JOURNAL OF BACrERIOLOGY, May 1978, P. 671-673 0021-9193/78/0134-0671$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 134, No. 2 Printed in U.S.A.
Chemoresponses of Chlamydomonas reinhardtii RONA HIRSCHBERG* AND SHARLYN RODGERS Department of Biology, University of Missouri-Kansas City, Kansas City, Missouri 64110 Received for publication 8 December 1977
Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.
There has been extensive study of phototaxis lation counting, and the average number of cells (3-5, 8) and geotaxis (2) in the eucaryotic green in the two capillaries was calculated. In each alga Chlamydomonas reinhardtii, but almost experiment, a chamber with capillaries containnothing is known about its ability to respond to ing only CAT buffer was included as a control chemical stimuli. While positive-mating-type ga- to determine the accumulation of cells due to metes of the closely related species C. moewusii random swimming. Since variability associated var. rotunda respond to unidentified substances with the assay is about 25%, only results that are produced by the negative-mating type (9), C. at least twice the control values are considered reinhardtii gametes do not exhibit this property. significant. All observations were repeated sevAs part of our ongoing investigation into the eral times on different days; representative exmolecular mechanisms of behavior in C. rein- periments are shown. hardtii, we have investigated responses to a wide The average velocity of cells in populations variety of chemicals; we report here that the was determined using the procedure described organism can respond to chemicals in two differ- by Ojakian and Katz (6) with the cells suspended ent ways. The first, chemotaxis, is a directed at a concentration of 3 x 105 to 6 x 105 cells per response of the cells to a chemical with no ml in CAT buffer. The assay is conducted by alteration in the average swimming speed of cells microscopically counting the number of cells in the population. Chemotaxis may be positive which swim through an 0.02-mm2 plane area in (cell accumulation) or negative (cell repulsion). a given time (usually 1 to 2 min). Red light is Chemokinesis, the second type of response, does used for illumination to prevent phototaxis. The involve changes in motility. Cells accumulate average velocity is then calculated from the because they swim more slowly, or not at all, in equation V = Nlt x 4/nA, where V = average the presence of the stimulus chemical. velocity, N = the number of cells passing To measure the response of cells to chemicals, through the plane, t = time, n = cell concentraa modification of the Adler capillary tube assay tion, and A = the defined plane area. Test subwas used (1). Exponentially growing cells were stances were added to the cell suspensions, and cultured and labeled with NaH'4CO3 as de- motility was assayed. Duplicate determinations scribed previously (8). Preparations with 1 to 2 were made; the average of the values is shown. cpm/cell were suspended to give 1 x 106 to 2 x Only values 20 ,im/s more or less than the con106 cells per ml in CAT buffer (0.02 M Tris- trols are considered significant. Each experiment hydrochloride, pH 7.0-0.1 M CaCl2-0.1 M was repeated on several days with different culNH4CI). Test substances were dissolved in the tures. Typical data are presented. same buffer. Duplicate 3-,tl capillaries containing The sodium salts of organic acids with one to about 1.5 ,ul of the test solution were incubated seven carbon atoms were tested for their ability in the chambers containing the radioactively to stimulate a chemoresponse. Those with four labeled cell suspension. Chambers were con- or more carbons caused chemokinesis. Valerate structed from microscope slides (1 by 3 inches (Fig. 1) is a good example. At concentrations [ca. 25.4 by 76.2 mm]), cover slips (22 mm2), and where accumulation occurred, motility was ala bent piece of Kimax no. 34502 capillary tubing most totally inhibited. Quite similar responses exactly as described (1). Assays were conducted were observed with hexanoate and heptanoate, for 30 min at room temperature in the dark (to although at somewhat lower concentrations. prevent phototaxis). The capillaries were re- The response with butyrate was less substantial, moved, rinsed, and wiped to remove adhering and formate, acetate, and propionate had no cells. The number of cells which had entered the effect. Since amino acids are good attractants for capillary was then determined by liquid scintil671
672
NOTES
J. BACTERIOL. 3
15
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E
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0
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10
(M)
FIG. 1. Chemokinesis with sodium valerate. Cells were assayed for their chemoresponse (0) and for motility (0) at various sodium valerate concentrations.
