SYNTHESIS AND DEGRADATION OF POLY-3-HYDROXYBUTYRIC ACID IN CONNECTION WITH SPORULATION OF BACILLUS MEGATERIUM

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1 SYNTHESIS AND DEGRADATION OF POLY-3-HYDROXYBUTYRIC ACID IN CONNECTION WITH SPORULATION OF BACILLUS MEGATERIUM RALPH A. SLEPECKY AND JOHN H. LAW Department of Biological Sciences, Northwestern University, Evanston, Illinois, and Department of Chemistry, Harvard University, Cambridge, Massachusetts Received for publication December 31, 196 ABSTRACT SLEPECKY, RALPH A. (Northwestern University, Evanston, 11.), AND JOHN H. LAW. Synthesis and degradation of poly-,b-hydroxybutyric acid in connection with sporulation of Bacillus megaterium. J. Bacteriol. 82: The production of poly-f-hydroxybutyrate has been followed in Bacillus megaterium, a sporulating strain, and B. megaterium strain KM, a nonsporulating strain, by an improved assay procedure and by the use of C14- acetate. The production of polymer in the KM strain follows the growth curve very slowly and reaches a peak at the time the cells are entering the stationary phase of growth. Slow utilization of polymer follows. When the sporulating strain is grown under conditions favorable for polymer production, no spores are formed; polymer production and utilization follow kinetics similar to those observed with asporogenous strains. When the sporulating strain is grown under conditions unfavorable for polymer production but favorable for sporulation, less polymer is produced and peak production occurs during the log phase of growth. Rapid utilization of the polymer precedes sporulation. If the medium is made favorable for polymer production by the addition of glucose and acetate and vigorous aeration conditions are used, sporulation can be obtained after good polymer production and subsequent utilization. Poly-f3-hydroxybutyric acid is the polymer that forms the major component of the lipid inclusions of some bacterial cells (Lemoigne, 1927; Williamson and Wilkinson, 1958; Law and Slepecky, 1961). Recent work indicates that the main role of the polymer is as an intracellular reserve of carbon and energy for some cells (Macrae and Wilkinson, 1958a), although it is important as an intracellular reserve of carbon and reducing power in the photosynthetic bacterium, Rhodospirillumt rubrum (Duodoroff and Stanier, 1959; Stanier et al., 1959), under certain growth conditions. The polymer has been particularly studied in the genus Bacillus; however, with the exception of the reports of Tinelli (1955a, b), who showed that the polymer is utilized in sporulating cultures, the behavior of this compound during the sporulation process has not been investigated. Using a more direct and sensitive specific assay for the polymer (Slepecky and Law, 196; Law and Slepecky, 1961), this paper presents data bearing on the role of poly-3-hydroxybutyric acid during sporulation. MATERIALS AND METHODS Bacillus megaterium strain KM, an asporogenous organism, was grown on Macrae-Wilkinson medium (Macrae and Wilkinson, 1958a). Fifty-milliliter portions of medium in 25-ml nephelometer flasks were inoculated with.5 ml of a 24-hr culture grown in one-half strength nutrient broth. Flasks were incubated at 3 C on a reciprocating shaking machine. B. megaterium, a sporogenous strain, was grown on Macrae-Wilkinson medium under exactly the same conditions as for strain KM. In some experiments this organism was grown on a chemically defined sucrose medium (Slepecky and Foster, 1959). A 2-ml amount of a 24-hr culture of one-half strength nutrient broth culture was inoculated into 5 ml of medium in 3-liter Fernbach flasks and incubated at 3 C on a shaker. In another experiment this organism was grown in a Biogen (American Sterilizer Company, Erie, Pa.) using a modified defined medium. In place of sucrose,.4% glucose was added and the medium was supplemented with.1% acetate. One liter of a 12-hr culture grown on one-half strength nutrient broth was inoculated into 4 liters of medium in the Biogen and grown at 3 C. 37 Downloaded from on October 15, 218 by guest

