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Single photon detection by an archaebacterium
J. Mol. Biol. (1988) 199, 663464
LETTERSTOTHEEDITOR
Single Photon Detection by an Archaebacterium Halobacteria are attracted by green and repelled by near ultraviolet or blue light. The photophobic response to blue light is mediated by the retinal protein P,,,. The analysis of stimulus response curves with Poisson statistical methods reveals that the photophobic response can be elicited at minimum by a single photon.
Halobacteria are propelled by a motor-driven right-handed flagellar bundle. Smooth swimming of the cells is interrupted by spontaneous motor switching events (Alam & Oesterhelt, 1984). After a switch, the motor rotates in the opposite sense and the cell reverses its direction of movement but does not necessarily retrace its original path. Through a signal transduction chain the stimulus causes a promot’ed (repulsion by blue light) or retarded (attraction by green light) motor switching event (Hildebrand & Dencher, 1975; Spudich & Stoeckenius, 1979). Thus, the cell carries out a random walk which becomes biased if light stimuli are applied to the cell. The cells sense blue and near ultraviolet light with at least two retinal proteins, sensory rhodopsin (Spudich B Bogomolni, 1984) and pigment P4s0 (Takahashi et al., 1985; Wolff et al., 1986; Marwan & Oesterhelt, 1987). Here we report on the photophobic response of cells to millisecond flashes of blue light. Individual cells are observed in infrared light, thus avoiding additional excitation of photoreceptors. While the time span between spontaneous reversals of the flagellar motor (t,) is about 20 seconds at 2O”C, saturating flashes of blue light reduce this time to about 1.2 seconds (tmin), but not less. This is evidence for rate-limiting dark reactions in signal formation (Marwan & Oesterhelt, 1987). The time between the flash and the stop response of the cell is called the response time, t,. It is a function of photon exposure (irradiance x time) and increases from tmin to about 2.5 seconds (t,,,) upon weakening of the flashes. Lowering the flash intensity further led to the observation of a new phenomenon, that a single cell does not always respond to a flash, thereby causing a split of the population of events into two groups with centers of frequency at t,,, and t,. Clearly, at this range of photon exposure light reception and the photophobic response of the cell appear as stochastic processes. Thus, photon exposure regulates the ratio of probabilities for a response with t,,, and for no photoresponse at all (t,). We measured the dependence of the percentage of photophobic events on the photon exposure and analyzed the results with the help of Poisson statistics. This approach was used first by Hecht et nl. (1942) to derive the minimal photon requirement in human vision and was recently applied to the (‘hlarn ydomonas photophobic response (P. Hegemann & W. Marwan, unpublished results). In Figure 1 the experimental data are compared 0022%2836/88/040663-o2
$03.00/O
with theoretical curves obtained for processes with different photon input requirement. The theoretical curves were calculated with the equation (Poisson distribution): P,(a)
a”eC’ = ~ n! ’
and are shown in the inset (upper left). P,(E) is the probability that in one experiment exactly n photons are absorbed and will produce a response upon a flash. The average number of photons contributing to a photophobic response in many experiments is OZ.The value of a is proportional to the photon exposure F, the photoreceptor number R, the absorption cross-section g and the quantum yield 4. cc=const.
FR (r 4 and log a=log F+x with x=log (R CJ4 const.). Since a cell responds when the minimum number n or more photons are effective, the comparison of experimental and theoretical data requires plots which describe the probability that n= 1 photon or more, n= 2 photons or more, and so on induce a response as a function of the logarithm of photon The probability p,,(a) that n or more exposure. photons are effective is 1 minus the probability that less than n photons are effective. n-1 .&,.-a P&Y) = 1 - I- ;. k=O
K!
pn(a) is plotted for the n values 1 to 9 versus log a (Fig. 1, inset, lower left) and experimental data for different or for individual cells fit only the curve for n= 1, which is evidence that at minimum the photophobic response of the halobacterial flagellar motor is elicited by one absorbed photon. The probability that exactly one photon elicits a response is maximal at log a = 0, which corresponds to a photon exposure of 8.12 x low6 mol m-’ in our experiment (Fig. 1). This value, together with that for the cross section of absorption (1.5 x 10m2’ m2 for ~~=40,000), yields the factor R 4 const. as 13. The average number of photoreceptor molecules in a cell was calculated to be approximately 250 (J. Otomo, personal communication), and therefore 4 const. must be 0.05. Assuming a quantum yield between 0.5 and 1, and assuming that any stray light effects are negligible in a single cell observa663 0
1988
Academic
Press
Limited
664
W. Marwan
et al.
