pM). (B) Bacterial suspension used in experiment 5 of Table 2 (bacteriochrolophyll concentration, 13 M). (E) Ethanol (19%, vollvol) added. (V) valinomycin (10 pM) added.
comitant inhibition of bacterial phototaxis. The effect of incubation time of the cells in EDTA solution on their phototactic response in the presence or absence of valinomycin was examined (Fig. 4). In the experiment, 10 mM CaCl2 was added to the cell suspension to stop the action of EDTA at the indicated time. It was shown that sensitivity ofthe cells to valinomycin increased with increased incubation time in EDTA solution; however, prolonged incubation resulted in inhibition of phototactic response even without addition of valinomycin. Valinomycin discharges membrane potential
only when appropriate monovalent cations such as K+ and Rb+ are present. To examine whether the inhibitory effects of valinomycin are dependent on the presence of these cations, the effects of valinomycin on phototactic response and light-induced absorbance change of carotenoid in the EDTA-treated cells suspended in buffer free from these cations were examined. For this purpose, cells of R. rubrum S-1 at the late exponential growth phase were harvested, suspended in 10 mM sodium phosphate buffer (pH 6.8), and incubated aerobically with shaking (120 strokes per min) at 28°C for 1 h; 0.3 mM
VOL. 131, 1977
PHOTOTAXIS IN R. RUBRUM
39
TABLE 3. Requirement of K+ or Rb+ for the inhibitory effect of valinomycin on both phototactic response and light-induced absorbance change of carotenoid
Additiona
R t\
E V E V E V
I~~~~9I'Jr
0
Monovalent cation added water (1%, vol/vol) water (1%, vol/vol)
10-2 M KCl 10-2 M KCl 10-2 M RbCl 10-2 M RbCl
b
41 36 44 100 41 70
Photo- AAB/M
(%)xi BChlc5 73 86 73 15
80 16
3.0 2.6 2.3 0.8 2.6 1.0
E, Ethanol (1%, vol/vol); V, valinomycin (10-5 M). Number of bacteria examined. Mean of 10 absorbance changes of carotenoid (A525510) per micromolar bacteriochrolophyll (BChl) 0.5 s after the actinic light was turned off. a '
c
Val 2
15 45 85 INCUBATION TIME IN EDTA(MIN)
FIG. 4. Effect of incubation time in EDTA solution on phototactic response. S-i cells harvested in the late exponential growth phase were suspended in 10-2 M Tris-hydrochloride buffer containing 10 mM KCl and were incubated aerobically at 280C for 1 h. EDTA (0.1 mM) and ethanol ( %, vollvol) or valinomycin (10 pM) were added to the bacterial suspensions, which were further incubated aerobically at 28°C. CaCl2 (1 mM) was added to stop the action of EDTA at a time, indicated on the figure, after the addition of EDTA, and then the phototactic response was observed 15 min afterwards.
EDTA and ethanol (1%, vol/vol) or valinomycin (10 AM) were added to the cell suspension and further incubated aerobically for 30 min. CaCl2 (2 mM) was added to stop the action of EDTA on the cells. Thirty minutes after the addition of CaCl2, 10 mM KCl, 10 mM RbCl, or water (final concentration, 1% [vol/vol]) was added to the cell suspensions, and phototactic activities of the cells in these suspensions were measured 20 to 35 min and 40 to 55 min afterwards. The mean of two measurements is presented in Table 3. The absorbance changes of carotenoid within 0.5 s after the actinic light was turned off were measured 60 to 65 min after the addition of KCl, RbCl, or water (Table 3). When the bacteria were suspended in the 10 mM sodium phosphate buffer containing no KCI, addition of valinomycin after EDTA treatment of the cells did not affect significantly their phototactic response or the light-induced absorbance change of carotenoid. Further addition of 10 mM KCl or RbCl to the bacterial suspension inhibited the phototactic response as well as the light-induced absorbance change of carotenoid (Table 3). The results clearly indicated that the inhibi-
tory effects of valinomycin on the phototactic response and light-induced absorbance change of carotenoid were dependent on the existence of appropriate cationic species such as K+ and Rb+, but not Na+.
