Accumulating Protoporphyrin IX

Proc. Natl. Acad. Sc. USA Vol. 92, pp. 7332-7336, August 1995 Biochemistry

Phototaxis away from blue light by an Escherichia coli mutant accumulating protoporphyrin IX HANJING YANG*t, HACHIRO INOKUCHIt, AND JULIUS ADLER*§¶ Departments of *Biochemistry and §Genetics, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706; and tDepartment of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606, Japan Contributed by Julius Adler, April 21, 1995

ABSTRACT The hemH gene of Escherichia coli encodes CH3 CH=CH2 ferrochelatase (EC 4.99.1.1), the enzyme that catalyzes the last step in the production' of heme, namely the synthesis of heme from protoporphyrin'IX plus Fe2t. The behavioral HG-C C0H responses to light were 'studied in E. coli carrying a hemH H3C-C-C N C= CH mutation. It was shown that the hemH mutant displayed a NH /H0N tumbling response upon illumination and a running response UH2-C%O N \ C=C-CH =CH2 el/ N\ upon removal of the light. The most effective light to induce a OH2 HG-C C=CH tumbling response in the hemH mutant was blue light (396- Hl H2H 450 nm). The chemotaxis machinery was needed for the light-induced tumbling response in the hemH mlutant. The bacterial defect is an analog of the human inherited disease lCH2 erythropoietic protoporphyria. Protoporphynn IX The porphyrias are a group of inherited human diseases, each showing a defect in a different step of heme synthesis (1). One Fe++ of these diseases is erythropoietic protoporphyria, which re- ferrochelatase -- mutant sults from, a defect in ferrochelatase (EC 4.99.1.1), the enzyme that inserts iron into protoporphyrin IX to make heme (1, 2) 2H+ (Fig. 1). We deal here with this same defect in hemH mutants of Escherichia coli bacteria (3-7), a parallel which was earlier recognized (5). CH3 CH=CH2 Human patients with erythropoietic protoporphyria are sensitive to light (1, 2, 8), and the mutant bacteria are killed by light when the intensity is sufficient (5-7). The patients actually II \N / avoid light: "...Even as a baby she had cried when brought H3C-C-C\ | C=C-CH3 into strong sunlight .. ." (p. 32 of ref. 8); "Many patients with | N-Fe8-N\ erythropoietic protoporphyria simply avoid sun exposure ..." HG-CN CH=C-CH=CH2 (p. 103 of ref. 2). Here we demonstrate in parallel fashion a OH2 HG-C O=OH negative phototaxis in the mutant E. coli. I-I H2 OH3

MATERIALS AND METHODS OH2 Strains. All bacterial strains are derivatives of E. coli K-12. The parental hemH mutant was VS101, also called hemHl or Heme visAl (5). From it we prepared (see below) AW804, the hemH mutant whose parent is the chemotactically Wild-type AW405 FIG. 1. The conversion of protoporphyrin IX to heme by insertion (9): These are the two strains most used in this study. of Fe+2 is catalyzed by ferrochelatase. This reaction, the final one in the synthesis of heme, is deficient owing to defective ferrochelatase in The hemH- chemotaxis transducer mutants were' as follows: human mutants having erythropoietic protoporphyria and in bacterial AW805, hemH- tsr- tar+ trg+ tap+, prepared from AW518 hemHmutants. Heme is needed for making cytochromes, catalase, and (10); AW806, hemH- tsr+ tar- trg+ tap+, prepared from peroxidase in humans and in E. coli, and for making hemoglobin and AW539 (11); AW807, hemH- tsr+ tar+ trg- tap+, prepared myoglobin in humans. from AW701 (12); AW808, hemH- tsr+ tar+ trg+ tap-, prepared from RP3525 (13); AW809, hemH- tsr- tar- trg- tap-, Parkinson; University of Utah); AW811, hemH- AcheB, pre- prepared from CP362 (14); AW827, hemH- tsr+ tar- trg- tap-, pared from RP4971 (J. S. Parkinson);' AW812, hemH- cheA, AW809 with tsr+ plasmid pCS5 (F. W. Dahlquist; University of prepared from AW690, previously called RP487 cheA, (18); Oregon); AW828, hemHl tsr- tar+ trg- tap, AW809 with tar+ AW813, hemH- AcheW, prepared from RP1078 (19); AW814, plasmid pNT201 (15); AW829, hemH- tsr- tar- trg+ tap-, hemH- AcheY, prepared from RP5232 (J. S. Parkinson); and AW809 with trg+ plasmid pGB1 (16); and AW830, hemH- tsr- AW815, hemH- AcheZ, prepared