JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 369-371 Copyright 0 1976 American Society for Microbiology
Vol. 125. No. 1
Printed in U.S.A.
Potassium Requirement for Cell Division in Anacystis nidulans' L. 0. INGRAM* AND E. L. THURSTON Department of Microbiology, University of Florida, Gainesville, Florida 32611,* and Biology Department, Texas A&M University, College Station, Texas 77843
Received for publication 18 July 1975
A potassium requirement for growth can be readily demonstrated in the autotrophic blue-green bacterium Anacystis nidulans strain TX20 equivalent to 0.7% of the cellular dry weight. Starvation of this organism for potassium partially dissociates growth from cell division, thereby inducing 50% of the population to form filaments. In previous studies in bacteria, a wide variety of treatments ranging from cell wall antibiotics to radiation (12) has been shown to uncouple growth from cell division and induce filament formation (1). The abundance of various metal ions in the growth medium also appears to alter cellular control of the division process. High levels of sodium chloride have been shown to
%/
A. nidulans strain TX20 was grown as described previously (8), except for modifications of the mineral salts medium CglO. Potassium nitrate and potassium phosphate were replaced by equimolar amounts of their respective sodium salts. Cultures were maintained in medium containing 10-3 M potassium chloride unless specified otherwise. Growth was mea-
B
I
Jf
I % tfE
10pm
10pm
FIG. 1. (A) Phase contrast micrographs of normal cells (10- 3M KCI) and (B) 72-h, K-starved cells (2 x 10 6 M KCI). Bar represents 10 ,um.
restore division ability to some temperatureconditional mutants of Escherichia coli (9). Starvation for magnesium selectivity blocks cell division in wild-type E. coli (3) and Aerobacter aerogenes (10), inducing the production of filaments. Calcium starvation has a similar effect on Lactobacillus bifidus (11). When Anacystis nidulans is starved for potassium, cell division is also uncoupled, and 50% of the population forms filaments (Fig. 1). ' Florida Agricultural Experiment Station publication no. 6014.
sured turbidimetrically at 450 nm with a Spectronic 70 colorimeter (Bausch & Lomb) and by dry weight determinations using pretared membrane filters. A potassium requirement in A. nidulans can be readily observed after the dilution of cultures into potassium-free media (Fig. 2). The addition of sodium chloride (10-3 M) does not alleviate this potassium requirement, thus eliminating the possibility of a chloride effect. Based upon the increase in cell yield accompanying the addition of various levels of potas369
370
J. BACTERIOL.
NOTES
.60
E
C
0~~~~~~~~~~~~~~
.30 o 0~~~~~~~~~~~~~ 0
.15
.07. 10
14
18
22
Time (hr) , 10-6MM; *, 5.0 x FIG. 2. Effect of K+ limitation on the growth of strain TX20. Symbols: A, 1.0 x 105M;
2.5 x 10-6 M; 0, 2.0 x 10-6 M. This work was supported by grant BMS 7.5-06525 from the sium chloride to starved cultures, a potassium ion requirement was found equivalent to 0.7% of National Science Foundation. its dry weight. This value compares favorably with the potassium content found in the leaves LITERATURE CITED of eukaryotic plants (2). 1. Adler, H. I., W. D. Fisher, A. A. Hardigree, and G. E. A. nidulans exhibits a novel response to Stapleton. 1966. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli. J. potassium limitations, namely the uncoupling Bacteriol. 91:737-742. of growth from cell division. In E. coli, potas2. Berslstein. B. I., S. Y. Ivanishcheva, E. M. Il'vashchuk, I. sium limitation is accompanied by the cessation I. Belous, A. K. Pshenichnaya, and A. S. O'Kanenko. of both protein synthesis and cell division while 1971. Photosynthesis, respiration and phosphorus metabolism in plants in connection with potassium distriribonucleic acid synthesis continues (13). Elecbution and K-deficient conditions. Sov. Plant. Physiol. tron micrographs of A. nidulans induced to form 18:436-442. filaments revealed no obvious envelope abnor3. Brock, T. D. 1962. Effects of magnesium ion deficiency on malities (Fig. 3). Multiple nuclear regions were Escherichia coli and possible relation to the mode of action of novobiocin. J. Bacteriol. 84:679-686. present throughout the filament length in the 4. Eagle, H. 1955. Nutritional needs of mammalian cells in absence of any cross wall initiation. tissue culture. Science 122:501-504. The concentration of potassium required for 5. Epstein, W., and M. Davies. 1970. Potassium-dependent A. nidulans in division normal growth and cell mutants of Escherichia coli K-12. .J. Bacteriol. 101:836-843. exceeds usual enzymatic cofactor requirements. 6. Ingram, L. O., and W. D. Fisher. 1973. Stimulation of cell Potassium has been implicated in bacterial division by membrane-active agents. Biochem. Bioosmoregulation (5). In bacteria, sodium, potasphys. Res. Commun. 50:200-210. beenall have magnesium and calcium, sium, 7. Ingram, L. O., and E. L. Thurston. 1970. Cell division in morphological mutants of Agmenellum quadruplicatum shown to effect cell division. The abundance strain BG-1. Protoplasma 71:55-75. and balance of these ions is particularly impor8. Ingram, L. O., C. Van Baalen, and J. A. Calder. 1973. tant in plasma membrane function (4). The Role of reduced exogenous organic compounds in the plasma membrane has been implicated as the physiology of the blue-green bacteria (algae): photoprimary site of regulation of cell division in both heterotrophic growth of an "autotrophic" blue-green bacterium. J. Bacteriol. 114:701-705. eukaryotes (14) and prokaryotes (6, 7). Interac9. Inouye, M. 1972. Reversal by sodium chloride of envelope tions with the polar membrane lipids could protein changes related to DNA replication and cell provide a common basis for the effects of these division of Escherichia coli. J. Mol. Biol. 63:597-600. 10. Kennell, D., and A. Kotoulas. 1967. Magnesium starvametal ions on the division process.