DISCUSSION Pigmented cells of R. rubrum reverse their swimming direction when the intensity of light impinging on them is abruptly decreased. Since the reversal of swimming direction is a key movement in attaining phototactic accumulation (6, 10), we examined the fraction of bacteria that reverse their swimming direction after a sudden decrease in light intensity to estimate their phototactic activity. Under the conditions of the experiments, very few cells showed repeated reversals within 0.5 s after a sudden decrease in actinic light intensity; i.e., almost all of the cells showed one or no reversals after the stimulus. Photoreceptor pigments responsible for the phototactic response of the bacteria have been identified as bacteriochlorophyll and carotenoids (4, 19). These are pigments driving photosynthesis. Thus, the hypothesis that phototaxis is induced by a transient disturbance of photosynthetic metabolism was proposed and widely accepted (6). A requirement of photosynthetic electron transfer for the manifestation of phototaxis has been claimed by Throm (32). He observed that inhibitors of photosynthetic electron transfer affected the maximum level of accumulation of cells in a light spot. However, the rate and the maximum level of bacterial accumulation in a light spot may be affected complexly by changes in the speed of the bacteria or the fraction of motile bacteria, as well as their phototactic activity. Therefore, it is preferable to observe the behavior of individual
40
J. BACTERIOL.
HARAYAMA AND IINO
cells rather than the mass accumulation of bacteria in a light spot for determination of their phototactic response. The conclusion of Throm was confirmed by the present experiments in which antimycin, an inhibitor of photosynthetic electron transfer, prevented phototactic response. Motility of the bacteria was not affected by the inhibitor under aerobic conditions, indicating that the energy for motility is provided by respiratory metabolism, which is not affected by antimycin (data now shown), as well as by photosynthetic metabolism. The inhibitory effect of antimycin on both phototactic response and photosynthetic electron transfer was annulled by PMS (Table 1). The effect of PMS has been interpreted as the formation of a bypass around the blocking site of antimycin in the electron transfer pathway (29). From these observations, it is inferred that the reversal of swimming direction after a sudden decrease in actinic light intensity is a consequence of the decrease in photosynthetic electron flow. Accompanying the decrease of electron flow is a change in the redox state of components of the electron transfer system and a decrease (depolarization) of membrane potential measured as an absorbance change of the carotenoid (Fig. 3). Both the reduction of cytochrome c2 and the decrease in membrane potential after the light was turned off were inhibited by antimycin. Thus, by considering only the action of antimycin on phototactic response, one cannot determine whether a redox change of electron transfer components or another event, such as depolarization of membrane potential, is important in the induction of a phototactic response in bacteria. Valinomycin is an ionophore that increases the permeability of biological membranes to certain cations (K+, Rb+, and Cs+). In the presence of these cations, the electrochemical gradient across the membrane is affected. Intact cells ofR. rubrum were insensitive to the antibiotic, presumably because of their intact outer membrane. However, when they were "properly" treated with EDTA, valinomycin plus 10 mM KCl inhibited both the phototactic response and the light-induced absorbance change of carotenoid (Table 2, Fig. 4) but not the redox changes of components in the photosynthetic electron transfer system (data not shown). The inhibitory effect of valinomycin on the bacterial phototactic response is probably due to depolarization of the membrane, not to permeability of any particular cation, since the phototactic response was affected by the ionophore only when the membrane was depolarized by the presence of both valinomycin and an interacting cation (Table 3).
The involvement of membrane potential in bacterial tactic response has been proposed by several authors (1). Recently, Szmelcman and Adler (31) reported that a chemotactic stimulus induces changes in membrane potential of E. coli cells. They discussed the possible role of ion fluxes in regulating flagellar rotation. The important role of ions in chemotactic responses was suggested by Ordal and Goldman (23-25). They reported that uncouplers of oxidative phosphorylation, inhibitors of electron transport, and permeable anions induce transient "tumbling"; that A23187, an ionophore for divalent cations, plus EDTA induces incessant "tumbling"; and that permeable cations inhibit "tumbling" in Bacillus subtilis. Ordal (23) proposed that binding of certain cations to the switch of a hypothetical chemotactic machinery inhibits "tumbling." Two possible roles of the membrane potential in the phototactic response of R. rubrum are conceivable: (i) the tactic signal inducing reversal of swimming direction is the depolarization of membrane potential; (ii) a normal membrane potential is required for flagellar reversal to occur. Since we could not determine the role of membrane potential in the phototactic response, we present three alternative possibilities for the signal that induces the response: a change in the redox state of the components in the photosynthetic electron transfer system; uptake of H+ (decrease in ApH across the membrane); and depolarization of the membrane potential. Bacterial motility was not paralyzed by valinomycin plus potassium (or rubidium). The relationship between membrane potential and cell motility will be discussed elsewhere (S. Harayama and T. Iino, manuscript in preparation). ACKNOWLEDGMENTS This work was supported by research grants 048068 and 178097 from the Japanese Ministry of Education, Science and Culture. LITERATURE CITED 1. Adler, J. 1975. Chemotaxis in bacteria. Annu. Rev.
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