from RP1616 (J. S. Parkin- tar- trg- tap+, AW809 with tap+ plasmid pVB8 (17). son). The hemH- intermediate chemotaxis mutants were as follows: AW810, hemH- AcheR, prepared from RP4944 (J. S. tPresent address: Department of Anesthesia, University of California, Veterans Administration Medical Center, San Francisco, CA 94121. The publication costs of this article were defrayed in part by page charge 1To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Biochemistry, University ofWisconsin, 420 Henry Mall, Madison, WI accordance with 18 U.S.C. §1734 solely to indicate this fact. 53706. 7332 Downloaded by guest on September 27, 2021 Biochemistry: Yang et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7333 The hemH mutant lacking four methyl-accepting chemo- error in these averages was ±30%, the minimum error was taxis proteins (MCPs) and six Che proteins was AW816, ±2%, and the average error was ±10%. prepared from HCB437 (20). The hemH mutants lacking four MCPs and six Che proteins but able to run and tumble were AW816 scy strains-i.e., hemH- derivatives of HCB482, RESULTS HCB483, and HCB484 (20). Taxis in E. coli includes increased tumbling when a repellent Genetic Manipulations. For construction of hemH strains, is encountered and increased running when a repellent is the transducing bacteriophage P1 vir (21) was used. First, a Tetr removed (26, 27). In this study, the behavior ofthe bacteria was genetic marker (11.5 min on the E. coli chromosome, ref. 22), observed and videotaped, and the videotape images were which was 0.4 min away from the hemH gene (11.1 min on the digitized and analyzed by computer to yield the angular speed E. coli chromosome, ref. 5) was introduced into VS101 (5) via of the population, which reflects its degree of tumbliness (29). P1 transduction by using P1 vir grown on CAG12154 (22) and Behavioral Response of hemH Mutant to Blue Light. The selecting a transductant, AW803, which was Tetr and sensitive hemH mutant AW804 tumbles upon exposure to white light to the presence of 150 ,umol of photons m-2's- of fluorescent (data not shown). We wished to determine the wavelength light. The cotransduction of hemH and Tetr was "60%. specificity of this light-induced tumbling response. The mini- Second, P1 vir grown on AW803 was used to transduce AW405, mum intensity of light required to elicit a threshold tumbling and the hemH transductant AW804 was isolated by screening response (a change in angular speed of about 10 degrees per for both Tetr and light sensitivity. The same procedure was frame) during 20 s of exposure was measured with filters used for transductions from AW803 into other recipient allowing the following wavelength ranges to pass through: strains. For plasmid transformations, the protocol described 355-380 nm, 396-450 nm, 426-472 nm, and 468-540 nm. An previously was used (23). action spectrum was constructed by plotting the reciprocal of Media. Vogel-Bonner minimal medium (24) with 25 mM these threshold intensities as a function of wavelength (30) DL-sodium lactate and 1 mM of each required amino acid was (Fig. 2). Light in the range of 396-450 nm is the most effective used in the behavioral experiments. Antibiotic plates were among the wavelengths of light tested; this suggests that the prepared with Luria broth (1% tryptone/0.5% yeast extract/ chromophore responsible for the light-induced tumbling be- 0.5% NaCl). Antibiotics were used at the following final in this concentrations: ampicillin at 50 ,ug/ml and tetracycline at 20 havior of hemH has its major absorption range. Proto- K&g/ml. porphyrin IX, known to accumulate in hemH (6), does indeed Growth and Preparation of the Bacteria. Cells were grown have a major absorption here, at 405 nm (6). at 35°C with shaking in 10 ml of medium in a flask wrapped in The behavioral response of the hemH mutant AW804 to aluminum foil. Cells were harvested when they reached mid- blue light (396-450 nm) was studied by using computer- exponential phase (OD590 = 0.4-0.6). The procedures