2 38 SLEPECKY AND LAW [VOL. 82 a. 15- cat ~~~~~~ ~-3 - I z E 6 ~~~~~ Idz 5- E ~4 d ~~~~~~~~I I ~1 CZ2 CD IL TIME,* HOURS FIG. 1. Growth, polymer production, and incorporation of acetate into Polymer for Bacillus megaterium strain KM in 6 ml Macrae-Wilkin-son medium, supplemented with 6 mg CH3C'4H, 1 i.ac/mg. In the preparation of all media, the phosphates were sterilized separately before addition to the other ingredients of the medium to prevent precipitation of phosphate salts. In some of the experiments the media were supp lemented with C'4-acetate as indicated in the figure legends. Sodium 1-C14-acetate (Nuclear- Chicago or Tracerlab) of specific activity 2,ug per mm was diluted to the desired specific activity with nonisotopic sodium acetate. Radioactivity measurements were made with a gas flow counter with mylar end window (Nuclear-Chicago) or with a liquid scintillation counter (Packard). At various intervals, growth was estimated directly in the nephelometer flasks or a sample w-as removed for growth determinations by measuiring turbidity in a photoelectric colorimeter equipped with a green filter (no. 54). Samples (.1 ml) were withdrawn for direct examination and counting of vegetative cells, sporangia, or spores in a Petroff-Hauser chamber and phase contrast microscope. Samples withdrawn at various time intervals were extracted and assayed for poly-,3-hydroxybutyric acid (Law and Slepecky, 1961). Dry weight determinations were made on samples of the cell suspensions; they were dried overnight at 1 C and weighed after cooling in a desiccator containing CaCl2. RESULTS To have a basis for comparison with the sporulating strain, growth and polymer production as given by chemical assay and acetate incorporation into poly-f-hydroxybutyric acid were followed in B. megaterium, the asporogenous organism, grown on Macrae-Wilkinson medium, a medium favorable for polymer production (Macrae and Wilkinson, 1958b) (Fig. 1). Peak polymer production, close to 5% on a dry weight basis, occurred at the time the cells entered the stationary phase of growth; as the cells aged, poly-f3-hydroxybutyric acid decreased in the cells, presumably due to endogenous utilization (Macrae and Wilkinson, 1958a; Doudoroff and Stanier, 1959). This organism under these conditions or on defined sucrose medium never formed spores. Polymer production on the latter medium was not followed since growth was very sparse when it occurred at all. When the sporulating strain of B. megaterium was grown on the medium favoring polymer production, Macrae-Wilkinson medium, essentially the same pattern of growth and polymer production occurred (Fig. 2) as with the asporogenous organism grown under these conditions. The amount of polymer was about one-fifth that formed in the asporogenous organism. However, peak production of polymer and utilization by cells in the stationary phase follows closely that obtained with the asporogenous organism. Less than 1 % spores were formed under these conditions. When the sporulating strain was grown under conditions unfavorable for the production of polymer, namely, in a defined sucrose medium Downloaded from on October 15, 218 by guest

3 19611 POLY-f-HYDROXYBUTYRATE AND SPORULATION I- f-- fi 214 < 1. SPORES 5, E TIME, HOURS FIG. 2. Growth and polymer production for Bacillus megaterium, the sporogenous strain, in 5 ml Macrae- Wilkinson medium. TIME (HOURS) 2 3 FIG. 3. Growth, polymer production, and incorporation of acetate into polymer of Bacillus megaterium, the sporogenous strain, in 5 ml defined sucrose medium supplemented with 225 mg CH3C'4H, 1 Ic/mg. containing little or no acetate (Fig. 3), much less polymer was formed (about 1% of that found under favorable polymer producing conditions) and peak polymer production occurred during the log phase of growth. Rapid decrease of the polymer level was observed several hours before sporulation ensued (greater than 9% under these conditions). Conditions were found where the cells could produce a high yield of polymer and still sporulate (Fig. 4). When glucose and acetate were added to the medium (ordinarily giving a low polymer yield) and vigorous conditions of aeration and agitation (as found in the Biogen) are employed, peak polymer production was obtained at the time of peak growth with polymer production being about 1% of the dry weight of the cells. Spores did not appear, however, until after 48 hr of growth (delineated by the dashed line in the figure) and after the polymer in the cells was being rapidly utilized. At 59 hr there was greater than 9% spores in the culture. Although under these conditions polymer utilization occurred after peak growth, as in the previous experiment Downloaded from on October 15, 218 by guest