CoCI2 (M)
FIG. 3. Positive chemotaxis with CoCl2. Accumulation of cells in capillaries (0) and cell motility (0) were measured at various concentrations of CoCl2.
concentrations of L-arginine. Interestingly, D-arginine did not cause negative chemotaxis or any type of response by the cells. L-cystine (data not shown) caused chemokinesis at 10-2 to 10'3 M. All other amino acids that were tested did not 15 k elicit a response. This is not entirely surprising 0 in view of the limited capacity of C. reinhardtii 0 to transport amino acids. 0 A variety of salts of divalent cations were tested. Chemokinesis was observed with CuS04, NiCl2, and ZnSO4. CoCl2 (Fig. 3) was a strong a. 10k attractant and stimulated positive chemotaxis at Cconcentrations where no effect on motility was observed. MnSO4 was also a chemotactic attracC tant at 10-2 to 10-3 M. MgCl2, BaCl2, and HgCl2 C._ did not elicit a response. 'U 5 The ability of C. reinhardtii to respond to several stimulus modes (light, chemicals, and gravity) underscores the complexity of its sensory apparatus. Additional study of chemores-5 -3 -64 -661 ponses should help to reveal the molecular basis for perceiving and responding to environmental Arginine (M) signals and define how several kinds of behavFIG. 2. Negative chemotaxis with L-arginine. Ac- ioral responses are integrated in a single-celled cumulation of cells in capillaries (0) and cell motility eucaryotic organism. (0) were measured at various L-arginine concentram
._
10
10
10
10
0
tions.
bacteria (1, 7), we tested all of the commonly occurring ones except tryptophan, aspartic acid, and glutamic acid. Both chemokinetic and chemotactic responses were observed. L-arginine was a strong repellent; at concentrations of 106 to 10-3 M, it elicited substantial negative chemotaxis (Fig. 2). Motility was not effected by these
This work was supported by the Research Council of the University of Missouri-Kansas City. The excellent technical assistance of William Hutchinson is greatly appreciated. LITERATURE CITED 1. Adler, J. 1973. A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J. Gen. Microbiol. 74:77-91. 2. Bean, B. 1977. Geotactic behavior of Chlamydomonas. J.
VOL. 134, 1978 Protozool. 24:394-401. 3. Feinleib, M. E. 1975. Phototactic responses of Chlamydomonas to flashes of light. I. Response of cell populations. Photochem. Photobiol. 21:351-354. 4. Hirschberg, R., and R. Stavis. 1977. Phototaxis mutants of Chlamydomonas reinhardtii. J. Bacteriol. 129:803-808. 5. Nultsch, W., and G. Throm. 1975. Effect of external factors on phototaxis of Chlamydomonas reinhardtii. I. Light. Arch. Microbiol. 103:175-179.
NOTES
673
6. Ojakian, G. K., and D. F. Katz. 1973. A simple technique for the measurement of swimming speed of Chlainydomonas. Exp. Cell Res. 81:487-490. 7. Ordal, G. W., D. P. Villani, and K. J. Gibson. 1977. Amino acid chemoreceptors of Bacillus subtilis. J. Bacteriol. 129:156-165. 8. Stavis, R., and R. Hirschberg. 1973. Phototaxis in Chiamydomonas reinhardtii. J. Cell Biol. 59:367-377. 9. Tsubo, Y. 1961. Chemotaxis and sexual behavior in Chiamydomonas. J. Protozool. 7:114-121.
Vol. 134, No. 2 Printed in U.S.A.