de- generated cell paths, as shown in Fig. 3. Before the blue light scribed below were carried out under dim light in a 30°C room. was turned on, AW804 ran and tumbled, which is represented For behavioral experiments, bacteria were washed twice with by smooth lines and by short erratic lines or dots, respectively 10-2 M Hepes-KOH, pH 7.0, containing 10-4 M potassium (Fig. 3A). When the blue light was on at an intensity of 10 ,mol EDTA (Hepes/EDTA buffer) and finally resuspended in the of photons m-2 s-1, the hemH mutant began to tumble within same buffer to an OD590 0.1 (about 7 x 107 cells per ml). The 1 s (Fig. 3B), and it kept tumbling during a 30-s exposure to the results of behavioral experiments done in chemotaxis medium light. After the blue light was turned off (Fig. 3C), the cells (10-2 M potassium phosphate, pH 7.0/10-4 M EDTA) were continued tumbling for 5-10 s. Then they gradually showed a comparable to those done in Hepes/EDTA buffer, but in the running response; 40 s after the blue light was off =90% of the latter case fewer cells became stuck on the glass slide, which cells were running (Fig. 3D). About 60 s after the blue light was was important for computerized motion analysis. For methi- turned off, the cells returned to the original unstimulated onine auxotrophs, 0.1 mM L-methionine was present in the behavior, now running and now tumbling (Fig. 3E). Hepes/EDTA buffer, since L-methionine is required for tumbling (25). 3. Bacterial Behavior. Taxis in E. coli consists of increased tumbling when the bacteria swim toward either an increasing concentration of a repellent or a decreasing concentration of attractant, and it consists of increased running when they swim toward either an increasing concentration of an attractant or a decreasing concentration of repellent: this is called a spatial 2 assay. In a temporal assay, the repellent is presented everywhere uniformly at once, or the attractant is removed every- .r4- where uniformly at once, which causes all the bacteria to tumble; or the attractant is presented everywhere uniformly at (I) once, or the repellent is removed everywhere uniformly at once, which causes all the bacteria to run (see refs. 26 and 27). 1* In the present work, we used the temporal assay: repellent light was presented everywhere uniformly at once or removed everywhere uniformly at once. Study of Light Response. The apparatus used here for observing bacterial responses to light is essentially similar to the one described by Sundberg et al. (28) (see Appendix). 350 Linear speed and angular speed are the two computerized 400 450 500 550 parameters used to describe the motion of E. coli cells (29); Wavelength, nm running causes an increase in linear speed and a decrease in FIG. 2. Action spectrum of the light-induced tumbling response of angular speed, while tumbling causes a decrease in linear speed the hemH mutant AW804. The unit on the ordinate is (,umol of and an increase in angular speed (29). Values of angular speed photons)-1.m2.s. The horizontal bars indicate the range of the light over every 2-s or 5-s period were obtained. Each experiment passing through the filters. Each point represents the average from two was done twice, and the averages are presented. The maximum experiments. Downloaded by guest on September 27, 2021 7334 Biochemistry: Yang et al. Proc. Natl. Acad. Sci. USA 92 (1995) A B C D E Light on 30 s; 1 s 1 s 40s 60 s Before after after after after FIG. 3. Computer-generated cell paths of the light light on light off light off light off hemH mutant AW804 in response to blue light. A 150 blue light stimulus (396-450 nm at an intensity of 10 ,umol of photons-m-2.s-1) was given for a period of § EE .[..' 0 -15 30 s. The cell paths were computed from digitized videoimages. Each panel contains cell paths for 2-s periods obtained at different time points (A-E, see text) during the experiment. Smooth lines represent 0 150 0 150 0 150 0 150 0 150 running behavior and short erratic lines or dots x-axis position, pm represent tumbling behavior.