0.4
0.2
Log a 0
I -I
I -0.0
1 -0.6
I -0.4
1
1 0
-0.2 Log F+X,
I 0.2
I 0.4
I 0.6
I 0.0
I I
log a
Figure 1. Minimal
photon requirement for the photophobic response in halobacteria. Halobacterium halobium strain Flx37 (bacteriorhodopsin-, halorhodopsin-deficient (8%), sensory rhodopsin+, P4so+) (Spudich & Spudich, 1982) derived from strain L33 (Wagner et al., 1981)was grown at 40°C in the dark (Oesterhelt t Krippahl, 1983) for 48 h using an inoculum of 1.5% of a stationary culture. For single cell measurements the culture was diluted 1 : 20 with fresh medium. Portions of 5 ~1 were placed on a precleaned slide and covered with a slip (20 mm x 20 mm). Before measurement, the specimen was incubated for 5 min in the dark at various temperatures (21 “C, 3O”C, 40°C). The results obtained were temperature independent. The methods used were described in detail by Marwan & Oesterhelt (1987). Flashes of 50 ms were applied 2 s after a spontaneous reversal and the relative frequency of the response R(F) was plotted versus log photon exposure. At log a=0 the photon exposure F was 8.12 x low6 mol m-‘, A=481 nm. Each cell was stimulated only once (O), each of the remaining symbols corresponds to one cell which had been repeatedly stimulated (about 15 times at each exposure). Each set of data was fitted using a least-squares procedure to one of the theoretical curves of the inset (lower right) by a linear shift on the abscissa. All data were fitting only the curve n= 1, but with slightly different values for x, indicating slight variations of the photoreceptor concentration or the constant.
tion experiment leads to the conclusion that out of 10 to 20 photoreceptor molecules only one causes
the motor to stop. All other signal chains started by excited photoreceptors vanish without success.
Hecht. S., Shlaer, S. & Pirenne, M. H. (1942). J. Gm. Physiol. 25, 819-840. Hildebrand, E. & Dencher, Ri. (1975). Nature (London), 257, 46-48. Marwan, W. & Oesterhelt, D. (1987). J. Mol. Hid. 195,
333-342. Wolfgang Manvan Peter Hegemann Dieter Oesterhelt Max-Planck-Institut D-8033 Martinsried,
fiir Biochemie W. Germany
Received 10 August
1987
References Alam, M. & Oesterhelt,
D. (1984). J. Mol. Biol. 176, 459-
475.
Oesterhelt, D. & Krippahl. G. (1983). Ann. Microbial. (In&. Pasteur), 134B, 137-150. Spudich, E. N. & Spudich, J. L. (1982). Proc. Nat. Acad. Sci., L’.S.A. 79, 4308-4312. Spudich. *J. 1,. & Bogomolni, R. A. (1984). Nature (London), 312, 509-513. Spudich, J. L. & Stoeckenius, W. (1979). Photobiochem. Photobiophys. 1, 43-53. Takahashi, T., Tomioka, H., Kamo, N. & Kobatake. Y. (1985). FEMS Microbial. Letters, 28. 161-164. Wagner, G., Oesterhelt, D., Krippahl, G. & Lanyi. ,J. K. (1981). FE&S Letters, 131, 341-345. Wolff: E. K., Bogomolni. R. A., Scherrer, P., Hess, B. & Stoeckenius. W. (1986). Proc. Nat. Acad. Sri., U.S.A.
