JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 843-849 Copyright C) 1977 American Society for Microbiology
Vol. 129, No. 2 Printed in U.S.A.
Evidence for Stable Messenger Ribonucleic Acid During Sporulation and Enterotoxin Synthesis by Clostridium perfringens Type A RONALD G. LABBE' AND CHARLES L. DUNCAN* Food Research Institute, Department ofFood Microbiology and Toxicology and Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 20 September 1976
Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens. When used at a concentration that inhibited [14C]uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amino acids by 3-h sporulating cells. At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation. When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence. Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat. The half-life of enterotoxin RNA was estimated to be at least 58 min. When cell extracts from 5h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained. The two peaks corresponded to enterotoxin and another spore coat protein(s). Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA. The presence of stable messenger ribonucleic acid (mRNA) in eucaryotic cells has been known for some time (15). In the case of bacterial systems, mRNA has a normal half-life of about 2 min (20, 22, 29). However, a role for long-lived mRNA in bacteria has been proposed for penicillinase production in Bacillus cereus (13, 28) and for the syntheses of flagellin in Bacillus subtilis (25) and of outer membranes of Escherichia coli minicells (23). There also have been numerous but conflicting reports regarding the existence of stable mRNA by sporulating bacteria. Aronson and del Vallee first proposed that a stable messenger functioned during sporulation of B. cereus (2, 7). A similar conclusion was reached by Sterlini and Mandelstam with B. subtilis (33). The calcium transport system in B. cereus also seemed to be independent of continued mRNA synthesis (26). On the other hand, Leighton and Doi concluded that, in the case of B. subtilis, post-logarithmic mRNA was unstable (18, 19). A similar conclu-
sion was reached by other workers with the same species (3, 34). The involvement of long-lived mRNA(s) in sporulation could be clarified by looking at spore specific gene product(s). Chasin and Szulmajster estimated that, in B. subtilis, mRNA coding for dipicolinic acid synthetase had a half-life of 15 min (4), although no long-lived messenger coding for this enzyme was found in Bacillus sphaericus (35). Horn et al. (14) showed that rifampin blocked production of coat protein when added just prior to phase whitening. Petit-Glatron and Rapoport reported that the mRNA coding for the crystalline parasporal protein of Bacillus thuringiensis has a half-life of 10 min (27). The enterotoxin of Clostridium perfringens is another example of a sporulation-specific protein (11, 12). This, together with the observation that this toxin continues to accumulate after the majority of sporulating cells have become heat resistant (8), suggested that enterotoxin formation by C. perfringens could be an I Present address: Department of Food Science and Nu- example of a protein coded for by a stable trition, University of Massachusetts, Amherst, MA 01002. mRNA species. This article presents evidence 843
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LABBE AND DUNCAN
J. BACTERIOL.
membrane (Amicon Corp., Lexington, Mass.) and then dialyzed against distilled water for 16 h. Protein content and radioactivity of the concentrate were determined to obtain specific activity (counts per minute/microgram of enterotoxin protein). Disc gel electrophoresis. Polyacrylamide disc gel MATERIALS AND METHODS C. perfringens NCTC 8798 (Hobbs serotype 9) was electrophoresis was performed by the method of used throughout. Antiserum against highly purified Davis (6). Gel tubes were loaded in triplicate with enterotoxin was prepared as described by Frieben approximately 20 gg of affinity chromatographyand Duncan (11). Protein was determined by the purified enterotoxin or cell extract and run at 3 mA method of Lowry et al. (24). Percent sporulation was per tube with bromophenol blue as a tracking dye. obtained by a previously described method (16). Ri- The acrylamide-separating gel was used at a confampin was purchased from Calbiochem (Los Ange- centration of 7% with tris(hydroxymethyl)amiles, Calif.), and actinomycin D and raffinose were nomethane-glycine buffer, pH 9.5, as the running obtained from Sigma Chemical Co. (St. Louis, Mo.). buffer. After the electrophoretic run, one gel was Streptolydigin was a gift from G. Whitfield, (Upjohn stained with Coomassie brilliant blue R-250 (5) and Co., Kalamazoo, Mich.). Uniformly 3H-labeled scanned with white light using a Zeineh soft laser amino acids and Phase Combining System (PCS) scanning