The level of the light-induced tumbling response of the nonmotile (data not shown). But adaptation occurred at bacteria depended upon the duration and the intensity of the relatively low intensities of the continuous blue light: After the light. Fig. 4 demonstrates that the hemH mutant responds to blue light was turned on at an intensity of 0.5 ,umol of blue light (396-450 nm) at an intensity of 10 ,umol of photons m-2-s-1, the angular speed of the population started photonsm-2.s-. Usually it required about 10 s to achieve to increase, then it dropped back to the original value even maximum tumbling after the light was turned on (Fig. 4). In though the light remained on (data not shown). contrast, the parental E. coli failed to respond to this intensity Requirement of Chemotaxis Components for the Tumbling of 396-450 nm light (Fig. 4). Response to Light by hemH Mutant. We studied the tumbling Compared to the tumbling response of hemH at 100% light responses to blue light (396-450 nm) by chemotaxis mutants intensity (10 ,umol of photons m-2 s-1) (Fig. 4), at 10% light lacking known transducers or other chemotaxis proteins in the intensity, the tumbliness of the hemH population was reduced background of the hemH mutation. by -75% and at 1% light intensity, tumbliness was not E. coli has four known transducers, also called sensory detected by computer analysis (data not shown). receptors: Tsr, Tar, Trg, and Tap mediate tactic responses to After removal of the light, the length of the running period of certain amino acids, sugars, dipeptides, pH, temperature, etc. hemH also depended upon the duration and the intensity of the (32). Strains with the hemH mutation and any one of the four illumination. After exposure to 100% light for 30 s (Fig. 4), transducer mutations, but with the other three transducers running is indicated at 60 to 100 s (here the angular speed is being normal, still responded to the blue light; strain AW809, smaller than before the light was turned on). A 2-s exposure to having the hemH mutation and missing all four known trans- 100% blue light (396-450 nm) generated about 12 s of running ducers (a relatively smooth strain), had only a slight response response after the light was turned off (data not shown). Similarly, to the blue light. This suggests that the transducers are upon dilution of chemical repellents (analogous to removal of necessary but that no one of them is essential. tumble-causing light), bacteria also run (31). We overexpressed each of the four transducers one at a time When E. coli cells are stimulated by a chemical repellent, in strain AW809 lacking all four of them; then we tested the they tumble exclusively for a period of time and then they tumbling response to blue light (396-450 nm) (Fig. 5). Strains return to running and tumbling as in the unstimulated state, overexpressing Tsr or Tar gave clear tumbling responses to even though the repellent is still present. This loss of response blue light, the strain that overexpressed Trg showed a weak in the continued presence of the stimulus (29) is referred to as tumbling response, and the strain that overexpressed Tap did sensory adaptation. Adaptation to blue light (396-450 nm) in not show a tumbling response more than did AW809 alone. the hemH mutant was studied. Cells exposed to continuous These results show that blue light at intensities around 1.0 ,umol of photons m-2.s-1 or Tsr, Tar, or Trg is required to stimulate higher did not the tumbling response of the hemH mutant to blue light. The adapt: they kept tumbling and then became reason for the reduced tumbling response to blue light in the cells could 100 overexpressing Tap be that the total amount of Tap hemH was less compared with the amounts of overexpressed Tsr, Tar, 0 or even Trg, or that the cells overexpressing Tap remained as E.E smooth as AW809 itself. 80 Those strains containing the four transducers and the hemH 0. mutation, together with the central processing cheW, cheA, cheY, or cheZ mutation, did not show any behavioral change $ 60 a in response to the blue light. The strain with the hemH 0) mutation and a cheR mutation did not tumble to the blue light, but with stronger light, such as white light at an intensity of 1.2 40 x 102 J-m-2 s-1, this strain had a weak tumbling response. The 0, strain with hemH and a cheB mutation tumbled all the time (due to the cheB mutation). After 10-4 M L-serine had been :, 20 added to this strain, it ran; under this condition it had a weak ht tumbling response to the blue light. These light responses of ight on Ught off the che mutants in the hemH background are similar to the 0 1 responses of the che mutants to chemical repellents (32). 