VOL. 125, 1976
NOTES
371
._~~~~~~~~~~~~~~~~-. ..
_3. . -. ,"-
_
. I~~~~~~~~~~~~~~~
03p
-7 ._~ 'I
"Il'Alilb'd4
Aft-
..
.0.. DN A-----.
-~N iF
C
FIG. 3. (A) Electron micrographs of normal cells (10-9M KC1) and (B, C) 72-h, K-starved cells (2 x 10- M KCI). Polyhedral body is shown by arrow. Bar represents 0.3 gm. Cells were prepared as described previously. tion of Aerobacter aerogenes. IV. Cytochemical
changes. J. Bacteriol. 93:367-378. 11. Kojima, M. S., S. Suda, S. Hotta, K. Hamada, and A. Suganuma. 1970. Necessity of calcium ion for cell division in Lactobacillus bifidus. J. Bacteriol. 104:1010-1013. 12. Loveless, L. E., E. Spoerl, and T. H. Weisman. 1954. A
survey of effects of chemicals on division and growth of Escherichia coli. J. Bacteriol. 68:637-644. 13. Lubin, M., and H. L. Ennis. 1964. The role of intracellular potassium in protein synthesis. Biochim. Biophys. Acta 80:614-631. 14. Pardee, A. B. 1971. The surface membrane as a regulator of animal cell division. In Vitro 7:95-104.
Vol. 125. No. 1
Printed in U.S.A.
Potassium Requirement for Cell Division in Anacystis nidulans' L. 0. INGRAM* AND E. L. THURSTON Department of Microbiology, University of Florida, Gainesville, Florida 32611,* and Biology Department, Texas A&M University, College Station, Texas 77843
Received for publication 18 July 1975
A potassium requirement for growth can be readily demonstrated in the autotrophic blue-green bacterium Anacystis nidulans strain TX20 equivalent to 0.7% of the cellular dry weight. Starvation of this organism for potassium partially dissociates growth from cell division, thereby inducing 50% of the population to form filaments. In previous studies in bacteria, a wide variety of treatments ranging from cell wall antibiotics to radiation (12) has been shown to uncouple growth from cell division and induce filament formation (1). The abundance of various metal ions in the growth medium also appears to alter cellular control of the division process. High levels of sodium chloride have been shown to
%/
A. nidulans strain TX20 was grown as described previously (8), except for modifications of the mineral salts medium CglO. Potassium nitrate and potassium phosphate were replaced by equimolar amounts of their respective sodium salts. Cultures were maintained in medium containing 10-3 M potassium chloride unless specified otherwise. Growth was mea-
B
I
Jf
I % tfE
10pm
10pm
FIG. 1. (A) Phase contrast micrographs of normal cells (10- 3M KCI) and (B) 72-h, K-starved cells (2 x 10 6 M KCI). Bar represents 10 ,um.
restore division ability to some temperatureconditional mutants of Escherichia coli (9). Starvation for magnesium selectivity blocks cell division in wild-type E. coli (3) and Aerobacter aerogenes (10), inducing the production of filaments. Calcium starvation has a similar effect on Lactobacillus bifidus (11). When Anacystis nidulans is starved for potassium, cell division is also uncoupled, and 50% of the population forms filaments (Fig. 1). ' Florida Agricultural Experiment Station publication no. 6014.