4 4 SLEPECKY AND LAW [VOL. 82 J I - N -J MD IL i C,) I-- 5 z '.-- w I 3 TIME, HOURS FIG. 4. Growth and polymer production for Bacillus megaterium, the sporogenous strain, in 4 liters of modified defined medium. Dashed line indicates spores in the culture starting at 48 hr, X = gravimetric determinations of polymer. poly-3-hydroxybutyric acid utilization preceded sporulation. DISCUSSION There are many early studies indicating the appearance and disappearance of lipid inclusions in cells preceding sporulation (Lewis, 1934; Knaysi, 1944). In recent studies by Young and Fitz-James (1959a, b) cytological changes during the growth and sporulation of B. cereus and B. cereus var. alesti were determined by phase contrast, brightfield, and electron microscopy. Although these studies were concerned primarily with the origin of the nuclear body of the spore and with the formation of protein inclusions, some attention was paid to the lipid inclusions. In those organisms lipid inclusions persisted in the spore up to the formation of the prerefractile spore. After this point lipid inclusions were absent. In the present study only three morphological stages as seen in the phase contrast microscope were used-vegetative cells; sporangia, cells containing a refractile body; and refractile spores. Lipid inclusions, presumably mostly poly-/3- hydroxybutyric acid (Lemoigne, 1927), were seen only in cells without spores. This was confirmed by chemical evidence which showed a decrease in polymer in the suspensions upon appearance of sporangia in the culture. No polymer was found in the spores in agreement with Tinelli (1955a); however, the possibility remains that polymer may be present in extremely small quantities. Although there have been many studies concerned with the occurrence of polymer in the genus Bacillus (Lemoigne, 1927; Lemoigne, Delaporte, and Croson, 1944; Weibull, 1953) and its role as a carbon and energy source in this genus (Macrae and Wilkinson, 1958a), these investigations, with the exception of the work of Tinelli (1955a, b), were performed either with asporogenous organisms or without consideration of the sporulation process. Tinelli found that complete oxidation of polymer occurred only in conjunction with sporulation, and deduced that the two processes were intimately connected. The present studies differ from Tinelli's in that polymer production and utilization during sporu- '43 1-J Downloaded from on October 15, 218 by guest

5 1961] POLY-3-HYDROXYBUTYRATE AND SPORULATION lation was followed over the complete growth cycle, whereas in the earlier studies growing cells were washed and resuspended in fresh medium without glucose or nitrate. Tinelli's experiments were essentially endotrophic sporulation experiments. The accumulation of poly-13-hydroxybutyric acid in cells grown in the medium favorable to polymer production may simply be due to the inability of the cells to utilize the polymer. High glucose and acetate concentrations, although favorable for good polymer production, will also lead to acid conditions in the medium. Macrae and Wilkinson (1958b) showed that both synthesis and breakdown of polymer have a sharp optimum around ph 7.5. Under favorable polymer producing conditions very few spores have been obtained and polymer was slowly utilized, but under poor polymer producing conditions, good crops of spores were obtained after rapid utilization of the polymer. This would tend to favor the hypothesis that the utilization of the polvmer is directly concerned in the sporulation process. In the Biogen experiments in which polymer production and sporulation were high, vigorous aeration and agitation not only would aid in the metabolism of polymer but also would prevent the appearance of acidic products of metabolism. The results in this paper suggest that if the bacteria are fortified with polymer or conditions are not favorable for the