Chemoresponses of Chlamydomonas reinhardtii RONA HIRSCHBERG* AND SHARLYN RODGERS Department of Biology, University of Missouri-Kansas City, Kansas City, Missouri 64110 Received for publication 8 December 1977
Cells of Chlamydomonas reinhardtii have been found to respond to chemicals in two ways: chemokinesis and chemotaxis. Several amino acids, fatty acids, and inorganic salts can stimulate these responses.
There has been extensive study of phototaxis lation counting, and the average number of cells (3-5, 8) and geotaxis (2) in the eucaryotic green in the two capillaries was calculated. In each alga Chlamydomonas reinhardtii, but almost experiment, a chamber with capillaries containnothing is known about its ability to respond to ing only CAT buffer was included as a control chemical stimuli. While positive-mating-type ga- to determine the accumulation of cells due to metes of the closely related species C. moewusii random swimming. Since variability associated var. rotunda respond to unidentified substances with the assay is about 25%, only results that are produced by the negative-mating type (9), C. at least twice the control values are considered reinhardtii gametes do not exhibit this property. significant. All observations were repeated sevAs part of our ongoing investigation into the eral times on different days; representative exmolecular mechanisms of behavior in C. rein- periments are shown. hardtii, we have investigated responses to a wide The average velocity of cells in populations variety of chemicals; we report here that the was determined using the procedure described organism can respond to chemicals in two differ- by Ojakian and Katz (6) with the cells suspended ent ways. The first, chemotaxis, is a directed at a concentration of 3 x 105 to 6 x 105 cells per response of the cells to a chemical with no ml in CAT buffer. The assay is conducted by alteration in the average swimming speed of cells microscopically counting the number of cells in the population. Chemotaxis may be positive which swim through an 0.02-mm2 plane area in (cell accumulation) or negative (cell repulsion). a given time (usually 1 to 2 min). Red light is Chemokinesis, the second type of response, does used for illumination to prevent phototaxis. The involve changes in motility. Cells accumulate average velocity is then calculated from the because they swim more slowly, or not at all, in equation V = Nlt x 4/nA, where V = average the presence of the stimulus chemical. velocity, N = the number of cells passing To measure the response of cells to chemicals, through the plane, t = time, n = cell concentraa modification of the Adler capillary tube assay tion, and A = the defined plane area. Test subwas used (1). Exponentially growing cells were stances were added to the cell suspensions, and cultured and labeled with NaH'4CO3 as de- motility was assayed. Duplicate determinations scribed previously (8). Preparations with 1 to 2 were made; the average of the values is shown. cpm/cell were suspended to give 1 x 106 to 2 x Only values 20 ,im/s more or less than the con106 cells per ml in CAT buffer (0.02 M Tris- trols are considered significant. Each experiment hydrochloride, pH 7.0-0.1 M CaCl2-0.1 M was repeated on several days with different culNH4CI). Test substances were dissolved in the tures. Typical data are presented. same buffer. Duplicate 3-,tl capillaries containing The sodium salts of organic acids with one to about 1.5 ,ul of the test solution were incubated seven carbon atoms were tested for their ability in the chambers containing the radioactively to stimulate a chemoresponse. Those with four labeled cell suspension. Chambers were con- or more carbons caused chemokinesis. Valerate structed from microscope slides (1 by 3 inches (Fig. 1) is a good example. At concentrations [ca. 25.4 by 76.2 mm]), cover slips (22 mm2), and where accumulation occurred, motility was ala bent piece of Kimax no. 34502 capillary tubing most totally inhibited. Quite similar responses exactly as described (1). Assays were conducted were observed with hexanoate and heptanoate, for 30 min at room temperature in the dark (to although at somewhat lower concentrations. prevent phototaxis). The capillaries were re- The response with butyrate was less substantial, moved, rinsed, and wiped to remove adhering and formate, acetate, and propionate had no cells. The number of cells which had entered the effect. Since amino acids are good attractants for capillary was then determined by liquid scintil671
672
NOTES
J. BACTERIOL. 3
15
0
100
cm
U
to
S
0~~~~
E
E
C2 C
0
-1
10 ~
10 10 0 ~ ~
were asae
-
-
~1
0
-1 aI
~1 ~
~
a
5
50=hi heoepne a n o 0 -
10p
-2
-3
10
10
-4
Valerste
0
10
10
(M)
FIG. 1. Chemokinesis with sodium valerate. Cells were assayed for their chemoresponse (0) and for motility (0) at various sodium valerate concentrations.