densitometer (Biomed Instruments, Inc., solubilizer were obtained from Amersham/Searle Chicago, Ill.). The second gel was sliced laterally at (Des Plaines, Ill.). Soluene 350 was obtained from 1.0-mm intervals with a repeating dispenser with Packard Instruments (Downers Grove, Ill.). Other plunger (MRA Corp., Boston, Mass.). Each slice was chemicals and radionuclides were from previously solubilized in a scintillation vial in the presence of 10 ml of a 5% solution of Soluene 350 in PCS solubilidescribed sources (17). a Macromolecular synthesis. Incorporation of la- zier. After 24 h, the vials were counted using third The counter. scintillation 2425 model Packard was uracil sporulation during or acids amino beled determined as previously described (17). The volume gel was subjected to disc immunoelectrophoresis as of Duncan and Strong (DS) sporulation medium (10) described by Stark and Duncan (32). routinely used was 25 ml (contained in a screw-cap RESULTS test tube [2.5 by 15.0 cm]) except as noted, in which case 50 ml of medium (in a 2.5- by 20.0-cm tube) was Effect of rifampin on uracil incorporation. employed. A 1% inoculum of an overnight culture of fluid thioglycollate medium (FTG) was used to inoc- One of the most direct methods of ascertaining ulate DS medium containing 0.4% raffinose in place the presence of stable mRNA during sporulaof starch. This carbohydrate improves the level of tion is to demonstrate the synthesis of sporulasporulation and enterotoxin formation in certain tion-associated protein(s) over an extended time period while preventing RNA synthesis, strains of C. perfringens (unpublished data). Radioactively labeled toxin was prepared as fol- specifically mRNA. The antibiotic rifampin can lows. A 50-ml portion of a 5-h sporulating culture be used for this purpose since it is a selective was treated for 10 min with a tritiated amino acid inhibitor of deoxyribonucleic acid (DNA)-demixture (final concentration, 1 ,uCi/ml). The culture was then poured over crushed 0.1% peptone (Difco, pendent RNA polymerase (21, 36). Figure 1 Detroit, Mich.)-ice and centrifuged 10,000 x g for 20 shows the inhibitory effect of this antibiotic on min, washed once with cold 0.1% peptone (Difco), incorporation of [14C]uracil by cells that had and centrifuged at 10,000 x g for 30 min. The pellet been growing in DS sporulation medium for 5 was suspended in 3 ml of cold distilled water. The h. Such cells were primarily at stage IV or V of cells, contained in a 15-ml polycarbonate centrifuge sporulation. Three-hour-old cells (primarily tube, were then sonically treated until at least 95% stage II and III) were similarly inhibited by of the sporangia had been disrupted and their spores rifampin (data not shown). The effectiveness of had been released. Cooling was effected by immer- this antibiotic was also confirmed again at the sion of the tube in an ice-water bath during sonic insure treatment. After breakage of the cells, the tube was end of the subsequent experiments tohad not centrifuged at 15,000 x g for 20 min, and the super- that the response of the culture natant fluid was removed and frozen until required changed. As determined by plate count methfor enterotoxin purification by affinity chroma- ods, rifampin at the concentrations used was not lethal to the cells after exposure for up to 60 tography. Affinity chromatography. Labeled enterotoxin in min. cell extracts was purified by affinity chromatograEffect of rifampin on protein and enterophy using the method of Scott and Duncan (30). A toxin synthesis. Having established that ri40-mg portion of anti-enterotoxin immunoglobulin fampin prevented RNA synthesis and was not was coupled to 1.6 g of CnBr-Sepharose (Pharmacia lethal to the cells, we then looked at protein Fine Chemicals, Uppsala, Sweden) for preparation of the affinity matrix. Toxin-containing fractions synthesis in the presence of this antibiotic. were identified by immunodiffusion as previously Such synthesis could be coded for by residual described (32). Positive fractions were concentrated mRNA. Figure 2 shows the incorporation of a to about 2 ml by ultrafiltration on a Diaflo PM10 14C-labeled amino acid mixture by 3-h and 5-h
that such a situation exists in this organism, and that the synthesis of not only enterotoxin but also another spore coat protein(s) may involve the functioning of a stable mRNA.
C. PERFRINGENS STABLE mRNA
VOL. 129, 1977
14
12
control
IC -a
0
6
+ RIF
A*~~~~ *
*
*
1S
20
~~*
~~*1 --
m
5
10
25
30
90
TIME (min)
FIG. 1. Rifampin (RIF) inhibition of [14C]uracil incorporation by 5-h sporulating cells. Rifampin (40 pg/mi), 2-deoxyadenosine (200 pg/mi), and labelI (0.3 ,uCilml) were added at zero time (after 5 min in DS sporulation medium). Vitamin-free Casamino Acids (4 mg/ml) was added 2 min prior to other components to enhance uracil incorporation.