20 40 60 80 100 120 AW816, a mutant lacking all four transducers and all six Che Time, s proteins (a "gutted" strain) but having a hemH mutation, is smooth since it lacks FIG. 4. Behavioral responses of hemH mutant AW804 and its wild- the chemotaxis machinery, and it did not type parent AW405 to blue light. A blue light stimulus (396-450 nm at tumble to light. Even when AW816 was converted into a an intensity of 10 ,umol of photons.m-2.s-1) was given for a period of 30 running-and-tumbling strain by changing it into various scy s, as indicated. Each point represents the average over a 5-s period and (flagellar motor switch) running-and-tumbling mutants (20) is the average from two experiments. 0, AW804; 0, AW405. (HCB482, scyA2; HCB483, scyA3; and HCB484, scyBlO), it still Downloaded by guest on September 27, 2021 Biochemistry: Yang et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7335 100 DISCUSSION ) A*Tsr only The hemH mutant tumbles upon exposure to blue light (396-450 nm) (Figs. 2, 3, and 4), and it runs in response to removal of the 80 iTar only *Trg only light (Fig. 4). At relatively low light intensities cells adapt to the a '~~~~~~~~Taponly light stimulus. The hemH mutant is at least 100-fold more sensitive to 396-450 nm blue light than its wild-type parent. 60 0 no Tsr, Tar, A transducer, Tsr, Tar, or Tap, is required to get a clear Trg, or Tap tumbling response in the hemH mutant (Fig. 5). The role of transducers here is not clear at this point; they most likely serve ~40 as receptors for the tumbling signal. The Che proteins of the chemotaxis machinery are important for this light-induced tumbling response. On the other hand, for those chemotaxis 20 mutants which did not respond to the light, their biased swimming behavior (too smooth or too tumbly) might prevent on Ught of them from showing the response. However, the scy mutants, 0 Ught which can run and tumble though lacking transducers and Che 0 20 40 60 80 proteins, failed to respond to light, so the chemotaxis machin- lime, s ery is clearly needed here. responses in general have been shown to be a FIG. 5. Behavioral responses to blue light in AW809 (hemHmutant Tumbling lacking all four transducers) and AW809 overexpressing each trans- protective device of E. coli cells, allowing them to run away ducer one at a time (AW827, which is tsr+; AW828, which is tar+; from harmful situations (34, 35). The fact that the light- AW829, which is tpg+; and AW830, which is tap+). Isopropyl ,3-D- induced tumbling response has a lower threshold than killing thiogalactoside (required to induce each transducer) was added to a by light in the hemH mutant suggests that the tumbling final concentration of 1 mM, 1.5 h before cells were harvested. A blue responses to light will potentially keep cells away from light light stimulus (396-450 nm at an intensity of 10 i.mol of which would have a lethal effect at higher intensities. This photons-m-2s-1) was given for a period of 30 s as indicated. Each killing results from the hemH mutant accumulating more than point represents the average over a 5-s period and is the average from a 100-fold greater amount of protoporphyrin IX than does the two experiments. wild type (6), a result which we have confirmed for the conditions used here (unpublished data). Protoporphyrin IX did not tumble to light. This suggests that the tumbling had already been found to be involved in lysis of myxobacteria response to light requires the chemotaxis machinery. by intense blue light when carotenoids were missing (36, 37). Effect of Oxygen on the Light-Induced Tumbling Response How does light generate a tumbling response in a hemH of hemH Mutant. The light-induced tumbling response of the mutant? Porphyrins are known photosensitizers. Several stud- hemH mutant AW804 was examined under anaerobic condi- ies have shown that, in the presence of light, porphyrins cause tions. The cells were grown anaerobically under nitrogen in damage to cells by producing reactive oxygen species and Vogel-Bonner minimal medium with required amino acids consequently bringing about lipid oxidation and protein and 25 mM D-fructose as a fermentable