JOURNAL OF BACTEIUOLOGY, July 1977, p. 34-41 Vol. 131, No. 1 Copyright © 1977 American Society for Microbiology Printed in U.S.A. Phototaxis and Membrane Potential in the Photosynthetic Bacterium Rhodospirillum rubrum SHIGEAKI HARAYAMA* AND TETSUO IINO Laboratory of Genetics, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan 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 metho- sulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic re- sponse; 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 antimy- cin 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 re- sponse. Bacterial phototaxis has been observed in systems. The chemotactic behavior of individ- photosynthetic bacteria (6, 22) and in Halobac- ual cells ofE. coli and S. typhimurium has also terium halobium (12). The photoreceptor pig- been examined (1-3, 16, 26, 30). The bacteria ments responsible for positive phototaxis (pho- modulate the frequency of change in direction totaxis towards light) have been identified as when they swim up or down a spatial gradient bacteriochlorophyll and carotenoids in photo- of a chemotactic stimulus; thus, their attraction synthetic bacteria (5, 19) and as bacteriorho- to or migration from it is attained. This modu- dopsin in H. halobium (12). The phototactic lation is also observed when they are exposed to behavior of individual cells of photosynthetic a temporal change in concentration of the stim- bacteria has been extensively examined (10): ulating chemicals. the bacteria show abrupt changes in direction Whereas cellular receptors of the tactic stim- when they swim from a light to a dark field, uli and swimming behaviors induced by the and some of them return to the light field; stimuli have been well documented in bacterial however, they fail to show such a response phototaxis, as well as in bacterial chemotaxis, when they swim from a dark to a light field. As how the stimuli are converted to a tactic signal a consequence of these responses, the number that modulates the frequency of change in their of organisms in a light spot projected onto a swimming direction is unclear. suspension of bacteria increases with time. The In this paper we show evidence that reversals change in swimming direction can be induced of the swimming direction of the photosynthetic by temporal decrease of an actinic light inten- bacterium Rhodospirillum rubrum induced by sity impinging on them. This observation sug- a sudden decrease in the actinic light intensity gested that the phototactic response is the re- are mediated by a sudden decrease of electron sult of sensing temporal, not spatial, differ- flow in the photosynthetic electron transfer sys- ences in light intensity. tem and that membrane potential plays an im- In chemotaxis ofEscherichia coli and of Sal- portant role in the response. monella typhimurium, chemoreceptor mole- cules of several attractants (chemicals that in- MATERIALS AND METHODS duce positive chemotaxis) have been identified Bacterial strains and growth of cells. Wild-type (1, 11, 18). The sugar chemoreceptors are pro- R. rubrum (S-1) was obtained from G. Soe, and the teins that are constituents of active transport carotenoid-less blue-green mutant (G-9) was from Y. 34 VOL. 131, 1977 PHOTOTAXIS IN R. RUBRUM 35 Kobayashi, to whom we are indebted. The bacteria were cultivated in light at 28°C in a 120-ml screw- capped bottle containing a synthetic medium whose composition was as described previously (8), except Li 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 II bacteria at the late exponential to stationary growth phase were harvested, centrifuged, washed at least IF c .' two times with 10 mM tris(hydroxymethyl)amino- methane (Tris)-hydrochloride buffer (pH 7.6), sus- * I Ch pended in the appropriate solution, and then used for experiments. In the experiments reported in Ta- ble 3, the bacteria were washed in 10 mM sodium phosphate buffer (pH 6.8). Measurement of phototactic response. To exam- ine quantitatively the phototactic response of the S bacteria, an apparatus for giving successive photo- H MI tactic stimuli to the bacteria was devised (Fig. 1). Two light beams, one for an actinic light and an- other for an observing light, were projected onto a I 12 L2 phase-contrast microscope condenser through a half IHo