The high zero-time incorpora I
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ment of a stable mRNA during sporulation by the organism. One significant difference between sporulating cells obtained at 3 h and at 5 h is the presence of large quantities of enterotoxin in the extracts of 5-h cells. Toxin is first detected in cell extracts of about 3-h-old cultures; by 5 h the level of toxin has increased tremendously, and it continues to accumulate in the sporangium until lysis occurs (8). Thus, we postulated that enterotoxin could be a specific protein coded for by stable mRNA and decided to investigate directly the synthesis of radioactive enterotoxin in the presence of rifampin. Labeled toxin was obtained by exposing 5-h sporulating cells to a mixture of labeled amino acids with or without rifampin. The newly synthesized radioactive enterotoxin was purified by affinity chromatography. Using this technique, homogeneous purified enterotoxin can be obtained as a single protein peak (staining for protein) with the radioactivity peak and the immunologically homogeneous material at the same position. Affinity chromatography was subsequently used to assess the extent of toxin synthesis in the presence of rifampin. Figure 3 shows that rifampin slightly decreased but did not prevent
cont rol
sporulating cells. tion of labeled amino acids by 3- and 5-h sporu lating cells mentioned in a previous report (17 was reduced by adding Casamino Acids 2 mir prior to label. Incorporation of amino acid labe ceased between 10 and 15 min after exposure ol 3 hr 3-h cells to rifampin. On the other hand, 5-E f +~~~~~~~~~~~ +RIF * * S~ ~ ~ ~ ~ ~ ~ ~3hr cells continued to synthesize protein in th( presence of rifampin during the 30-min obser j00 vation period, although at a reduced rate ac compared to the non-rifampin-containing con * trol. Since mRNA normally has a reported half L life of about 2 min, less than 1% of the mRNA present at zero time would remain 20 min aftei initial inhibition. Although some incorporatior of label occurred during the first 5 min, chlor amphenicol was effective in preventing genera r protein synthesis. Results similar to those obtained with rifam pin were obtained using streptolydigin, also ar'I inhibitor of RNA polymerase (31). When added TIME (min) together with labeled amino acids, this anti FIG. 2. Effect of rifampin (RIF; 40 pg/ml) and biotic, like rifampin, did not completely inhibii chloramphenicol (CAP; 150 pg/mi) on incorporation protein synthesis by 5-h cells during a 30-mir of a "IC-labeled amino acid mixture (0.2 Ci/ml) by 3-h and 5-h sporulating cells. Casamino Acids (4 period (data not shown). These results strongly suggested the involve mg/ml) was added 2 min before label. I
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LABBE AND DUNCAN
J. BACTERIOL.
z 0 I-
Um ML
*
*
*
+cap
TIME (min)
FIG. 3. Enterotoxin synthesis by 5-h sporulating cells in the presence of rifampin (rif; 40 pg/ml) or chloramphenicol (cap; 150 pg/ml). Tritiated enterotoxin was purified by affinity chromatography from cell extracts of 50 ml of culture. Antibiotics and tritiated amino acid mixture were added to 5-h cells (zero time).
enterotoxin synthesis. Chloramphenicol did prevent enterotoxin synthesis. This suggested direct involvement of a stable mRNA in C. perfringens enterotoxin formation. Half-life of enterotoxin mRNA. The approach we took to estimate the half-life of enterotoxin mRNA was to measure radioactive toxin
formation by 5-h cells that had been preexposed to rifampin for 0, 30, 60, and 90 min before addition of labeled amino acids. The specific activity (counts per minute per microgram of
toxin) of toxin obtained decreased with increasing lengths of time of exposure of cells to rifampin, with a half-life of approximately 58 min (Fig. 4). Five-hour cells that had been pretreated with rifampin for 90 min before the addition of label were in effect 6.5-h-old cells. Untreated 6.5-h cells normally incorporate, within 10 min, about half the amount of labeled amino acids as 5-h cells (data not shown). Assuming that label incorporation into enterotoxin decreases proportionally to total incorporation, then, if incorporation levels had been similar between 5- and 6.5-h cells, the specific activity of the synthesized toxin (Fig. 4) would
have decreased even less during the 90-min observation periods. This would have resulted in a much longer half-life for mRNA. However, enterotoxin continues to accumulate during this time period, although at a reduced rate (8). In this case, 58 min would be an overestimate. These opposing factors could cancel each other. Thus, 58 min is likely an accurate estimation of mRNA half-life. Characterization of proteins synthesized in vitro by stable mRNA. The presence of a relatively stable mRNA for synthesis of enterotoxin does not preclude the existence of other stable messenger species. Figure 5 shows that in the absence of rifampin two distinct labeled peaks were formed by 5-h cells labeled for 10 min with an amino acid mixture. Including rifampin for 1 h before labeling gave essentially the same pattern (Fig. 5B) as without the antibiotic, indicating the involvement of stable mRNA in the synthesis of the major and minor peak of radioactively labeled protein. The Rf (mobility relative to the migration of bromophenol blue) of the minor peak (0.51) is in agreement with the Rf of the precipitin line obtained by gel immunodiffusion of 5-h cell extracts against anti-enterotoxin sera. Thus the minor peak of radioactivity is toxin. The Rf (0.83) of the major peak of radioactivity corresponds to the Rf of
mRNA half life:
58 min
60
Z 0 I-.
TIME OF PREEXPOSURE TO RIFAMPIN (min)
FIG. 4. Decay of enterotoxin messenger RNA activity. Cultures from 50 ml of 5-h sporulating cells were exposed to rifampin (40 pg/ml) for varying periods before the addition of a 3H-labeled amino acid mixture (final concentrations 1 p]CiIml) for 10 min. Labeled enterotoxin was purified from cell extracts and counted.
VOL. 129, 1977
C. PERFRINGENS STABLE mRNA
A 6
4
2
+
10
30
20
GEL
SLICE
40-
NO.
