Photoattractant response was measured in a relatively carotenoid-poor strain derived from the mutant of Halobacterium halobium that lacks both bacteriorhodopsin and halorhodopsin (strain Fix3). No photoat- tractant response was observed in the cells at logarithmic growth stage, coinciding with the fact that there was no sensory rhodopsin in membrane fraction prepared from the cells in logarithmic growth stage as measured by flash photolysis experiment. When all-tram-retinal was added to the cell suspension or the membrane suspension, the phototactic activity or the photocycling due to sensory rhodopsin appeared rapidly. This indicates that apoprotein of sensory rhodopsin had been formed in the cells at the growth stage, and suggests that the photoattractant response was mediated by sensory rhodopsin. The action spectrum of the photoattractant response resembled sensory rhodopsin absorption at wavelengths longer than 600 nm, but was distorted at shorter wavelengths by the photorepellent system that was found recently.
Introduction
Halobacterium halobium (H. halobium), a rod- shaped, extremely halophilic bacterium, is at- tracted to orange-red light and repelled by blue- ultraviolet light [1]. The photoattractant or the photorepellent response is often called positive or negative phototaxis, respectively, by analogy with the behavioral responses in bacterial chemotaxis [2]. The bacterium swims along its long axis. It stops the movement for a moment and starts swimming in an almost opposite direction when it senses either an increase in intensity of blue-ultra- violet light or a decrease in orange-red light [1].
Correspondence address: Department of Biophysics, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan.
Therefore, the response in halobacteria is classi- fied into the categories of 'photoclinokinesis' or 'photophobic response' [3], according to a termi- nology of behavioral responses of motile micro- organisms [4]. Conversely, decrease in blue-ultra- violet light or increase in orange-red light causes a decrease in frequency of spontaneous reversal of the swimming direction of the cells [5]. The sensory photosystem for negative photoresponse is called PS-370, because of the wavelength where maxi- mum sensitivity appears [1]. The photobehavioral response requires retinal [6].
At least four pigments containing retinal have so far been identified spectrophotometrically in the cell membrane of H. halobium [7-14,17]. Two of them act as light-driven ion pumps: bacteriorhodopsin ejects protons from the cell [8] and halorhodopsin transports chloride ions from external medium into the cell [10]. The third rhodopsin-like pigment was found in a
bacteriorhodopsin-deficient mutant strain in the course of a study on the flash-induced absorbance change of halorhodopsin [11]. Independently, this pigment was identified in a bacteriorhodopsin- halorhodopsin double-deficient mutant strain [12,13] and was named sensory rhodopsin [15].
Sensory rhodopsin is not an electrogenic light- driven pump [13]. Because the double-mutant strain also shows phototaxis similar to that of the wild-type strain, sensory rhodopsin is most likely a receptor pigment for both the repellent and the attractant photoresponse [13]. An alternative view is that there are two spectrophotometrically un- identified pigments, each of which mediates the signal of the photoattractant system or that of PS-370 [16]. A spectrophotometrically identified fourth rhodopsin-like pigment was found quite recently [14], but can explain only the additional peak that has occasionally appeared in photorepel- lent action spectra [6]. This pigment seemed to be the receptor for the independent photorepellent system whose sensitivity is maximal at around 480 nm [14]. The flash-induced absorbance change of the pigment is also maximal at this wavelength [14,17].
Recent behavioral studies strongly suggested that the receptor molecule for PS-370 is the photo- chemical intermediate of sensory rhodopsin whose absorption maximum is at 373 nm [15,18,19]. However, some results concerning the photoat- tractant system were inconsistent with each other [19-21].
Halobacterial cells in the early stage of their growth have no apparent photoattractant activity [6], but the cells seem to synthesize sensory rhodopsin constitutively [22]. When the cells were growing in stationary phase, first the repellent and then the attractant responses appeared (Refs. 6, 21, and Takahashi and Kobatake, unpublished result). These observations prompted us to study the relationship between biosynthesis of sensory rhodopsin and photoattractant sensitivity of the cells.
In this report, we show that the absence of photoattractant response in the cells at logarith- mic growth phase is not due to the absence of the biosynthesis of the protein but due to insufficient synthesis of retinal. The masking effect of a pho- torepellent system other than PS-370 on the pho-
579
toattractant system was also demonstrated in the cells. Taking these into account, we can explain most of the discrepant observations so far ob- tained. In addition, the relation between the pho- toattractant sensitivity and the absorbance of sensory rhodopsin is also discussed on the basis of photobehavioral action spectroscopy.
