Spore Formation Induced by Glycerol, Dimethyl Sulfoxide,

JOURNAL OF BACTERIOLOGY, Dec. 1980, p. 1076-1082 0021-9193/80/12-1076/07$2.00/0 Vol. 144, No. 3 Patterns of Protein Production in Myxococcus xanthus During Spore Formation Induced by Glycerol, Dimethyl Sulfoxide, and Phenethyl Alcohol TERUYA KOMANO, SUMIKO INOUYE, AND MASAYORI INOUYE* Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, N.Y. 11794 Spore formation of Myxococcus xanthus can occur not only on agar plates during fruiting body formation, but also in a liquid culture by simply adding glycerol, dinethyl sulfoxide, or phenethyl alcohol to the culture. This chemicallyinduced spore formation occurs synchronously and much faster than that occurring during fruiting body formation. Dramatic changes in patterns of protein synthesis were observed during chemically-induced spore formation, as had previously been observed during fruiting body formation (Inouye et al., Dev. Biol. 68:579-591, 1979). However, the production of protein S, one of the major development-specific proteins during fruiting body formation, was not detected at all, although protein U, another development-specific protein, was produced in a late stage of spore formation as in the case of fruiting body formation. This indicates that the control of the gene expression during chemically-induced spore formation is significantly different from that during fruiting body formation. It was also found that during spore formation, every cell seems to have a potential to form a spore regardless of its age, since smaller cells as well as larger cells separated by sucrose density gradient centrifugation could equally form spores upon the addition of glycerol. Patterns of protein synthesis were almost identical for all the three chemicals. However, the final yield of spores was significantly different depending upon the chemicals used. When phenethyl alcohol was added with glycerol or dimethyl sulfoxide, the final yields were determined by the multiple effect of the two chemicals added. This suggests that although these chemicals are able to induce the gene functions required for spore formation, they may have inhibitory effects on some of the gene functions or the processes of spore formation. 1076 Although myxobacteria are procaryotes, they can undergo cellular differentiation (for a review, see reference 8). When myxococcal cells are placed on starvation agar, they aggregate and form mounds which eventually convert to fruiting bodies. Rod-shaped vegetative cells change to round or ovoid spores within the fruiting bodies. On the other hand, spore formation can be directly induced in liquid cultures by some chemicals, such as glycerol, propanol, ethyleneglycol, dimethyl sulfoxide, and phenethyl alcohol (1, 4, 10). The process of chemically-induced spore formation is shown to be synchronous and much more rapid than that of fruiting body formation (4). Recently, we have studied the patterns of protein synthesis during the development of Myxococcus xanthus (5, 6) and Stigmatella aurantiaca (7), and we have demonstrated that gene expression during the development is complex and tightly regulated. In this report, we investigated gene expression during spore formation induced by glycerol, dimethyl sulfoxide, and phenethyl alcohol. It was found that gene expression during chemicallyinduced spore formation is substantially different from that during fruiting body formation. However, the present results indicate that gene expression during chemically-induced spore formation is also complex and tightly regulated. MATERIALS AND METHODS Bacterium and growth conditions. M. xanthus strain FB (DZF1) was used. Vegetative cultures were grown at 30 C writh vigorous aeration in methioninelimited Al medium containing 2.5,ug of methionine per ml (2). The doubling time of FB in these conditions was 22 to 24 h. Induction of spore formation. To an exponentially growing culture (ca. 2.5 x 108 cells per ml), glycerol, dimethyl sulfoxide, or phenethyl alcohol was added at a final concentration of 0.5, 0.7, or 0.01 M, respectively. After the addition, portions of the culture were taken at time intervals and sonicated for 30 s to break vegetative cells. Sonication-resistant spores were then