83, 7272-7276. Edited
by A. Kluy
LETTERSTOTHEEDITOR
Single Photon Detection by an Archaebacterium Halobacteria are attracted by green and repelled by near ultraviolet or blue light. The photophobic response to blue light is mediated by the retinal protein P,,,. The analysis of stimulus response curves with Poisson statistical methods reveals that the photophobic response can be elicited at minimum by a single photon.
Halobacteria are propelled by a motor-driven right-handed flagellar bundle. Smooth swimming of the cells is interrupted by spontaneous motor switching events (Alam & Oesterhelt, 1984). After a switch, the motor rotates in the opposite sense and the cell reverses its direction of movement but does not necessarily retrace its original path. Through a signal transduction chain the stimulus causes a promot’ed (repulsion by blue light) or retarded (attraction by green light) motor switching event (Hildebrand & Dencher, 1975; Spudich & Stoeckenius, 1979). Thus, the cell carries out a random walk which becomes biased if light stimuli are applied to the cell. The cells sense blue and near ultraviolet light with at least two retinal proteins, sensory rhodopsin (Spudich B Bogomolni, 1984) and pigment P4s0 (Takahashi et al., 1985; Wolff et al., 1986; Marwan & Oesterhelt, 1987). Here we report on the photophobic response of cells to millisecond flashes of blue light. Individual cells are observed in infrared light, thus avoiding additional excitation of photoreceptors. While the time span between spontaneous reversals of the flagellar motor (t,) is about 20 seconds at 2O”C, saturating flashes of blue light reduce this time to about 1.2 seconds (tmin), but not less. This is evidence for rate-limiting dark reactions in signal formation (Marwan & Oesterhelt, 1987). The time between the flash and the stop response of the cell is called the response time, t,. It is a function of photon exposure (irradiance x time) and increases from tmin to about 2.5 seconds (t,,,) upon weakening of the flashes. Lowering the flash intensity further led to the observation of a new phenomenon, that a single cell does not always respond to a flash, thereby causing a split of the population of events into two groups with centers of frequency at t,,, and t,. Clearly, at this range of photon exposure light reception and the photophobic response of the cell appear as stochastic processes. Thus, photon exposure regulates the ratio of probabilities for a response with t,,, and for no photoresponse at all (t,). We measured the dependence of the percentage of photophobic events on the photon exposure and analyzed the results with the help of Poisson statistics. This approach was used first by Hecht et nl. (1942) to derive the minimal photon requirement in human vision and was recently applied to the (‘hlarn ydomonas photophobic response (P. Hegemann & W. Marwan, unpublished results). In Figure 1 the experimental data are compared 0022%2836/88/040663-o2
$03.00/O
with theoretical curves obtained for processes with different photon input requirement. The theoretical curves were calculated with the equation (Poisson distribution): P,(a)
a”eC’ = ~ n! ’
and are shown in the inset (upper left). P,(E) is the probability that in one experiment exactly n photons are absorbed and will produce a response upon a flash. The average number of photons contributing to a photophobic response in many experiments is OZ.The value of a is proportional to the photon exposure F, the photoreceptor number R, the absorption cross-section g and the quantum yield 4. cc=const.
FR (r 4 and log a=log F+x with x=log (R CJ4 const.). Since a cell responds when the minimum number n or more photons are effective, the comparison of experimental and theoretical data requires plots which describe the probability that n= 1 photon or more, n= 2 photons or more, and so on induce a response as a function of the logarithm of photon The probability p,,(a) that n or more exposure. photons are effective is 1 minus the probability that less than n photons are effective. n-1 .&,.-a P&Y) = 1 - I- ;. k=O
K!
pn(a) is plotted for the n values 1 to 9 versus log a (Fig. 1, inset, lower left) and experimental data for different or for individual cells fit only the curve for n= 1, which is evidence that at minimum the photophobic response of the halobacterial flagellar motor is elicited by one absorbed photon. The probability that exactly one photon elicits a response is maximal at log a = 0, which corresponds to a photon exposure of 8.12 x low6 mol m-’ in our experiment (Fig. 1). This value, together with that for the cross section of absorption (1.5 x 10m2’ m2 for ~~=40,000), yields the factor R 4 const. as 13. The average number of photoreceptor molecules in a cell was calculated to be approximately 250 (J. Otomo, personal communication), and therefore 4 const. must be 0.05. Assuming a quantum yield between 0.5 and 1, and assuming that any stray light effects are negligible in a single cell observa663 0
1988
Academic
Press
Limited
664
W. Marwan
et al.