mirror. The actinic light beam from a tungsten- halogen lamp (Ushio, JC-24V-150W) was passed RD 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 c7 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 halfband width of 14 nm) and then continuously projected on the bacte- rial suspension. The intensity of the observing light FIG. 1. Apparatus for observation of bacterial was 3 W/m2 or below. phototactic response. Abbreviations: Li, tungsten The tactic response of R. rubrum induced by a lamp (Nikon, 30 W); L2, tungsten-halogen lamp sudden decrease in actinic light intensity is simple: (Ushio, JC-24V-150W); IF, interference filter (To- the bacteria reverse their swimming direction after shiba, KL-56) plus thermo-cut-off filter (Hoya, HA- being stimulated (6, 10). Therefore, as an index of 50); 11 and 12, collector lens; H20, 4 cm of water; their phototactic activity, we measured the fraction HM, half-reflecting mirror; Ch, rotary chopper cut- of bacteria that showed reversal(s) of swimming ting actinic light for 0.5 s every 2 s; RD, phase ring direction within 0.5 s after the actinic light was diaphragms; C, condenser lens; S, specimen; and 0, turned off in repeated "on" (1.5 s) and "off" (0.5 s) 40 x phase objective (Nikon, long-working distance). circles, by direct microscopic observations made at an ambient temperature of300C. When more than 50 bacteria were counted, the standard deviation ofthe the measuring beam of the spectrophotometer. The measured values from the same sample did not ex- intensity of the actinic light was 1.3 kW/m2. Be- ceed 20% of the mean value. tween the cuvette and a photomultiplier tube, two Measurement of an absorbance change in bacte- filters (Corning 9782 and HA-50) were placed to rial suspension. Measurement of light-induced ab- protect the photomultiplier tube from the cross-illu- sorbance (A) change was carried out with a Hitachi minating actinic light. Intermittent illumination of 356 double-beam spectrophotometer. The wave- the actinic light (1.5 s on and 0.5 s off) was attained length used for measuring the absorbance change of by insertion or removal of a sector connected to a carotenoid in wild-type cells (S-1) was 525 nm, with rotary solenoid operated by an autotimer. 510 nm as a reference. Those used for measuring Estimation of cellular content of bacteriochloro- that of cytochrome c2 and reaction center bacterio- phyll. Bacteriochlorophyll was extracted with ace- chlorophyll in cells ofthe carotenoid-less mutant (G- tone-methanol (7:2, vol/vol), and then the cellular 9) were 552 and 600 nm, with 540 and 585 nm, content of the pigment was determined spectropho- respectively, as references. Actinic light from a tometrically by using the absorption coefficient tungsten-halogen lamp (Ushio, JC-24V-150W) fil- given by Clayton (5). tered through an R-72 filter (Hoya), allowing trans- Chemicals. Valinomycin and antimycin were mission of light only above 700 nm, was projected purchased from Boehringer Manheim. Other re- onto a cuvette (light path, 1 cm) at a right angle to agents used were of analytical grade. 36 HARAYAMA AND IINO J. BACTERIOL. RESULTS its oxidation (decrease in A552-540) by light was Effect of antimycin and PMS on phototac- inhibited, and its reduction after the light was tic response and photosynthetic electron turned off was prevented by the inhibitor. Re- transfer in the bacteria. Cells of R. rubrum dox changes of reaction center bacteriochloro- reverse their swimming direction when the ac- phyll, measured as A605585, were hardly af- tinic light intensity impinging onto them is fected by the inhibitor. Changes in carotenoid abruptly decreased. Since the phototactic phe- absorbance, which reflects changes in mem- nomenon, i.e., accumulation ofthe bacteria in a brane potential (14, 15), were affected by the light spot projected onto a bacterial suspension, inhibitor: an increase in membrane potential is a result of this response (6, 10), we estimated (hyperpolarization) was observed after the light the phototactic activity ofthe bacteria by count- was first turned on, but a decrease in mem- ing the fraction that reversed swimming direc- brane potential (depolarization) after the light tion in a 0.5-s dark period after the actinic light was turned off was prevented by the inhibitor.