VOL. 144, 1980 spread, after an appropriate dilution, on Casitoneyeast extract plates with the use of soft agar. After 4 days at 350 C, the number of colonies was counted. The plating efficiency was about 90%. Radioactive labeling experiments. Portions of an induced or noninduced culture were pulse-labeled at intervals for 30 mm at 30 C with 40,uCi of [3S]- methionine (Amersham Corp.) unless otherwise noted. At the end of the incubation, cells were harvested by centrifugation and washed once with 0.01 M phosphate buffer, ph 7.1. From the washed cells, the soluble, membrane, and spore fractions were prepared as described previously (5). Polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described previously (5). Protein patterns were examined by autoradiography. RESULTS Time course of spore formation. Glycerol was added at a final concentration of 0.5 M into a culture of M. xanthus in methionine-limited Al medium. The number of sonication-resistant spores started to increase 2 h after the addition of glycerol and reached a plateau at 4 h (Fig. 1). The final yield of sonication-resistant spores was about 40% of the initial cells. Under the phase contrast microscope, cell shapes did not change for the first 1.5 h. However, after 1.5 h, the cells started to shorten, and at 2.5 h most of the cells became ovoid. Refractile spores started to increase from 2 h, and over 90% of the cell population became refractile spores at 4 h. This indicates that about 50% of the refractile spores were sonication-sensitive. When dimethyl sulfoxide was added as an inducer, cells became spores with a longer lag period than when glycerol was added (Fig. 1). The final yield of sonication-resistant spores was E en 0. co UA. 0 z TIME AFTER ADDITION (hrs) FIG. 1. Time course ofspore formation induced by glycerol (0), dimethyl sulfoxide (*), and phenethyl alcohol (A). The initial cell density was 2.54 x 10' cells per ml. At intervals, sonication-resistant spores were counted as described in the text. SPORE FORMATION OF M. XANTHUS 1077 also less than in the case of glycerol (about 15%, Fig. 1). Under the microscope, about 60% of the cell population was found to be converted into refractile spores after 6 h. When phenethyl alcohol was added, the lag period was even longer than in the case of dimethyl sulfoxide, and the final yield of spores was much less (about 5%, Fig. 1). Under the microscope, about 30% of the cell population was converted into refractile spores after 6 h. Patterns of soluble protein synthesis during spore formation. After induction of spore formation by glycerol, cells were pulse-labeled at time intervals with [3S]methionine. Figure 2 shows the patterns of soluble protein synthesis during spore formation induced by glycerol together with that of exponentially growing cells. Remarkable changes can be observed. The production of many proteins actively synthesized in log phase gradually decreases after induction (for example, proteins c and h in Fig. 2). Some proteins appear to be synthesized at a constant rate throughout the period of spore formation (for example, protein d). On the other hand, there is a group of proteins which are synthesized after induction of spore formation, as had been previously observed during fruiting body formation (5, 7). These proteins induced after spore induction can be classified into the following three groups according to the stages of their production. (i) Early proteins. Proteins such as protein e which are synthesized mainly at a very early stage of induction. (ii) Peak proteins. The proteins such as proteins a and b which are synthesized most actively between 1 and 2 h after induction. Protein j may be classified in this group, although its production is also seen in log phase as well, which is not the case with proteins a and b. The rate of production of protein j increased to reach maximum at around 1 h after induction and then diminished almost completely 3 h after induction. (iii) Late proteins. The proteins such as proteins f, g, and i which are synthesized in a late stage of induction. The production of protein g and i started at around 2.5 h after induction, and appears to be parallel to the appearance of sonication-resistant spores (Fig. 1). On the other hand, the production of protein f started at 3.5 h when the number of sonication-resistant spores reached almost the maximum level (Fig. 1). Although remarkable changes in the patterns of protein synthesis were found (Fig. 2), no significant change in the Coomassie brilliant bluestaining bands was observed during the spore formation induced by glycerol. Essentially the same patterns as shown in Fig. 2 were obtained when spore formation was in-

1078 KOMANO, INOUYE, AND INOUYE duced by dimethyl sulfoxide (Fig. 3). The production of an early protein e occurred immediately after the induction. Peak proteins a and b as weli as late proteins f, g, and i were also synthesized in a very similar fashion as observed for spore induction by glycerol (Fig. 2). Similar patterns of protein synthesis were also observed in the case of spore formation induced by phenethyl alcohol (Fig. 4). However, in addition to early protein e, the production of two other early proteins x and y were clearly observed immediately after induction. It should be noticed that the patterns of soluble protein synthesis during spore formation induced by different chemicals are remarkably similar in spite of substantial differences in the yields of sonication-resistant spores (Fig. 1). Patterns of membrane protein synthesis during spore formation. Figure 5 shows the patterns of membrane protein synthesis during spore formation induced by glycerol. Synthesis of many proteins (proteins 1, 4, 9, 12, 13, and 14) diminished after glycerol induction. There are again three classes of proteins according to modes of their synthesis. Protein. 