Materials and Methods
Bacterial strain and chemicals. The strain used was Flx3-9, a derivative obtained by long-lasting (approx. 3 months) semi-continuous cultivation of strain Flx 3 under illumination. Strain Flx 3 was a generous gift from John. L. Spudich. Flx 3 and Flx3-9 lack both bacteriorhodopsin and halorho- dopsin [12]. Flx3-9 contained less carotenoid and sensory rhodopsin than Flx 3. All-trans-retinal was purchased from Sigma.
Biosynthesis of sensory rhodopsin. Cells were grown at 38°C in a 20 1 carboy containing pep- tone complex medium [23]. The density of the cells was monitored spectrophotometrically at 660 nm. Cell suspension at A 0.3 corresponds to a cell density of approx. 10 s cells/ml. During the growth of the cells, 2-6 1 portions of the culture were introduced to an ice-cold vessel, and the cooled cells were harvested. The cells were washed and resuspended in approx. 100 ml 4 M NaC1, then the suspension was frozen completely with liquid nitrogen and thawed slowly in a cold room. The viscosity of the specimen was reduced using an electric juicer (model M J-C2, Matsusita Electric Co., Osaka) for 30 s three times: After debris were removed by centrifugation at 12000 X g for 10 min, the membrane fraction was collected and washed with 4 M NaC1 by repeating centrifuga- tion at 100000 × g for 30 min until the super- natant became clear. The amount of sensory rhodopsin in the membrane fraction was de- termined by flash photolysis, the methods and the apparatus of which were as described previously [24]. The pH of the sample solution was adjusted to 7.0 with Pipes/NaOH (final 20 mM). Protein concentration was determined by the method of Lowry et al. [27] using bovine serum as a stan- dard.
Phototactic assay. Routinely, the cells were grown in 11 ral peptone medium containing 25
580
m M P i p e s / N a O H (pH 6.9). The cell suspension was diluted about ten times with the medium, and incubated prior to measurement with or without addit ion of all-trans-retinal in ethanolic solution. The final concentra t ion of ethanol in bacterial suspension never exceeded 0.3%. Phototaxis was measured as the percentage of the cells that re- versed within 3 s after an abrupt interruption of the actinic light, using a computer- l inked auto- mated method described previously [18,25]. A tungsten-halogen lamp was used as the light source for actinic irradiation. All experiments were done at 38°C.
Results
Presence of sensory rhodopsin apoprotein in the cells in early logarithmic growth phase
Spudich and Spudich [22] showed that sensory rhodopsin apoprote in is a constitutive protein, using retinal-deficient mutan t strains FIx3R and OD2R. They also observed the restoration of pho- totaxis and sensory rhodopsin absorpt ion after addit ion of retinal to the cell suspension of non- phototact ic OD2R. However, they did not conf i rm the presence of sensory rhodopsin apoprote in at logari thmic growth phase. Since experiments with a retinal-deficient strain cannot provide informa- t ion about the inducibili ty of phototaxis during growth, we used the strain Fix3-9. First, we con-
7:> X <
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1 Red flash /
!
I
300 900 1500
Time (ms) Fig. 1. Flash-induced absorbance changes of membrane frac- tions from Flx3-9 cells at early logarithmic growth phase. Monitoring wavelength was 590 nm. Red actinic flash (~, > 620 nm) was selected by use of a short cut-off filter. (A) Membrane fraction of a protein concentration of 2 mg/ml without retinal. B) Measurement 3 h after addition of all-trans-retinal to the same specimen. Final concentration of retinal was 4.4 #M.
f irmed the presence of sensory rhodopsin apo- protein in this strain at early logarithmic growth phase.
Fig. 1 shows typical flash-induced absorbance changes of the membrane fractions prepared from the cells in early logarithmic growth phase. The solid line indicates the kinetic trace of the speci- men without retinal. Almost no detectable amount of photosensit ive sensory rhodopsin was contained in the specimen. The presence of sensory rhodop- sin apoprote in in this growth stage was demon- strated by the broken line, which indicates that a flash-induced absorbance change due to sensory rhodopsin appeared after the addit ion of retinal.
We moni tored the change in the amount of sensory rhodopsin and sensory rhodopsin apo- protein in this strain in the early stages of culture growth. The turbidity of the cell culture and the relative ampli tude of the flash-induced absorbance change of the membrane fraction with and without the addit ion of retinal are plotted in Fig. 2 as a funct ion of time after inoculation.