FIG. 5. Radioactive gel profile from cell extracts of 5-h sporulating cells. (A) culture labeled for 10 min with 3H-amino acid mixture (1 CXiml); (B) culture exposed to rifampin (40 pg/ml) for 60 min before addition oflabel; and (C) culture exposed to rifampin for 60 min before addition oflabel and chloramphenicol (300 pg/ml).
of five precipitin bands obtained by gel immunodiffusion of 5-h cell extracts against antisera prepared using spore coat protein extracted from whole spores (data not shown). Thus the major peak of Fig. 5A and B probably represents coat protein and may indicate a role for stable mRNA in general coat protein synthesis. Chloramphenicol added together with labeled amino acids partially inhibited synthesis of the major protein peak of radioactivity and eliminated the minor peak (Fig. 50). Older sporulating cells of some Bacillus species are known to become increasingly resistant to chloramphenicol (2), which could account for continued synthesis of some protein in the presone
of the antibiotic. This may also be the case in C. perfringens.
ence
DISCUSSION Results reported here strongly suggest that stable mRNA is involved in synthesizing enterotoxin and spore coat protein(s). Rifampin was effective in preventing RNA synthesis and pre-
847
vented prolonged (greater than 10 to 15 min) protein synthesis by 3-h but not by 5-h sporulating cells. If one assumes that continued protein synthesis was due to residual mRNA, then long-lived messenger(s) must be functioning during the late stages of sporulation by C. perfringens. Apparently this is not the case with 3h cells. The differences between 3-h and 5-h cells can perhaps be explained if one considers the major biosynthetic events occurring at these two times. Three-hour cells (primarily stage II and III) are probably synthesizing a variety of proteins, only a few of which are sporulation specific. By 5 h (primarily stage IV and V), it is likely that the sporulating cell is synthesizing spore products, e.g., enzymes involved in dipicolinic acid and perhaps cortex synthesis. A major spore-specific protein at this time would also be coat protein. Frieben and Duncan (11) found that 20 to 30% of the total spore protein of C. perfringens could be solubilized by methods known to remove spore coat. From the results reported here, it appears that biosynthesis of coat protein is coded for by stable mRNA. Whether synthesis of all spore coat proteins involves stable mRNA is not clear at this time. Since C. perfringens enterotoxin has been shown to be a structural component of spore coat (11), our finding that it is probably coded for by a long-lived messenger is not unusual and might be expected. B. thuringiensis is similar to C. perfringens in that it is a spore former that produces a parasporal crystalline protein structurally similar to its coat protein. However, the exact relationship between the paracrystalline inclusions produced by C. perfringens (9) and coat protein has yet to be determined. The mRNA coding for B. thuringiensis crystal formation is long lived, with a half-life of 10 min. We found that enterotoxin mRNA had a minimum half-life of 58 min. Dipicolinic acid synthetase mRNA of B. subtilis was reported to have a half-life of 15 min (4). The previous longest estimated mRNA half-life reported in bacterial systems is 40 to 80 min from E. coli minicells (23). Considering that the normal half-life of mRNA is about 2 min, it is obvious that degradation of messenger is effected by different mechanisms in these various systems. Aronson showed that stable mRNA in sporulating B. cereus was tightly bound to cytoplasmic membrane and that membrane-bound polysomes from sporulating cells were more resistant to ribonuclease than those from young cells (1). Similarly, Petit-Glatron and Rapoport reported that the stable mRNA coding for B. thuringiensis crystalline parasporal protein produced, in the presence of rifampin, crystal
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LABBE AND DUNCAN
proteins that appeared to be attached to membrane fragments (27). The complexing of sporulation mRNA with membrane-associated polysomes may confer varying degrees of stability to the messenger. It must be noted that this same membrane-polysome-mRNA association may result in a compartmentalization effect, which could allow continued turnover of uridine into an unstable mRNA fraction that is refractory to rifampin action. The question of involvement of stable mRNA in sporulation has been controversial. It is highly probable that stable messenger codes for both enterotoxin, which is a defined spore coat structural protein, and for other spore coat protein(s) in C. perfringens. This may imply that such coat proteins are similarly coded for by stable messenger in other sporulating organisms. The recent evidence (18) against stable mRNA during sporulation in B. subtilis appears convincing for the gene products that were examined. However, in that study latestage sporulation gene products were not examined. Instead, only extracellular proteolytic activity and alkaline phosphatase activity were tested, and these were found to require continual synthesis of mRNA. If continued synthesis of coat proteins in the presence of rifampin were looked for; stable mRNA could conceivably be found. The development of spore refractility was also found to require continual synthesis of mRNA in B. subtilis (18). This may not be surprising, since several gene products may be necessary for development of refractility, not all of which would involve stable mRNA. From our results, it would appear that functional stable mRNA must be considered to achieve an understanding of developmental regulation in C. perfringens and possibly other spore-forming bacteria. ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, by research grant FD-00203-06 from the Food and Drug Administration, by Public Health Service research grant AI-11865-06 from the National Institute of Allergy and Infectious Diseases, and by contributions to the Food Research Institute by member industries. C.L.D. is the recipient of Public Health Service Research Career Development Award AI-70721-03 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Aronson, A. 1965. Membrane bound messenger RNA and polysomes in sporulating bacteria. J. Mol. Biol.
