Vol. 124, No. 3 Printed in U.S.A.
JOURNAL OF BACERMOLOGY, Dec. 1975, p. 1635-1636 Copyright 0 1975 American Society for Microbiology
Carbohydrate Accumulation During Myxospore Formation in Myxococcus xanthus KAREN BACON,' DONALD CLUTTER, RANDALL H. KOTTEL, MICHAEL ORLOWSKI, WHITE* Department of Microbiology, Indiana University, Bloomington, Indiana 47401
AND
DAVID
Received for publication 29 August 1975
During glycerol-induced myxospore formation in Myxococcus xanthus, cellular neutral polysaccharide increases by approximately 200%, respiration decreases by 80%, and net phospholipid synthesis ceases. Previous reports from this laboratory have established that increased levels of the glyoxylate cycle enzymes can be measured in cell-free extracts prepared from early stages of glycerolinduced myxospores of Myxococcus xanthus (3, 11). Since myxospore formation takes place in complex solutions of peptides or amino acids, many of which can de degraded to pyruvic acid and acetyl-coenzyme A, it is possible that the increased activities of the glyoxylate cycle enzymes reflect a shift in flow of carbon from the tricarboxylic acid cycle into gluconeogenesis. Such a shift in carbon flow may be a part of the developmental process and reflect metabolic control central to myxospore differentiation. To assess the degree to which the developing myxospore directs the net flow of carbon into carbohydrate, the cellular contents of neutral polysaccharide and lipoprotein were measured and compared to previously published data on deoxyribonucleic acid, ribonucleic acid, and protein. Rates of respiration of developing myxospores were also measured, since a major shift in flow of carbon out of the tricarboxylic acid cycle into carbohydrate should result in a concomitant decrease in rates of oxygen uptake. Vegetable cells of M. xanthus FB (6) were grown and induced to form myxospores with glycerol as previously described (1, 7, 13). By 80 min after the introduction of the glycerol, the rod-shaped vegetable cells had completed the morphological alteration to spheres and had begun to synthesize an extracellular coat composed primarily of carbohydrate (lOa). It was found that, although the pattern of neutral polysaccharide was variable, there always occurred a 150 to 200% increase over a 24-h period (Table 1). By 12 h this accumulation amounted to 20% of the dry weight of the cells (unpublished data). However, it is clear that the bulk IPresent address: Biology sity, New York, N.Y. 10033.
of this material is not in the coat, since total neutral polysaccharide in the coat at 12 h is less than 1% of the cellular dry weight (unpulished data). Preliminary experiments with broken cell extracts indicated that approximately 50% of TABLE 1. Neutral carbohydrate, phospholipid, respiration during myxospore formation Time after addition of glycerol (h)
Carbohydratea (mg/ml)
0 1 2 4 6 8 10 12 24
0.087 0.086 0.087 0.112
Phos-
pholipid5 (#g/ml)
Qomg(Ahof
3.1 3.0
243d 197 176
OJmg/h)
3.3 2.8
0.145
61 46
0.180 0.262
46
Cells were harvested, washed once with cold distilled water, and assayed by the phenol-sulfuric acid method as described by Hodge and Hofreiter (10) for neutral polysaccharide. Glucose was used as a standard. The dry weight of the culture at 0 time was 0.576 mg/ml. b Cells were taken directly from the induction medium and lipids were extracted using the Bligh and Dyer procedure (4). Phosphorus was analyzed by a modification of the procedure by Chen (2, 5). Analysis of the culture medium alone during induction yielded no lipid phosphorus. The dry weight of the culture at 0 time was 0.576 mg/ml. c Portions of an inducing culture were transferred to the sample holder of a YSI biological oxygen monitor (Yellow Springs Instrument Co. Inc., Yellow Springs, Ohio), and temperature was equilibrated for 5 min at 28 C prior to rate measurements. A control consisting of boiled vegetative cells showed no oxygen consumption. The dry weight of the culture at 0 time was 0.266 a
mg/ml.
dThese rates are substantially higher than those reported by Dworkin and Niederpruem (8). 1635
Department, Yeshiva Univer-
1636
NOTES
the accumulated neutral polysaccharide remains soluble after centrifugation at 41,000 x g for 30 min. Additionally, more than 90% of the neutral polysaccharide could not be removed by repeated washing of the myxospores with 1.0 M KCl. A decrease of approximately 50% in the rate of respiration was detected during the first 3 to 4 h of induction (Table 1). This is the period during which high glyoxylate enzyme activity can be measured in cell-free extract (11) and when myxospore coat is being deposited (lOa). It has been previously reported that net synthesis of deoxyribonucleic acid, ribonucleic acid, and protein stops during early stages of myxospore formation (1, 11-13). Measurements of phospholipid content indicate that this material also ceases to accumulate very early during myxospore formation (Table 1). Thus, gluconeogenesis becomes the major net biosynthetic activity of developing myxospores and reflects controls presumably critical to the differentiating process. This investigation was supported by grant GB-20516 from the Developmental Biology Program of the National Science Foundation. Karen Bacon and Randall H. Kottel received support from U.S. Public Health Service grant GM 503-13 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bacon, K., and E. Rosenberg. 1967. Ribonucleic acid synthesis during morphogenesis in Myxococcus xanthus. J. Bacteriol. 94:1883-1889. 2. Bacon, K.. and D. White. 1974. Phospholipid metabolism and membrane synthesis during sporulation in Bacillus megaterium. J. Bacteriol. 118:225-230.