10 is classified as an early protein, which is synthesized immediately after induction. Many peak proteins (proteins 2, 3, 5, 6, 8, 11, 15, and 17) are observed. The rates of synthesis became highest from 0.5 to 1.5 h for proteins 8 and 11, from 1 to 2 h for 5, 6, 15, and 17, and from 1.5 to 2.5 h for 2 and 3. Synthesis of proteins 7 and 16 (late proteins) were induced after 2.5 h when spores started to appear. Figure 5 also shows the pattern of protein synthesis in the sonication-resistant spore fraction at 4 h (SP in Fig. 5). Almost identical patterns were observed between the membrane fraction and the spore fraction. Essentially the same patterns of membrane protein synthesis were observed when spore formation was induced by dimethyl sulfoxide except for the synthesis of protein 17 which did not decrease even at a late stage of the spore induction (Fig. 6). In the case of phenethyl alcohol, the patterns of membrane protein synthesis were somewhat different from the glycerol and dimethyl sulfoxide patterns (Fig. 7). The production of early protein 10 was very low. On the other hand, a new protein 18 was produced actively at an early stage. Production of early proteins. It is interesting to examine how soon cells respond to the d. b T i 11t II J. BACTERIOL. II~~~~~~SA FIG. 3. Pattern of soluble protein synthesis during spore formation induced by dimethyl sulfoxide. The experiments were performed as described in Fig. 2. The large arrows and LOG are described in Fig. 2. r i m, e ( h1 r s!1: i!,r- 1 1.5 2 OS3 3 335 E; "E - HMi.A,...4..._K I 'J.'S FIG. 2. Pattern of soluble protein synthesis during spore formation induced by glycerol Cells were pulselabeled for 30 min with [35SJmethionine at time intervals as indicated in the figure. The soluble proteins were then prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large arrows indicate the positions of the molecular weight standards. Bovine serum albumin, BSA; and hen egg white lysozyme, LYS; LOG, Pattern of soluble protein synthesis of vegetative cells.

- z < l, L~~~~~~~~~J; VOL. 144, 1980 SPORE FORMATION OF M. XANTHUS 1079 addition of spore inducers. For this purpose, that many development-specific proteins including proteins S and U are produced during fruit- glycerol-induced cells were pulse-labeled for 10 min at 30 C with [3S]methionine, and then ing body formation (5). Synthesis of protein S is soluble (Fig. 8a) and membrane (Fig. 8b) fractions were analyzed by sodium dodecyl sulfate- for up to 15% of the total soluble protein synthe- induced early in development, and it accounts gel electrophoresis. sis at the stage of mound formation. On the In the case of soluble proteins (Fig. 8a), we other hand, protein U is induced at a later stage can see some proteins (such as protein e) were of development. Therefore, it is of great interest induced immediately after the induction, to examine whether these development-specific whereas the other proteins (such as protein j) proteins S and U are produced during spore were induced somewhat later. In the case of formation induced by glycerol. membrane proteins (Fig. 8b), the production of Glycerol-induced cells were labeled with protein 10 was induced only in a very early stage [3S]methionine between 2 and 3.5 h and between 3.5 and 5 h after the addition of glycerol. of spore induction (0 to 10 min), whereas the production of many other membrane proteins The soluble fractions from the labeled cells were (for example protein 17) was induced a little mixed with the purified preparation of protein later. S. Then, anti-protein S serum was added to the Detection of development-specific proteins. In the early experiments, we have shown Time ( hrs) LOG 0 0.75 1.5 2.25 3 3.75 SP Tinie ( hrs) LOG 0 1 2 3 4 5 6 _. _ ~~~~~~~~~~~~~~~~~~~~~~~~... 4-13 --a BSA iwas - --K f -......ssfs: :4..,. L.YS3_3i.m-ju.o -a P, 17B-2l = LYS...... FIG. 6. Pattern of membrane protein synthesis during spore formation induced by dimethyl sulfoxide. The experiments were carried out as described FIG. 4. Pattern ofsoluble protein synthesis during spore formation induced by phenethyl alcohol. The in Fig. 3. SP, Spore proteins prepared from cells experiments were carried out as described in Fig. 2. pulse-labeled at 3.75 h after induction. The large The large arrows and LOG are described in Fig. 2. arrows and LOG are described in Fig. 2. Time (hr. ) LOG 0 0.5 1 1.5 2 2.5 3 3.5 4 SP.1OW Z m r ' e flfs} N.. FIG. 5. Pattern ofmembrane protein synthesis during spore formation induced by glycerol. The membrane fractions were prepared from pulse-labeled cells as described in Fig. 2 and analyzed by sodium dodecyl sulfate-gel electrophoresis. SP, Spore proteins prepared from cells pulse-labeled at 4 h after induction. The large arrows and LOG are described in Fig. 2.