13:92-104. 2. Aronson, A., and M. del Vallee. 1964. RNA and protein synthesis required for bacterial spore formation. Biochim. Biophys. Acta 87:267-276. 3. Balassa, G. 1966. Synthese et fonction des ARN messa-
J. BACTERIOL. gers au cours de la sporulation de Bacillus subtilis. Ann. Inst. Pasteur (Paris) 110:175-191. 4. Chasin, L., and J. Szulmajster. 1969. Enzymes of dipicolinic acid biosynthesis in Bacillus subtilis, p. 133167. In L. Campbell (ed.), Spores IV. American Society for Microbiology, Bethesda, Md. 5. Chrambach, A., R. Reisfeld, M. Wyckoff, and J. Zaccan. 1967. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal. Biochem. 20:150-154. 6. Davis, B. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121:404-427. 7. del Vallee, M., and A. Aronson. 1962. Evidence for the synthesis of stable informational RNA required for bacterial spore formation. Biochem. Biophys. Res. Commun. 9:421-425. 8. Duncan, C. 1973. Time of enterotoxin formation and release during sporulation ofClostridium perfringens type A. J. Bacteriol. 113:932-936. 9. Duncan, C., G. King, and W. Frieben. 1973. A paracrystalline inclusion formed during sporulation of enterotoxin-producing strains of Clostridium perfringens type A. J. Bacteriol. 114:845-859. 10. Duncan, C., and D. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82-89. 11. Frieben, W., and C. Duncan. 1973. Homology between enterotoxin protein and spore structural protein in Clostridium perfringens type A. Eur. J. Biochem. 39:393-401. 12. Frieben, W., and C. Duncan. 1975. Heterogeneity of enterotoxin-like protein extracted from spores of Clostridium perfringens type A. Eur. J. Biochem. 55:455-463. 13. Harris, H., and L. Sabath. 1964. Induced enzyme synthesis in the absence of concomitant ribonucleic acid synthesis. Nature (London) 202:1078-1080. 14. Horn, D., A. Aronson, and S. Golub. 1974. Development of a quantitative immunological assay for the study of spore coat synthesis and morphogenesis. J. Bacteriol. 113:313-321. 15. Kafatos, F., and R. Gelinas. 1974. mRNA stability and the control of specific protein synthesis in highly differentiated cells, p. 223. In J. Paul (ed.), Biochemistry of cell differentiation, vol. 9. Butterworths, London. 16. Labbe, R., and C. Duncan. 1974. Sporulation and enterotoxin production by Clostridium perfringens type A under conditions of controlled pH and temperature. Can. J. Microbiol. 20:1493-1501. 17. Labbe, R., and C. Duncan. 1976. Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens. J. Bacteriol. 125:444 452. 18. Leighton, T. 1974. Further studies on the stability of sporulation messenger ribonucleic acid in Bacillus subtilis. J. Biol. Chem. 249.7808-7812. 19. Leighton, T., and R. Doi. 1971. The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J. Biol. Chem. 246:3189-3195. 20. Leive, L. 1965. RNA degradation and the assembly of ribosomes in actinomycin treated Ewcherichia coli. J. Mol. Biol. 13:862-875. 21. Lester, W. 1972. Rifampin: a semisynthetic derivative of rifamycin-a prototype for the future. Annu. Rev. Microbiol. 26:85-102. 22. Levinthal, C., A. Keynan, and A. Higa. 1962. Messenger RNA turnover and protein synthesis in Bacillus subtilis inhibited by actinomycin D. Proc. Natl. Acad. Sci. U.S.A. 48:1631-1638. 23. Levy, S. 1975. Very stable procaryotic messenger RNA in chromosomeless Escherichia coli minicells. Proc.
VOL. 129, 1977 Natl. Acad. Sci. U.S.A. 72:2900-2904. 24. Lowry, O., N. Rosebrough, A. Farr, and R. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 25. Martinez, R. 1966. The formation of bacterial flagella. II. The relative stability of messenger RNA for flagellin 1iosynthesis. J. Mol. Biol. 17:10-17. 26. Pearce, S., and P. Fitz-James. 1971. Spore refractility in variants of Bacillus cereus treated with actinomycin D. J. Bacteriol. 107:337-344. 27. Petit-Glatron, M., and G. Rapoport. 1975. In vivo and in vitro evidence for the existence df stable messenger ribonucleic acids in sporulating cells ofBacillus thuringiensis, p. 255-264. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. 28. Pollock, M. 1963. The differential effect of actinomycin D on the biosynthesis of enzymes in Bacillus subtilis andBacillus cereus. Biochim. Biophys. Acta 76:80-93. 29. Schaechter, M., E. Previc, and M. Gillespie. 1965. Messenger RNA and polyribosomes in Bacillus megaterium. J. Mol. Biol. 12:119-129.
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30. Scott, V., and C. Duncan. 1975. Affinity chromatography purification of Clostridium perfringens enterotoxin. Infect. Immun. 12:536-543. 31. Siddhiko, C., J. Erbstoeszer, and B. Weisblum. 1969. Mode of action of streptolydigin. J. Bacteriol. 99:151155. 32. Stark, R., and C. Duncan. 1971. Biological characteristics of Clostridium perfringens type A enterotoxin. Infect. Immun. 4:89-96. 33. Sterlini, J., and J. Mandelstam. 1969. Commitment to sporulation in Bacillus subtilis and its relationship to development of actinomycin resistance. Biochem. J. 113:29-37. 34. Szulmajster, J., R. Canfield, and J. Blicharska. 1963. Action de l'actinomycine D sur la sporulation de Bacillus subtilis. C. R. Acad. Sci. 256:2057-2060. 35. Tipper, D., and I. Pratt. 1970. Cell wall polymers of Bacillus sphaericus 9602. II. Synthesis of the first enzyme unique to cortex synthesis during sporulation. J. Bacteriol. 103:305-317. 36. Wehrli, W., and M. Staehelin. 1971. Actions of the rifamycins. Bacteriol. Rev. 35:290-309.