J. BACTERIOL. 3. Bland, J., W.-K. Yeh, D. White, and A. Hendricks. 1971. Increase in glyoxylate shunt enzymes during cellular morphogenesis in Myxococcus xanthus. Can. J. Microbiol. 17:209-211. 4. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 5. Chen, P. S., T. Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. 28:1756-1758. 6. Dworkin, M. 1963. Nutritional regulation of morphogenesis in Myxococcus xanthus. J. Bacteriol. 91:1520-1525. 7. Dworkin, M., and S. M. Gibson. 1964. A system for studying microbial morphogenesis: rapid formation of microcysts in Myxococcus xanthus. Science 146: 243-244. 8. Dworkin, M., and D. J. Niederpruem. 1964. Electron transport system in vegetative cells and microcysts of Myxococcus xanthus. J. Bacteriol. 87:316-322. 9. Dworkin, M., and W. Sadler. 1966. Induction of cellular morphogenesis in Myxococcus xanthus. I. General description. J. Bacteriol. 91:1516-1519. 10. Hodge, J. E., and B. T. Hofreiter. 1962. Determination of reducing sugars and carbohydrates, p. 380-394. In R. L. Whistler and W. L. Wolfrom (ed.), Methods in carbohydrate chemistry, vol. 1. Academic Press Inc., New York. 10a. Kottel, R. H., K. Bacon, D. Clutter, and D. White. 1975. Coats from Myxococcus xanthus: characterization and synthesis during myxospore differentiation. J. Bacteriol. 124:550-557. 11. Orlowski, M., P. Martin, D. White, and M. C.-W. Wong. 1972. Changes in activity of glyoxylate cycle enzymes during myxospore development in Myxococcus xanthus. J. Bacteriol. 11:784-790. 12. Rosenberg, I., M. Katarski, and P. Gottlieb. 1967. Deoxyribonucleic acid synthesis during exponential growth and microcyst formation in Myxococcus xanthus. J. Bacteriol. 93:1402-1408. 13. Sadler, W., and M. Dworkin. 1966. Induction of cellular morphogenesis in Myxococcus xanthus. II. Macromolecular synthesis and mechanism of inducer action. J. Bacteriol. 91:1520-1525.
JOURNAL OF BACERMOLOGY, Dec. 1975, p. 1635-1636 Copyright 0 1975 American Society for Microbiology
Carbohydrate Accumulation During Myxospore Formation in Myxococcus xanthus KAREN BACON,' DONALD CLUTTER, RANDALL H. KOTTEL, MICHAEL ORLOWSKI, WHITE* Department of Microbiology, Indiana University, Bloomington, Indiana 47401
AND
DAVID
Received for publication 29 August 1975
During glycerol-induced myxospore formation in Myxococcus xanthus, cellular neutral polysaccharide increases by approximately 200%, respiration decreases by 80%, and net phospholipid synthesis ceases. Previous reports from this laboratory have established that increased levels of the glyoxylate cycle enzymes can be measured in cell-free extracts prepared from early stages of glycerolinduced myxospores of Myxococcus xanthus (3, 11). Since myxospore formation takes place in complex solutions of peptides or amino acids, many of which can de degraded to pyruvic acid and acetyl-coenzyme A, it is possible that the increased activities of the glyoxylate cycle enzymes reflect a shift in flow of carbon from the tricarboxylic acid cycle into gluconeogenesis. Such a shift in carbon flow may be a part of the developmental process and reflect metabolic control central to myxospore differentiation. To assess the degree to which the developing myxospore directs the net flow of carbon into carbohydrate, the cellular contents of neutral polysaccharide and lipoprotein were measured and compared to previously published data on deoxyribonucleic acid, ribonucleic acid, and protein. Rates of respiration of developing myxospores were also measured, since a major shift in flow of carbon out of the tricarboxylic acid cycle into carbohydrate should result in a concomitant decrease in rates of oxygen uptake. Vegetable cells of M. xanthus FB (6) were grown and induced to form myxospores with glycerol as previously described (1, 7, 13). By 80 min after the introduction of the glycerol, the rod-shaped vegetable cells had completed the morphological alteration to spheres and had begun to synthesize an extracellular coat composed primarily of carbohydrate (lOa). It was found that, although the pattern of neutral polysaccharide was variable, there always occurred a 150 to 200% increase over a 24-h period (Table 1). By 12 h this accumulation amounted to 20% of the dry weight of the cells (unpublished data). However, it is clear that the bulk IPresent address: Biology sity, New York, N.Y. 10033.