The effect of retinal on sensory rhodopsin synthesis The amount of sensory rhodopsin increased
significantly when the culture grew into stationary phase (Fig. 2). To test whether the increase in sensory rhodopsin was induced by growth condi- tions such as low 0 2 tension, or by endogenous retinal which was present in the cells in stationary growth phase, we added retinal to culture medium of the cells in early logarithmic growth phase. The addition of retinal results in an increase in sensory rhodopsin content (broken line in Fig. 2), suggest- ing the enhancement of sensory rhodopsin synthe- sis by retinal. It is not known whether the con- stitutive apoprotein of sensory rhodopsin was a different species from the inducible sensory rhodopsin. However, the absorption difference spectra obtained after a flash are essentially the same in the whole range of wavelengths examined (Fig. 3).
Photoattractant response This strain did not show a photoattractant re-
sponse in the logarithmic growth phase. However, the addition of retinal results in the appearance of photoattractant activity in a concentration-depen- dent manner (Fig. 4). The time required for the development of the response (20 min) was shorter than that for the induction of protein synthesis (see also Fig. 2).
Fig. 4. Photoattractant response of Flx3-9 cells after addition of various concentration of retinal. Cells at logarithmic growth phase were diluted to approx. 107 cells/ml with the medium containing varying concentrations of all-trans-retinal, and incubated 20 rain prior to measurement. Each point represents the percentage of the cells that reversed within 3 s after abrupt interruption of actinic irradiation. Total of the cells at each point were 100-200. Actinic light was 608_+ 8 nm, intensity 10 TM h~/mm2.s. Arrow indicates the level of spontaneous reversals measured without actinic light.
Action spectrum of the photoattractant system To confirm that the development of photoat-
tractant sensitivity upon addition of retinal is re- lated to the generation of sensory rhodopsin, we measured the action spectrum of the photoattrac- tant response. We determined the intensity of the
70 f 565nm • e%
. . so - ' " "
30 / m io ~
so :N.
3 ," d' ld'
Light Zntensity
. , : . [ . ; • t.:?" f ..,'-, • , , : .
. . . . . . . , . . . . . . . . i i
I@ i@ d' d'
662nm F 68Ohm
%o
ld' i@ ( h0 /md$ )
Fig. 5. Dependence of photoattractant response of the cells at early logarithmic growth phase on intensity of actinic light at various wavelengths. Actinic light was selected with a narrow band interference filter (half-width 10-15 nm). Each point represents the results with 100-200 cells. The results from six experiments are shown. Each experiment covers the measure- ments at nine different wavelength with a single culture of the cell.
582
O i f i i
,,rE / -- d" o ro 3.0 /.-" "',. ,~
1 0 .
5 0 600 650 700 Wavelength ( nm )
Fig. 6. Dependence of photoattractant sensitivity on actinic wavelength. Sensitivity was determined as the inverse of the intensity of actinic light at which 50% of the cells showed the response. Data were obtained from the points shown in Fig. 5 by a least-squares method. Vertical bars represent standard errors. The broken line is the absorption spectrum of sensory rhodopsin obtained from Fig. 3.
actinic light, the ceasing of which caused half of the cells to reverse their swimming direction. First, photoat tractant response was plotted against the light intensity in a restricted range where an ap- proximately linear relation was expected (Fig. 5). The relationships at various wavelengths should be parallel to each other if a single photoreceptor molecule is concerned. The data in Fig. 5 seem to fit this criterion, except for the relationship at 565 nm. Sensitivity for 50% reversal at each wave- length was obtained with a least-squares method and plotted in Fig. 6.
Interference by a photorepellent system Actinic light at wavelengths shorter than 560
nm could not cause 50% reversal of the cells (Fig. 7). Moreover, Fig. 7 shows a drop in the response
550nm
6 (
i i.-:. • .:... 201 • • •
| . . . . . . . a . ,
557nm
~ 10 4
565nm # : - %
. . . . . . . . ! , ,
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Fig. 7. Dependence on light intensity of photoattractant re- sponse to the actinic light shorter than 570 nm. At 565 nm, the data in Fig. 5 are included. Details are as in Fig. 5.
at higher actinic intensity. When the behavioral response was measured upon turning on the actinic light (step-up photoresponse) instead of upon turning off the light, the cells showed a significant response to the high intensity light at X < 560 nm. Because the cells in stationary growth phase did not show the step-up photoresponse and showed a simple sigmoidal fluence-response relationship of photoattractant response at 530-570 nm, we con- cluded that the photoattractant response at below 570 nm interfered with the other photorepellent system which is most active in the cells in logarith- mic growth phase [14]. The photorepellent system has maximum sensivity at around 480 nm and shows steep fluence-response curves [14].