Vol. 129, No. 2 Printed in U.S.A.
Evidence for Stable Messenger Ribonucleic Acid During Sporulation and Enterotoxin Synthesis by Clostridium perfringens Type A RONALD G. LABBE' AND CHARLES L. DUNCAN* Food Research Institute, Department ofFood Microbiology and Toxicology and Department ofBacteriology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 20 September 1976
Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens. When used at a concentration that inhibited [14C]uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amino acids by 3-h sporulating cells. At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation. When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence. Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat. The half-life of enterotoxin RNA was estimated to be at least 58 min. When cell extracts from 5h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained. The two peaks corresponded to enterotoxin and another spore coat protein(s). Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA. The presence of stable messenger ribonucleic acid (mRNA) in eucaryotic cells has been known for some time (15). In the case of bacterial systems, mRNA has a normal half-life of about 2 min (20, 22, 29). However, a role for long-lived mRNA in bacteria has been proposed for penicillinase production in Bacillus cereus (13, 28) and for the syntheses of flagellin in Bacillus subtilis (25) and of outer membranes of Escherichia coli minicells (23). There also have been numerous but conflicting reports regarding the existence of stable mRNA by sporulating bacteria. Aronson and del Vallee first proposed that a stable messenger functioned during sporulation of B. cereus (2, 7). A similar conclusion was reached by Sterlini and Mandelstam with B. subtilis (33). The calcium transport system in B. cereus also seemed to be independent of continued mRNA synthesis (26). On the other hand, Leighton and Doi concluded that, in the case of B. subtilis, post-logarithmic mRNA was unstable (18, 19). A similar conclu-
sion was reached by other workers with the same species (3, 34). The involvement of long-lived mRNA(s) in sporulation could be clarified by looking at spore specific gene product(s). Chasin and Szulmajster estimated that, in B. subtilis, mRNA coding for dipicolinic acid synthetase had a half-life of 15 min (4), although no long-lived messenger coding for this enzyme was found in Bacillus sphaericus (35). Horn et al. (14) showed that rifampin blocked production of coat protein when added just prior to phase whitening. Petit-Glatron and Rapoport reported that the mRNA coding for the crystalline parasporal protein of Bacillus thuringiensis has a half-life of 10 min (27). The enterotoxin of Clostridium perfringens is another example of a sporulation-specific protein (11, 12). This, together with the observation that this toxin continues to accumulate after the majority of sporulating cells have become heat resistant (8), suggested that enterotoxin formation by C. perfringens could be an I Present address: Department of Food Science and Nu- example of a protein coded for by a stable trition, University of Massachusetts, Amherst, MA 01002. mRNA species. This article presents evidence 843
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LABBE AND DUNCAN
J. BACTERIOL.
membrane (Amicon Corp., Lexington, Mass.) and then dialyzed against distilled water for 16 h. Protein content and radioactivity of the concentrate were determined to obtain specific activity (counts per minute/microgram of enterotoxin protein). Disc gel electrophoresis. Polyacrylamide disc gel MATERIALS AND METHODS C. perfringens NCTC 8798 (Hobbs serotype 9) was electrophoresis was performed by the method of used throughout. Antiserum against highly purified Davis (6). Gel tubes were loaded in triplicate with enterotoxin was prepared as described by Frieben approximately 20 gg of affinity chromatographyand Duncan (11). Protein was determined by the purified enterotoxin or cell extract and run at 3 mA method of Lowry et al. (24). Percent sporulation was per tube with bromophenol blue as a tracking dye. obtained by a previously described method (16). Ri- The acrylamide-separating gel was used at a confampin was purchased from Calbiochem (Los Ange- centration of 7% with tris(hydroxymethyl)amiles, Calif.), and actinomycin D and raffinose were nomethane-glycine buffer, pH 9.5, as the running obtained from Sigma Chemical Co. (St. Louis, Mo.). buffer. After the electrophoretic run, one gel was Streptolydigin was a gift from G. Whitfield, (Upjohn stained with Coomassie brilliant blue R-250 (5) and Co., Kalamazoo, Mich.). Uniformly 3H-labeled scanned with white light using a Zeineh soft laser amino acids and Phase Combining System (PCS) scanning densitometer (Biomed Instruments, Inc., solubilizer