1080 KOMANO, INOUYE, AND INOUYE B SA- 5 Time (hrs) LOG 0 1 2 3 4 5 6 SP 14u~ LYS j i " * \ FIG. 7. Pattern of membrane protein synthesis during spore formation induced byphenethyl alcohol. The experiments were carried out as described in Fig. 4. SP, Spore proteins prepared from cells pulselabeled at 5 h after induction. The large arrows and LOG are described in Fig. 2. a a dime 10 Tim e ( nm i it LOG 0 10 20 30 b.y,,s,,,~ ~.....,,.,,,,... Im- A - km _~i j" 8 w BSA- am- 11 13 135-1 6 --BSA I.,YS --BSA - LYS FIG. 8. Pattern of soluble (a) and membrane (b) protein synthesis during a very early stage of spore formation induced by glycerol. Cells were pulse-labeled for 10 min with 100 ttci of /85S]methionine at the time intervals indicated for both (a) and (b) in the figure. The soluble and membrane proteins were analyzed as in Fig. 2. The large arrows and LOG are described in Fig. 2...-r_ a J. BACTERIOL. mixture. The immunoprecipitates thus formed were analyzed by the sodium dodecyl sulfate-gel electrophoresis (Fig. 9). The same experiment was also carried out with protein U and antiprotein U serum and the results are also shown in Fig. 9. Proteins S and U which were added as carrier are clearly detected by staining as shown in Fig. 9a. However, in the case of protein S, no radioactivity was found in the protein S band (Fig. 9b, C and D). In contrast, radioactive bands were clearly detected in the protein U bands (Fig. 9b, A and B). It should be noticed that protein U is synthesized more actively at the later stage of spore formation. Effect of chemicals on the final yield of spores. Final yields of sonication-resistant spores were considerably different depending upon the chemicals used as shown in Fig. 1. To elucidate the factors which determine the final yield of spores, we examined the effect of various combinations of chemicals on the final yields of spores. Table 1 summarizes the results of this experiment. When glycerol was added with dimethyl sulfoxide, the final yield appeared to be LYS-- ].. b A B C D d" 5011 14 ig m FIG. 9. Detection of the production of proteins S and U during spore formation induced by glycerol. The cells were labeled with ['3S]methionine during two different periods: one from 2 to 3.5 h and the other from 3.5 to 5 h after induction. Immunoprecipitation with anti-protein S serum or anti-protein U serum was carried out as described in the text. The immunoprecipitates were analyzed by sodium dodecyl sulfate-gel electrophoresis. (a) Coomassie brilliant blue-staining and (b) autoradiography of the gel. Lane A, immunoprecipitate from the cells labeled between 2 and 3.5 h with anti-protein U serum; lane B, immunoprecipitate from the cells labeled between 3.5 and 5 h with anti-protein U serum; lane C, immunoprecipitate from the cells labeled between 2 and 3.5 h with anti-protein S serum; lane D, immunoprecipitate from the cells labeled between 3.5 and 5 h with anti-protein S serum. The small arrows indicate the positions ofproteins S and U. The large arrows are described in Fig. 2..-

VOL. 144, 1980 determined by dimethyl sulfoxide. On the other hand, when phenethyl alcohol was added with glycerol or dimethyl sulfoxide, the final yields were determined by the multiple effect of the two chemicals used. DISCUSSION The present results reveal that there is a tightly controlled program of gene expression during spore formation induced by chemicals. Immediately after the addition of glycerol, the production of some proteins (for example, protein e in Fig. 2, and protein 10 in Fig. 5) was induced. However, the production of many other soluble and membrane proteins was started later at different stages for different proteins. Furthermore, the production of some proteins continued for a long time, whereas the production of the other proteins was shut-off in a short period of time. Such a tightly controlled regulation of protein synthesis was also observed during fruiting body formation (5). However, there seem to be considerable differences in gene expression between spore formation induced by glycerol and fruiting body formation. Although in both cases, cells were converted into spores, spores are formed much earlier in the case of induction by glycerol (3 to 4 h) than in fruiting body formation (35 to 60 h). Furthermore, there are substantial differences in the ultrastructures of thin sections between glycerol spores and fruiting body spores (6). Another clear difference can be seen in gene expression of protein S. During fruiting body formation, as much as 15% of the total cellular protein synthesis is devoted to protein S, whereas protein S was not detectable at all throughout spore formation induced by glycerol (Fig. 9). On the other hand, protein U was found to be produced during