CoCI2 (M)
FIG. 3. Positive chemotaxis with CoCl2. Accumulation of cells in capillaries (0) and cell motility (0) were measured at various concentrations of CoCl2.
concentrations of L-arginine. Interestingly, D-arginine did not cause negative chemotaxis or any type of response by the cells. L-cystine (data not shown) caused chemokinesis at 10-2 to 10'3 M. All other amino acids that were tested did not 15 k elicit a response. This is not entirely surprising 0 in view of the limited capacity of C. reinhardtii 0 to transport amino acids. 0 A variety of salts of divalent cations were tested. Chemokinesis was observed with CuS04, NiCl2, and ZnSO4. CoCl2 (Fig. 3) was a strong a. 10k attractant and stimulated positive chemotaxis at Cconcentrations where no effect on motility was observed. MnSO4 was also a chemotactic attracC tant at 10-2 to 10-3 M. MgCl2, BaCl2, and HgCl2 C._ did not elicit a response. 'U 5 The ability of C. reinhardtii to respond to several stimulus modes (light, chemicals, and gravity) underscores the complexity of its sensory apparatus. Additional study of chemores-5 -3 -64 -661 ponses should help to reveal the molecular basis for perceiving and responding to environmental Arginine (M) signals and define how several kinds of behavFIG. 2. Negative chemotaxis with L-arginine. Ac- ioral responses are integrated in a single-celled cumulation of cells in capillaries (0) and cell motility eucaryotic organism. (0) were measured at various L-arginine concentram
._
10
10
10
10
0
tions.
bacteria (1, 7), we tested all of the commonly occurring ones except tryptophan, aspartic acid, and glutamic acid. Both chemokinetic and chemotactic responses were observed. L-arginine was a strong repellent; at concentrations of 106 to 10-3 M, it elicited substantial negative chemotaxis (Fig. 2). Motility was not effected by these
This work was supported by the Research Council of the University of Missouri-Kansas City. The excellent technical assistance of William Hutchinson is greatly appreciated. LITERATURE CITED 1. Adler, J. 1973. A method for measuring chemotaxis and use of the method to determine optimum conditions for chemotaxis by Escherichia coli. J. Gen. Microbiol. 74:77-91. 2. Bean, B. 1977. Geotactic behavior of Chlamydomonas. J.
VOL. 134, 1978 Protozool. 24:394-401. 3. Feinleib, M. E. 1975. Phototactic responses of Chlamydomonas to flashes of light. I. Response of cell populations. Photochem. Photobiol. 21:351-354. 4. Hirschberg, R., and R. Stavis. 1977. Phototaxis mutants of Chlamydomonas reinhardtii. J. Bacteriol. 129:803-808. 5. Nultsch, W., and G. Throm. 1975. Effect of external factors on phototaxis of Chlamydomonas reinhardtii. I. Light. Arch. Microbiol. 103:175-179.
NOTES
673
6. Ojakian, G. K., and D. F. Katz. 1973. A simple technique for the measurement of swimming speed of Chlainydomonas. Exp. Cell Res. 81:487-490. 7. Ordal, G. W., D. P. Villani, and K. J. Gibson. 1977. Amino acid chemoreceptors of Bacillus subtilis. J. Bacteriol. 129:156-165. 8. Stavis, R., and R. Hirschberg. 1973. Phototaxis in Chiamydomonas reinhardtii. J. Cell Biol. 59:367-377. 9. Tsubo, Y. 1961. Chemotaxis and sexual behavior in Chiamydomonas. J. Protozool. 7:114-121.