were obtained from Amersham/Searle Chicago, Ill.). The second gel was sliced laterally at (Des Plaines, Ill.). Soluene 350 was obtained from 1.0-mm intervals with a repeating dispenser with Packard Instruments (Downers Grove, Ill.). Other plunger (MRA Corp., Boston, Mass.). Each slice was chemicals and radionuclides were from previously solubilized in a scintillation vial in the presence of 10 ml of a 5% solution of Soluene 350 in PCS solubilidescribed sources (17). a Macromolecular synthesis. Incorporation of la- zier. After 24 h, the vials were counted using third The counter. scintillation 2425 model Packard was uracil sporulation during or acids amino beled determined as previously described (17). The volume gel was subjected to disc immunoelectrophoresis as of Duncan and Strong (DS) sporulation medium (10) described by Stark and Duncan (32). routinely used was 25 ml (contained in a screw-cap RESULTS test tube [2.5 by 15.0 cm]) except as noted, in which case 50 ml of medium (in a 2.5- by 20.0-cm tube) was Effect of rifampin on uracil incorporation. employed. A 1% inoculum of an overnight culture of fluid thioglycollate medium (FTG) was used to inoc- One of the most direct methods of ascertaining ulate DS medium containing 0.4% raffinose in place the presence of stable mRNA during sporulaof starch. This carbohydrate improves the level of tion is to demonstrate the synthesis of sporulasporulation and enterotoxin formation in certain tion-associated protein(s) over an extended time period while preventing RNA synthesis, strains of C. perfringens (unpublished data). Radioactively labeled toxin was prepared as fol- specifically mRNA. The antibiotic rifampin can lows. A 50-ml portion of a 5-h sporulating culture be used for this purpose since it is a selective was treated for 10 min with a tritiated amino acid inhibitor of deoxyribonucleic acid (DNA)-demixture (final concentration, 1 ,uCi/ml). The culture was then poured over crushed 0.1% peptone (Difco, pendent RNA polymerase (21, 36). Figure 1 Detroit, Mich.)-ice and centrifuged 10,000 x g for 20 shows the inhibitory effect of this antibiotic on min, washed once with cold 0.1% peptone (Difco), incorporation of [14C]uracil by cells that had and centrifuged at 10,000 x g for 30 min. The pellet been growing in DS sporulation medium for 5 was suspended in 3 ml of cold distilled water. The h. Such cells were primarily at stage IV or V of cells, contained in a 15-ml polycarbonate centrifuge sporulation. Three-hour-old cells (primarily tube, were then sonically treated until at least 95% stage II and III) were similarly inhibited by of the sporangia had been disrupted and their spores rifampin (data not shown). The effectiveness of had been released. Cooling was effected by immer- this antibiotic was also confirmed again at the sion of the tube in an ice-water bath during sonic insure treatment. After breakage of the cells, the tube was end of the subsequent experiments tohad not centrifuged at 15,000 x g for 20 min, and the super- that the response of the culture natant fluid was removed and frozen until required changed. As determined by plate count methfor enterotoxin purification by affinity chroma- ods, rifampin at the concentrations used was not lethal to the cells after exposure for up to 60 tography. Affinity chromatography. Labeled enterotoxin in min. cell extracts was purified by affinity chromatograEffect of rifampin on protein and enterophy using the method of Scott and Duncan (30). A toxin synthesis. Having established that ri40-mg portion of anti-enterotoxin immunoglobulin fampin prevented RNA synthesis and was not was coupled to 1.6 g of CnBr-Sepharose (Pharmacia lethal to the cells, we then looked at protein Fine Chemicals, Uppsala, Sweden) for preparation of the affinity matrix. Toxin-containing fractions synthesis in the presence of this antibiotic. were identified by immunodiffusion as previously Such synthesis could be coded for by residual described (32). Positive fractions were concentrated mRNA. Figure 2 shows the incorporation of a to about 2 ml by ultrafiltration on a Diaflo PM10 14C-labeled amino acid mixture by 3-h and 5-h
that such a situation exists in this organism, and that the synthesis of not only enterotoxin but also another spore coat protein(s) may involve the functioning of a stable mRNA.
C. PERFRINGENS STABLE mRNA
VOL. 129, 1977
14
12
control
IC -a
0
6
+ RIF
A*~~~~ *
*
*
1S
20
~~*
~~*1 --
m
5
10
25
30
90
TIME (min)
FIG. 1. Rifampin (RIF) inhibition of [14C]uracil incorporation by 5-h sporulating cells. Rifampin (40 pg/mi), 2-deoxyadenosine (200 pg/mi), and labelI (0.3 ,uCilml) were added at zero time (after 5 min in DS sporulation medium). Vitamin-free Casamino Acids (4 mg/ml) was added 2 min prior to other components to enhance uracil incorporation.