rapid utilization of polymer, the cells slowly utilize the endogenous polymer as a reserve energy and carbon source and are not prone to sporulate; however, if the polymer is in low supply or conditions for its rapid utilization are favorable, the organisms rapidly utilize this source of endogenous nutrient in the energy requiring sporulation process. Not all spore formers make polymer (Lemoigne et al., 1944); therefore another alternative is that the polymer is not directly concerned with sporulation but it is a ready source of carbon and energy in those species producing it. It would be expected that during the energy demanding process of sporulation available compounds would be metabolized. Perhaps an analogous situation is that found in the study of bacitracin biosynthesis and spore formation in Bacillus licheniformis in which bacitracin was utilized during sporulation (Bernlohr and Novelli, 196). Although rapid polymer utilization is observed in sporulating organisms, there is no evidence to suggest that a polymer reserve is a mandatory prerequisite for sporulation. Rather it appears that polymer can serve, if present, as an endogenous carbon and energy source to fuel the sporulation process. ACKNOWLEDGMENTS This work was supported by grants from the National Science Foundation (NSF G7597; G152) and the U. S. Public Health Service (RG787). LITERATURE CITED BERNLOHR, R. W., AND G. D. NOVELLI Uptake of bacitracin by sporangia and its incorporation into the spores of B. licheniformis. Biochim. et Biophys. Acta 41: DOUDOROFF, M., AND R. Y. STANIER Role of poly-,3-hydroxybutyric acid in the assimilation of organic carbon by bacteria. Nature 183: KNAYSI, G Elements of bacterial cytology. Comstock Publishing Co., Inc., Ithaca, N. Y. LAW, J. H., AND R. A. SLEPECKY Assay of poly-3-hydroxybutyric acid. J. Bacteriol. 82: LEMOIGNE, M etudes sur l'autolyse microbienne. Origine de l'acide f-oxy-butyrique forme par autolyse. Ann. inst. pasteur 41: LEMOIGNE, M., B. DELAPORTE, AND M. CROSON Contribution a l'6tude botanique et biochimique des bacteries du genre Bacillus. 2. Valeur du test des lipides,b-hydroxybutyrique pour la characterisation des especes. Ann. inst. Pasteur 7: LEWIS, I. M Cell inclusions and endospore formation in Bacillus mycoides. J. Bacteriol. 28: MACRAE, R. M., AND J. F. WILKINSON. 1958a. Poly-,3-hydroxybutyrate metabolism in washed suspensions of Bacillus cereus and Bacillus megaterium. J. Gen. Microbiol. 19: MACRAE, R. M., AND J. F. WILKINSON. 1958b. The influence of cultural conditions on poly-(3- hydroxybutyrate synthesis in Bacillus megaterium. Proc. Roy. Soc. Edinburgh 27: SLEPECKY, R. A., AND J. W. FOSTER Alterations in metal content of spores of Bacillus megaterium and the effect on some spore properties. J. Bacteriol. 78: SLEPECKY, R. A., AND J. H. LAW A rapid 41 spectrophotometric acids and 3-hydroxy acids. Anal. Chem. 32: assay of a,,b-unsaturated Downloaded from on October 15, 218 by guest

6 42 SLEPECKY AND LAW [VOL. 82 STANIER, R. Y., M. DOUDOROFF, R. KUNISAWA, AND R. CONTOPOULOU The role of organic substrates in bacterial photosynthesis. Proc. Natl. Acad. Sci. U. S. 45: TINELLI, R. 1955a. Etude de la biochimie de la sporulation chez Bacillus megaterium. I. Composition des spores obtenues par carence des differents substrats carbones. Ann. inst. Pasteur 88: TINELLI, R. 1955b..tude de la biochimie de la sporulation chez Bacillus megaterium. II. Modifications biochimiques et echanges gazeux accompagnant la sporulation provoquee par carence de glucose. Ann. Inst. Pasteur 88: WEIBULL, C Characterization of the protoplasmic constituents of Bacillus megaterium. J. Bacteriol. 66: WILLIAMSON, D. M., AND J. F. WILKINSON The isolation and estimation of poly-13-hydroxybutyrate inclusions of Bacillus species. J. Gen. Microbiol. 19: YOUNG, I. E., AND P. C. FITZ-JAMES. 1959a. Chemical and morphological studies of bacterial spore formation. I. The formation of spores in Bacillus cereus. J. Biophys. Biochem. Cytol. 6: YOUNG, I. E., AND P. C. FITZ-JAMES. 1959b. Chemical and morphological studies of bacterial spore formation. II. Spore and parasporal protein formation in Bacillus cereus var. alesti. J. Biophys. Biochem. Cytol. 6: Downloaded from on October 15, 218 by guest