Limit of the influence of a photorepellent system To compare an absorption spectrum of sensory
rhodopsin to the action spectrum obtained, it be- came necessary to determine the maximal wave- length where the photorepellent system affected the photoattractant response. The influence of the photorepellent system was examined at several wavelengths as follows. We tested whether the drop of the photoattractant response appeared on increasing the actinic light up to 1015 pho tons / m m 2. s. At above 590 nm, we could not detect a decrease of the photoattractant response. Second, the effectiveness of the photorepellent response was examined using the mutant strain [14] which has the photorepellent system but shows no pho- toattractant response. The mutant strain showed no response at above 590 nm. Therefore, we con- cluded that the repellent system does not interfere with the action spectrum of the photoattractant system at wavelengths longer than 600 nm. The action spectrum in the corresponding region was compared with the absorption spectrum of sensory rhodopsin in Fig. 6. The two spectra agree well with each other.
D i s c u s s i o n
What is the photoreceptor pigment for photoat- tractant response? In a series of the earliest works on halobacterial phototaxis [1,3], Dencher men- tioned that the development of the photoattrac- tant response is as slow as if it parallels the formation of purple membrane in a growing c u l -
583
ture of the cells [3]. Although the idea that bacteriorhodopsin mediates the phototaxis dis- agreed with the experiments using a bacteriorho- dopsin-deficient mutant strain [12,13] or by recon- stitution studies with a retinal analogue [26], the inducibility of the photosensory function was in- consistent with the finding that sensory rhodopsin, a most probable sensory photoreceptor, is a con- stitutive protein [22]. Hildebrand and Schimz [21] reported that the photoattractant system could be observed separately from a photorepellent system (PS-370) by carefully choosing the stages of cul- ture growth or by using an inhibitor of protein synthesis. Because recent works have proved that the receptor pigment for PS-370 is a photochem- ically activated state of sensory rhodopsin [15,18,19], another photoattractant receptor is nec- essary for the explanation of these observations. Nevertheless, a corresponding photoreactive pig- ment has so far not been found spectrophotomet- rically.
Our data presented in this paper, however, clearly interpret the earlier observations without assuming an unidentified photoreceptor pigment. First, it was demonstrated that the inducible fac- tor for the photoattractant response is not a recep- tor protein but retinal. Therefore, if we obtain the strain of halobacteria which can produce retinal constitutively, the photoattractant response may be observed at all stages of culture growth as Spudich and Stoeckenius mentioned [5]. Second, there is an unexpectedly large contribution of the second photorepellent system to the photobehav- ior of halobacteria-at early growth phase. The system was found quite recently [14]. Therefore, earlier works should be reinterpreted. Because the photorepellent system is more active in earlier growth phase [14], the appearance of the photore- pellent response seems to precede that of the photoattractant response. This holds especially when the photoattractant response is observed at shorter wavelengths than 590 nm, as in the experi- ment by Hildebrand and Schimz [21]. In fact, their action spectrum of PS-370 obtained at an early stage of the culture growth shows a very broad activity maximum approx. 450 nm [21]. The recep- tor of the photorepellent system was found flash- spectrophotometrically in the cell membrane of halobacteria, and the content of the pigment de-
pends on stages of culture growth [14,17]. It may be a question as to whether the photoat-
tractant response that develops after the addition of retinal is due to sensory rhodopsin apoprotein or not. Our conclusion is yes, based on the follow- ing findings: (1) flash-induced difference spectra obtained from exponentially grown cells are close to that of sensory rhodopsin; (2) the photoattrac- tant response appears much faster (approx. 15 min) after the addition of retinal than induction of the protein synthesis; (3) the phototactic action spectrum agreed well with the absorption spec- trum of sensory rhodopsin at wavelengths longer than 600 nm; (4) the sensitivity of the photoat- tractant response was relatively low at logarithmic growth phase, showing correspondence with the content of sensory rhodopsin in the cell mem- brane (see Fig. 2).
We have tried to resolve the confusion about the photoreceptor pigments in halobacterial pho- totaxis, rearranging the problem as follows. (1) Is sensory rhodopsin a photoreceptor for photorepel- lent response? (2) Is there any" other receptor pigment for photorepellent response? (3) Is sensory rhodopsin a receptor for photoattractant re- sponse? (4) Is there any other receptor for photo- attractant response? The first and the second problems were answered already [14,15,18,19]. The work presented here deals with the third. How- ever, the fourth is an open question at this point.
Acknowledgements
The authors wish to give their thanks to John L. Spudich for providing Fix 3 strain and reading the manuscript. This work was supported by a grant from the Ministry of Education, Science and Culture of Japan.
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