0.4
0.2
Log a 0
I -I
I -0.0
1 -0.6
I -0.4
1
1 0
-0.2 Log F+X,
I 0.2
I 0.4
I 0.6
I 0.0
I I
log a
Figure 1. Minimal
photon requirement for the photophobic response in halobacteria. Halobacterium halobium strain Flx37 (bacteriorhodopsin-, halorhodopsin-deficient (8%), sensory rhodopsin+, P4so+) (Spudich & Spudich, 1982) derived from strain L33 (Wagner et al., 1981)was grown at 40°C in the dark (Oesterhelt t Krippahl, 1983) for 48 h using an inoculum of 1.5% of a stationary culture. For single cell measurements the culture was diluted 1 : 20 with fresh medium. Portions of 5 ~1 were placed on a precleaned slide and covered with a slip (20 mm x 20 mm). Before measurement, the specimen was incubated for 5 min in the dark at various temperatures (21 “C, 3O”C, 40°C). The results obtained were temperature independent. The methods used were described in detail by Marwan & Oesterhelt (1987). Flashes of 50 ms were applied 2 s after a spontaneous reversal and the relative frequency of the response R(F) was plotted versus log photon exposure. At log a=0 the photon exposure F was 8.12 x low6 mol m-‘, A=481 nm. Each cell was stimulated only once (O), each of the remaining symbols corresponds to one cell which had been repeatedly stimulated (about 15 times at each exposure). Each set of data was fitted using a least-squares procedure to one of the theoretical curves of the inset (lower right) by a linear shift on the abscissa. All data were fitting only the curve n= 1, but with slightly different values for x, indicating slight variations of the photoreceptor concentration or the constant.
tion experiment leads to the conclusion that out of 10 to 20 photoreceptor molecules only one causes
the motor to stop. All other signal chains started by excited photoreceptors vanish without success.
Hecht. S., Shlaer, S. & Pirenne, M. H. (1942). J. Gm. Physiol. 25, 819-840. Hildebrand, E. & Dencher, Ri. (1975). Nature (London), 257, 46-48. Marwan, W. & Oesterhelt, D. (1987). J. Mol. Hid. 195,
333-342. Wolfgang Manvan Peter Hegemann Dieter Oesterhelt Max-Planck-Institut D-8033 Martinsried,
fiir Biochemie W. Germany
Received 10 August
1987
References Alam, M. & Oesterhelt,
D. (1984). J. Mol. Biol. 176, 459-
475.
Oesterhelt, D. & Krippahl. G. (1983). Ann. Microbial. (In&. Pasteur), 134B, 137-150. Spudich, E. N. & Spudich, J. L. (1982). Proc. Nat. Acad. Sci., L’.S.A. 79, 4308-4312. Spudich. *J. 1,. & Bogomolni, R. A. (1984). Nature (London), 312, 509-513. Spudich, J. L. & Stoeckenius, W. (1979). Photobiochem. Photobiophys. 1, 43-53. Takahashi, T., Tomioka, H., Kamo, N. & Kobatake. Y. (1985). FEMS Microbial. Letters, 28. 161-164. Wagner, G., Oesterhelt, D., Krippahl, G. & Lanyi. ,J. K. (1981). FE&S Letters, 131, 341-345. Wolff: E. K., Bogomolni. R. A., Scherrer, P., Hess, B. & Stoeckenius. W. (1986). Proc. Nat. Acad. Sri., U.S.A.
83, 7272-7276. Edited
by A. Kluy