sured turbidimetrically at 450 nm with a Spectronic 70 colorimeter (Bausch & Lomb) and by dry weight determinations using pretared membrane filters. A potassium requirement in A. nidulans can be readily observed after the dilution of cultures into potassium-free media (Fig. 2). The addition of sodium chloride (10-3 M) does not alleviate this potassium requirement, thus eliminating the possibility of a chloride effect. Based upon the increase in cell yield accompanying the addition of various levels of potas369
370
J. BACTERIOL.
NOTES
.60
E
C
0~~~~~~~~~~~~~~
.30 o 0~~~~~~~~~~~~~ 0
.15
.07. 10
14
18
22
Time (hr) , 10-6MM; *, 5.0 x FIG. 2. Effect of K+ limitation on the growth of strain TX20. Symbols: A, 1.0 x 105M;
2.5 x 10-6 M; 0, 2.0 x 10-6 M. This work was supported by grant BMS 7.5-06525 from the sium chloride to starved cultures, a potassium ion requirement was found equivalent to 0.7% of National Science Foundation. its dry weight. This value compares favorably with the potassium content found in the leaves LITERATURE CITED of eukaryotic plants (2). 1. Adler, H. I., W. D. Fisher, A. A. Hardigree, and G. E. A. nidulans exhibits a novel response to Stapleton. 1966. Repair of radiation-induced damage to the cell division mechanism of Escherichia coli. J. potassium limitations, namely the uncoupling Bacteriol. 91:737-742. of growth from cell division. In E. coli, potas2. Berslstein. B. I., S. Y. Ivanishcheva, E. M. Il'vashchuk, I. sium limitation is accompanied by the cessation I. Belous, A. K. Pshenichnaya, and A. S. O'Kanenko. of both protein synthesis and cell division while 1971. Photosynthesis, respiration and phosphorus metabolism in plants in connection with potassium distriribonucleic acid synthesis continues (13). Elecbution and K-deficient conditions. Sov. Plant. Physiol. tron micrographs of A. nidulans induced to form 18:436-442. filaments revealed no obvious envelope abnor3. Brock, T. D. 1962. Effects of magnesium ion deficiency on malities (Fig. 3). Multiple nuclear regions were Escherichia coli and possible relation to the mode of action of novobiocin. J. Bacteriol. 84:679-686. present throughout the filament length in the 4. Eagle, H. 1955. Nutritional needs of mammalian cells in absence of any cross wall initiation. tissue culture. Science 122:501-504. The concentration of potassium required for 5. Epstein, W., and M. Davies. 1970. Potassium-dependent A. nidulans in division normal growth and cell mutants of Escherichia coli K-12. .J. Bacteriol. 101:836-843. exceeds usual enzymatic cofactor requirements. 6. Ingram, L. O., and W. D. Fisher. 1973. Stimulation of cell Potassium has been implicated in bacterial division by membrane-active agents. Biochem. Bioosmoregulation (5). In bacteria, sodium, potasphys. Res. Commun. 50:200-210. beenall have magnesium and calcium, sium, 7. Ingram, L. O., and E. L. Thurston. 1970. Cell division in morphological mutants of Agmenellum quadruplicatum shown to effect cell division. The abundance strain BG-1. Protoplasma 71:55-75. and balance of these ions is particularly impor8. Ingram, L. O., C. Van Baalen, and J. A. Calder. 1973. tant in plasma membrane function (4). The Role of reduced exogenous organic compounds in the plasma membrane has been implicated as the physiology of the blue-green bacteria (algae): photoprimary site of regulation of cell division in both heterotrophic growth of an "autotrophic" blue-green bacterium. J. Bacteriol. 114:701-705. eukaryotes (14) and prokaryotes (6, 7). Interac9. Inouye, M. 1972. Reversal by sodium chloride of envelope tions with the polar membrane lipids could protein changes related to DNA replication and cell provide a common basis for the effects of these division of Escherichia coli. J. Mol. Biol. 63:597-600. 10. Kennell, D., and A. Kotoulas. 1967. Magnesium starvametal ions on the division process.
VOL. 125, 1976
NOTES
371
._~~~~~~~~~~~~~~~~-. ..
_3. . -. ,"-
_
. I~~~~~~~~~~~~~~~
03p
-7 ._~ 'I
"Il'Alilb'd4
Aft-
..
.0.. DN A-----.
-~N iF
C
FIG. 3. (A) Electron micrographs of normal cells (10-9M KC1) and (B, C) 72-h, K-starved cells (2 x 10- M KCI). Polyhedral body is shown by arrow. Bar represents 0.3 gm. Cells were prepared as described previously. tion of Aerobacter aerogenes. IV. Cytochemical
changes. J. Bacteriol. 93:367-378. 11. Kojima, M. S., S. Suda, S. Hotta, K. Hamada, and A. Suganuma. 1970. Necessity of calcium ion for cell division in Lactobacillus bifidus. J. Bacteriol. 104:1010-1013. 12. Loveless, L. E., E. Spoerl, and T. H. Weisman. 1954. A
survey of effects of chemicals on division and growth of Escherichia coli. J. Bacteriol. 68:637-644. 13. Lubin, M., and H. L. Ennis. 1964. The role of intracellular potassium in protein synthesis. Biochim. Biophys. Acta 80:614-631. 14. Pardee, A. B. 1971. The surface membrane as a regulator of animal cell division. In Vitro 7:95-104.