of this material is not in the coat, since total neutral polysaccharide in the coat at 12 h is less than 1% of the cellular dry weight (unpulished data). Preliminary experiments with broken cell extracts indicated that approximately 50% of TABLE 1. Neutral carbohydrate, phospholipid, respiration during myxospore formation Time after addition of glycerol (h)
Carbohydratea (mg/ml)
0 1 2 4 6 8 10 12 24
0.087 0.086 0.087 0.112
Phos-
pholipid5 (#g/ml)
Qomg(Ahof
3.1 3.0
243d 197 176
OJmg/h)
3.3 2.8
0.145
61 46
0.180 0.262
46
Cells were harvested, washed once with cold distilled water, and assayed by the phenol-sulfuric acid method as described by Hodge and Hofreiter (10) for neutral polysaccharide. Glucose was used as a standard. The dry weight of the culture at 0 time was 0.576 mg/ml. b Cells were taken directly from the induction medium and lipids were extracted using the Bligh and Dyer procedure (4). Phosphorus was analyzed by a modification of the procedure by Chen (2, 5). Analysis of the culture medium alone during induction yielded no lipid phosphorus. The dry weight of the culture at 0 time was 0.576 mg/ml. c Portions of an inducing culture were transferred to the sample holder of a YSI biological oxygen monitor (Yellow Springs Instrument Co. Inc., Yellow Springs, Ohio), and temperature was equilibrated for 5 min at 28 C prior to rate measurements. A control consisting of boiled vegetative cells showed no oxygen consumption. The dry weight of the culture at 0 time was 0.266 a
mg/ml.
dThese rates are substantially higher than those reported by Dworkin and Niederpruem (8). 1635
Department, Yeshiva Univer-
1636
NOTES
the accumulated neutral polysaccharide remains soluble after centrifugation at 41,000 x g for 30 min. Additionally, more than 90% of the neutral polysaccharide could not be removed by repeated washing of the myxospores with 1.0 M KCl. A decrease of approximately 50% in the rate of respiration was detected during the first 3 to 4 h of induction (Table 1). This is the period during which high glyoxylate enzyme activity can be measured in cell-free extract (11) and when myxospore coat is being deposited (lOa). It has been previously reported that net synthesis of deoxyribonucleic acid, ribonucleic acid, and protein stops during early stages of myxospore formation (1, 11-13). Measurements of phospholipid content indicate that this material also ceases to accumulate very early during myxospore formation (Table 1). Thus, gluconeogenesis becomes the major net biosynthetic activity of developing myxospores and reflects controls presumably critical to the differentiating process. This investigation was supported by grant GB-20516 from the Developmental Biology Program of the National Science Foundation. Karen Bacon and Randall H. Kottel received support from U.S. Public Health Service grant GM 503-13 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bacon, K., and E. Rosenberg. 1967. Ribonucleic acid synthesis during morphogenesis in Myxococcus xanthus. J. Bacteriol. 94:1883-1889. 2. Bacon, K.. and D. White. 1974. Phospholipid metabolism and membrane synthesis during sporulation in Bacillus megaterium. J. Bacteriol. 118:225-230.
J. BACTERIOL. 3. Bland, J., W.-K. Yeh, D. White, and A. Hendricks. 1971. Increase in glyoxylate shunt enzymes during cellular morphogenesis in Myxococcus xanthus. Can. J. Microbiol. 17:209-211. 4. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 5. Chen, P. S., T. Y. Toribara, and H. Warner. 1956. Microdetermination of phosphorus. Anal. Chem. 28:1756-1758. 6. Dworkin, M. 1963. Nutritional regulation of morphogenesis in Myxococcus xanthus. J. Bacteriol. 91:1520-1525. 7. Dworkin, M., and S. M. Gibson. 1964. A system for studying microbial morphogenesis: rapid formation of microcysts in Myxococcus xanthus. Science 146: 243-244. 8. Dworkin, M., and D. J. Niederpruem. 1964. Electron transport system in vegetative cells and microcysts of Myxococcus xanthus. J. Bacteriol. 87:316-322. 9. Dworkin, M., and W. Sadler. 1966. Induction of cellular morphogenesis in Myxococcus xanthus. I. General description. J. Bacteriol. 91:1516-1519. 10. Hodge, J. E., and B. T. Hofreiter. 1962. Determination of reducing sugars and carbohydrates, p. 380-394. In R. L. Whistler and W. L. Wolfrom (ed.), Methods in carbohydrate chemistry, vol. 1. Academic Press Inc., New York. 10a. Kottel, R. H., K. Bacon, D. Clutter, and D. White. 1975. Coats from Myxococcus xanthus: characterization and synthesis during myxospore differentiation. J. Bacteriol. 124:550-557. 11. Orlowski, M., P. Martin, D. White, and M. C.-W. Wong. 1972. Changes in activity of glyoxylate cycle enzymes during myxospore development in Myxococcus xanthus. J. Bacteriol. 11:784-790. 12. Rosenberg, I., M. Katarski, and P. Gottlieb. 1967. Deoxyribonucleic acid synthesis during exponential growth and microcyst formation in Myxococcus xanthus. J. Bacteriol. 93:1402-1408. 13. Sadler, W., and M. Dworkin. 1966. Induction of cellular morphogenesis in Myxococcus xanthus. II. Macromolecular synthesis and mechanism of inducer action. J. Bacteriol. 91:1520-1525.