energy source and crosslinkage (38, 39). It has been suggested (7) that in the 0.025% L-cysteine as a reductant and then transferred in an hemH mutant light produces some kinds of reactive toxic anaerobic glove box to a glass slide under a coverslip sealed by oxygen species, probably singlet oxygen (102), although su- using vaseline to keep the cells anaerobic. The bacteria did not peroxide anion (O-) and hydrogen peroxide (H202) were also tumble in response to blue light (396-450 nm, 10 ,mol of discussed, and hydroxyl radical (-OH) is also possible; these are photons.m-2-s-1) or to white light (1.9 X 102 J.m-2-s-1) under discussed further in refs. 40 and 41. We found that the anaerobic conditions, even though they were motile. phototaxis by hemH failed under anaerobic conditions; oxygen The same, results were obtained with cells grown anaerobi- is required here (see Results). It seems likely that an oxygen cally in the presence of 25 mM D-fructose with the addition of species generated by photosensitization of porphyrin may be 30 mM sodium fumarate or 30 mM sodium nitrate as terminal the direct signal for the light-induced tumbling response. electron acceptors (33). This indicates that functional electron Recently, Benov and Fridovich (personal communication) transport was not enough for getting a light-induced tumbling have shown that hydrogen peroxide is a repellent for E. coli. response in the hemH mutant. Because of this and because of our finding that aerobic conditions are required for porphyrin action, we suggest as one To find out if the lack of light-induced tumbling responses possibility the following mechanism for the negative photo- in these anaerobically grown cells was due to absence of taxis described here: oxygen, their light-induced tumbling responses were examined following exposure to air for 15 min. The oxygen indicator protoporphyrin IX activated protoporphyrin IX resazurin (0.002%) had now turned from colorless to pink, and the cells displayed a clear tumbling response to the blue light, speeded up by as well as to white light. From those experiments we conclude 2 superoxide anion, superoxide dismutase that something in the air, most likely oxygen, is needed for the reductant 0 light-induced tumbling response in the hemH mutant. Relationship Between Behavioral Responses and Light- H202, goes across cell envelope to outside -> tumbling Killing Effect in hemH Mutant. Light kills the hemH mutants to H202 chemotaxis). (5, 7). We found that 30 s of illumination did not cause any (negative killing of the hemH mutant AW804 at the light intensities used Wild-type E. coli and wild-type Salmonella typhimurium are in the behavioral studies described here (data not shown). also negatively phototactic, but strong light is required here: However, after a 10-min exposure of the hemH mutant to blue they tumble upon short exposure (1 s) to intense blue light light (396-450 nm) at an intensity of 10 ,umol of (350-530 nm); after tumbling to this light they then run, then photons'm-2.s, instead of a 30-s exposure, --20% of the they become paralyzed, and finally they die (33, 42-45). These hemH cells were killed. These results show that the behavioral cells show similar effects at lower intensities of light when response to light has a lower threshold than the killing by light. certain chemicals, such as proflavine or methylene blue, are Downloaded by guest on September 27, 2021 7336 Biochemistry: Yang et aL Proc. Natl. Acad. Sci. USA 92 (1995) present in the extracellular medium (33, 42-44). The relation- 10. Hazelbauer, G. L., Mesibov, R. E. & Adler, J. (1969) Proc. Natl. ship, if any, between the mechanism of behavioral response of Acad. Sci. USA 64, 1300-1307. the hemH mutant and the wild-type cells to light remains to be 11. Mesibov, R. & Adler, J. (1972) J. Bacteriol. 112, 315-326. investigated. A further study ofthe effect ofblue light onE. coli 12. Kondoh, H., Ball, C. B. & Adler, J. (1979) Proc. Natl. Acad. Sci. cells may help in understanding the mechanism of blue-light USA 76, 260-264. 13. Slocum, M. K. & Parkinson, J. S. (1985) J. Bacteriol. 163, 586- action elsewhere in biology (30, 46-48). 594. 14. Park, C. & Hazelbauer, G. L. (1986) J. Bacteriol. 