a late stage of spore formation in both cases (Fig. 9). The patterns for glycerol and dimethyl sulfoxide induction were almost the same, whereas the patterns for phenethyl alcohol induction were somewhat different, especially in an early stage. This suggests that phenethyl alcohol works on induction of spore formation in a different fashion from glycerol and dimethyl sulfoxide. This is consistent with the fact that mutants insensitive to glycerol induction are also insensitive to dimethyl sulfoxide but still sensitive to phenethyl alcohol induction (3). It should be noted that in the case of mutants insensitive to glycerol induction, there were no significant alterations in the patterns of protein synthesis upon the addition of 0.5 M glycerol (data not shown). It should be noted that chemically induced spore formation is much faster than that occur- SPORE FORMATION OF M. XANTHUS 1081 ring during fruiting body formation. In spite of a doubling time of 22 to 24 h in Al medium, as many as 90% of exponentially growing cells can form refractile spores synchronously within 4 h upon the addition of glycerol. This suggests that gene expression of every cell is immediately altered when glycerol is added to a culture and that every cell is synchronously converted into a spore regardless of its age. This was confirmed by the fact that smaller (younger) as well as larger (older) cells which were separated by sucrose density gradient centrifugation were equally able to form spores upon the addition of glycerol (data not shown). This is also consistent with the fact that the spore formation induced by glycerol is independent from DNA replication of the cells (9). Differences of the effects of the three chemicals are noteworthy. Although patterns of protein synthesis were almost identical for all three chemicals, the final yields of spores were significantly different depending upon the chemical used (Table 1). Furthermore, when phenethyl alcohol was added with glycerol or dimethyl sulfoxide, the final yield appeared to be determined by the multiple effect of the two chemicals. This indicates that although these chemicals are able to induce the gene functions required for spore formation, they may have inhibitory effects on some of the gene functions or the processes of spore formation. TABLE 1. Effect of various inducer combinations' Sonica- tion-re- Inducer sistant spores (spores/ ml) Glycerol (0.5 M)... 8.5 x 107 (35)b Dimethyl sulfoxide (0.7 M)... 4.4 x 107 (18) Phenethyl alcohol (0.01 M)..... 3.1 x 107 (13) Glycerol + dimethyl sulfoxide 3.9 x 107 (16) Glycerol + phenethyl alcohol... 1.7 x 107 (7) Dimethyl sulfoxide + phenethyl alcohol.. 0.6 x 107 (2) Inducers were added to vegetative cultures ofm. xanthus. The initial cell density was 2.42 x 108 cells per ml. After 8 h, the number of sonication-resistant spores was counted as described in the text. b The values in parentheses represent percentages calculated from the initial cell density. ACKNOWLEDGMENTS The present work was supported by Public Health Service grant GM-26843 from the National Institutes of Health. We thank Thomas Yee for critical reading of the manuscript.

1082 KOMANO, INOUYE, AND INOUYE LITERATURE CITED 1. Bacon, K., and E. Rosenberg. 1967. Ribonucleic acid synthesis during morphogenesis in Myxococcus xanthus. J. Bacteriol. 94:1883-1889. 2. Bretscher, A. P., and D. Kaiser. 1978. Nutrition of Myxococcus xanthus, a fruiting myxobacterium. J. Bacteriol. 133:763-768. 3. Burchard, R. P., and J. H. Parish. 1975. Mutants of Myxococcus xanthus insensitive to glycerol-induced myxospore formation. Arch. Microbiol. 104:289-292. 4. Dworkin, M., and S. M. Gibson. 1964. A system for studying microbial morphogenesis: rapid formation of microcysts in Myxococcus xanthus. Science 146:243-244. 5. Inouye, M., S. Inouye, and D. R. Zusman. 1979. Gene expression during development of Myxococcus xanthus: pattern of protein synthesis. Dev. Biol. 68:579-591. J. BACTERIOL. 6. Inouye, M., S. Inouye, and D. R. Zusman. 1979. Biosynthesis and self-assembly of protein S, a development-specific protein of Myxococcus xanthus. Proc. Natl. Acad. Sci. U.S.A. 76:209-213. 7. Inouye, S., D. White, and M. Inouye. 1980. Development of Stigmatella aurantiaca: effects of light and gene expression. J. Bacteriol. 141:1360-1365. 8. Kaiser, D., C. Manoil, and M. Dworkin. 1979. Myxobacteria: cell interactions, genetics, and development, p. 595-639. In M. P. Starr, J. L. Ingraham, and S. Raffel (ed.), Annual Review of Microbiology, vol. 33. Annual Reviews Inc., Palo Alto, Calif. 9. Kimchi, A., and E. Rosenberg. 1976. Linkages between deoxyribonucleic acid synthesis and cell division in Myxococcus xanthus. J. Bacteriol. 128:69-79. 10. 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.