167, 101-109. APPENDIX 15. Borkovich, K. A., Kaplan, N., Hess, J. F. & Simon, M. I. (1989) Proc. Natl. Acad. Sci. USA 86, 1208-1212. The apparatus used for observing and measuring bacterial 16. Burrows, G. G., Newcomer, M. E. & Hazelbauer, G. L. (1989) J. responses to light is described here (see also ref. 28). A 10-ptl Biol. Chem. 264, 17309-17315. drop ofbacterial suspension was placed on a glass slide, and the 17. Nara, T., Lee, L. & Imae, Y. (1991)J. Bacteriol. 173, 1120-1124. behavior of the cells was observed on a television monitor 18. Szupica, C. J. & Adler, J. (1985) J. Bacteriol. 162, 451-453. through a phase-contrast microscope (Zeiss Axioskop) that 19. Liu, J. & Parkinson, J. S. (1989) Proc. Natl. Acad. Sci. USA 86, was connected to an infrared-sensitive videocamera (68 series 8703-8707. quantitative measurement camera; Dage-MTI, Michigan City, 20. Wolfe, A. J., Conley, M. P., Kramer, T. J. & Berg, H. C. (1987) IN). The behavior of the bacteria was recorded on videotape J. Bacteriol. 169, 1878-1885. a 21. Silhavy, T. J., Berman, M. L. & Enquist, L. W. (1984) Experi- for study with computerized motion-analysis system (29). All ments with Gene Fusions (Cold Spring Harbor Lab. Press, Plain- the studies were done, at an infrared background provided by view, NY). a 12-V, 100-W tungsten lamp and a filter which passes only 22. Singer, M., Baker, T. A., Schnitzler, G., Deischel, S. M., Goel, M., infrared light (LL-700-f; Corion, Holliston, MA). Dove, W., Jaacks, K J., Grossman, A. D., Erickson, J. W. & Light stimuli were provided by a mercury lamp (HBO 50 W; Gross, C. A. (1989) Microbiol. Rev. 53, 1-24. Osram, Berlin) which was directed to the bacteria from above 23. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular through a X40 objective lens. A heat filter (HR 750F; Corion) Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, was present during all experiments to minimize the heat Plainview, NY), 2nd Ed. generated by the light from the mercury lamp. Different 24. Vogel, H. J. & Bonner, D. M. (1956) J. Biol. Chem. 218, 97-106. wavelengths of light were produced by use of various visible 25. Springer, M. S., Kort, E. N., Larsen, S. H., Ordal, G. W., Reader, light bandpass filters ("color filters") (40-nm bandwidth; Co- R. W. & Adler, J. (1975) Proc. Natl. Acad. Sci. USA 72, 4640- rion). The intensity of the light was varied different 4644. by using 26. Berg, H. C. & Brown, D. A. (1972) Nature (London) 239, 500- neutral-density filters (GD series; Corion). The light intensity 504. was measured by using an LI-1800 Spectroradiometer (LI-Cor, 27. Macnab, R. M. & Koshland, D. E., Jr. (1972) Proc. Natl. Acad. Lincoln, NB). Delivery of light stimuli was controlled by an Sci. USA 69, 2509-2512. electronic shutter coupled to a timer (model T132 shutter 28. Sundberg, S. A., Alam, M. & Spudich, J. L. (1986) Biophys. J. 50, driver/timer and 25-mm shutter; Uniblitz, Vincent Associates). 895-900. Once the control button was pushed, a 500-Hz tone maker 29. Sager, B. M., Sekelsky, J. J., Matsumura, P. & Adler, J. (1988) generated a tone sound, which was recorded on videotape, and Anal. Biochem. 173, 271-277. after 20 s the light automatically turned on for an amount of 30. Lipson, E. D. (1991) in Biophysics ofPhotoreceptors and Photo- time determined in each experiment. The tone sound recorded movements in Microorganisms, eds. Lenci, F., Ghetti, F., Colom- on the videotape triggered the start of digitization of video- betti, G., Hader, D.-P. & Song, P.-S. (Plenum, New York), NATO images on the computer. ASI Series, Vol. 211, pp. 293-309. 31. Tsang, N., Macnab, R. & Koshland, D. E., Jr. (1973) Science 181, We thank Howard Berg, Rick Dahlquist, Carol Gross, Gerald 60-63. Hazelbauer, and Sandy Parkinson for providing bacterial strains. We 32. Armitage, J. P. (1992) Annu. Rev. Physiol. 54, 683-714. also thank T. J. Frank and Ellen Lake for help in measurement oflight 33. Taylor, B. L., Miller, J. B., Warrick, H. M. & Koshland, D. E., Jr. intensities. We are grateful to Irwin Fridovich for sharing his knowl- (1979) J. Bacteriol. 140, 567-573. edge of oxygen chemistry. We are highly indebted to John Spudich for 34. Larsen, S. H., Reader, R. W., Kort, E. N., Tso, W.-W. & Adler, generously teaching us how to use and build his apparatus for J. (1974) Nature (London) 249, 74-77. observing bacterial responses to light. This work was supported by 35. Tso, W.-W. & Adler, J. (1974) J. Bacteriol. 118, 560-576. National Institutes of Health Grant AI08746. Research at Kyoto was 36. Burchard, R. P. & Dworkin, M. (1966) J. 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