The high zero-time incorpora I
845
ment of a stable mRNA during sporulation by the organism. One significant difference between sporulating cells obtained at 3 h and at 5 h is the presence of large quantities of enterotoxin in the extracts of 5-h cells. Toxin is first detected in cell extracts of about 3-h-old cultures; by 5 h the level of toxin has increased tremendously, and it continues to accumulate in the sporangium until lysis occurs (8). Thus, we postulated that enterotoxin could be a specific protein coded for by stable mRNA and decided to investigate directly the synthesis of radioactive enterotoxin in the presence of rifampin. Labeled toxin was obtained by exposing 5-h sporulating cells to a mixture of labeled amino acids with or without rifampin. The newly synthesized radioactive enterotoxin was purified by affinity chromatography. Using this technique, homogeneous purified enterotoxin can be obtained as a single protein peak (staining for protein) with the radioactivity peak and the immunologically homogeneous material at the same position. Affinity chromatography was subsequently used to assess the extent of toxin synthesis in the presence of rifampin. Figure 3 shows that rifampin slightly decreased but did not prevent
cont rol
sporulating cells. tion of labeled amino acids by 3- and 5-h sporu lating cells mentioned in a previous report (17 was reduced by adding Casamino Acids 2 mir prior to label. Incorporation of amino acid labe ceased between 10 and 15 min after exposure ol 3 hr 3-h cells to rifampin. On the other hand, 5-E f +~~~~~~~~~~~ +RIF * * S~ ~ ~ ~ ~ ~ ~ ~3hr cells continued to synthesize protein in th( presence of rifampin during the 30-min obser j00 vation period, although at a reduced rate ac compared to the non-rifampin-containing con * trol. Since mRNA normally has a reported half L life of about 2 min, less than 1% of the mRNA present at zero time would remain 20 min aftei initial inhibition. Although some incorporatior of label occurred during the first 5 min, chlor amphenicol was effective in preventing genera r protein synthesis. Results similar to those obtained with rifam pin were obtained using streptolydigin, also ar'I inhibitor of RNA polymerase (31). When added TIME (min) together with labeled amino acids, this anti FIG. 2. Effect of rifampin (RIF; 40 pg/ml) and biotic, like rifampin, did not completely inhibii chloramphenicol (CAP; 150 pg/mi) on incorporation protein synthesis by 5-h cells during a 30-mir of a "IC-labeled amino acid mixture (0.2 Ci/ml) by 3-h and 5-h sporulating cells. Casamino Acids (4 period (data not shown). These results strongly suggested the involve mg/ml) was added 2 min before label. I
846
LABBE AND DUNCAN
J. BACTERIOL.
z 0 I-
Um ML
*
*
*
+cap
TIME (min)
FIG. 3. Enterotoxin synthesis by 5-h sporulating cells in the presence of rifampin (rif; 40 pg/ml) or chloramphenicol (cap; 150 pg/ml). Tritiated enterotoxin was purified by affinity chromatography from cell extracts of 50 ml of culture. Antibiotics and tritiated amino acid mixture were added to 5-h cells (zero time).
enterotoxin synthesis. Chloramphenicol did prevent enterotoxin synthesis. This suggested direct involvement of a stable mRNA in C. perfringens enterotoxin formation. Half-life of enterotoxin mRNA. The approach we took to estimate the half-life of enterotoxin mRNA was to measure radioactive toxin
formation by 5-h cells that had been preexposed to rifampin for 0, 30, 60, and 90 min before addition of labeled amino acids. The specific activity (counts per minute per microgram of
toxin) of toxin obtained decreased with increasing lengths of time of exposure of cells to rifampin, with a half-life of approximately 58 min (Fig. 4). Five-hour cells that had been pretreated with rifampin for 90 min before the addition of label were in effect 6.5-h-old cells. Untreated 6.5-h cells normally incorporate, within 10 min, about half the amount of labeled amino acids as 5-h cells (data not shown). Assuming that label incorporation into enterotoxin decreases proportionally to total incorporation, then, if incorporation levels had been similar between 5- and 6.5-h cells, the specific activity of the synthesized toxin (Fig. 4) would
have decreased even less during the 90-min observation periods. This would have resulted in a much longer half-life for mRNA. However, enterotoxin continues to accumulate during this time period, although at a reduced rate (8). In this case, 58 min would be an overestimate. These opposing factors could cancel each other. Thus, 58 min is likely an accurate estimation of mRNA half-life. Characterization of proteins synthesized in vitro by stable mRNA. The presence of a relatively stable mRNA for synthesis of enterotoxin does not preclude the existence of other stable messenger species. Figure 5 shows that in the absence of rifampin two distinct labeled peaks were formed by 5-h cells labeled for 10 min with an amino acid mixture. Including rifampin for 1 h before labeling gave essentially the same pattern (Fig. 5B) as without the antibiotic, indicating the involvement of stable mRNA in the synthesis of the major and minor peak of radioactively labeled protein. The Rf (mobility relative to the migration of bromophenol blue) of the minor peak (0.51) is in agreement with the Rf of the precipitin line obtained by gel immunodiffusion of 5-h cell extracts against anti-enterotoxin sera. Thus the minor peak of radioactivity is toxin. The Rf (0.83) of the major peak of radioactivity corresponds to the Rf of
mRNA half life:
58 min
60
Z 0 I-.
TIME OF PREEXPOSURE TO RIFAMPIN (min)
FIG. 4. Decay of enterotoxin messenger RNA activity. Cultures from 50 ml of 5-h sporulating cells were exposed to rifampin (40 pg/ml) for varying periods before the addition of a 3H-labeled amino acid mixture (final concentrations 1 p]CiIml) for 10 min. Labeled enterotoxin was purified from cell extracts and counted.
VOL. 129, 1977
C. PERFRINGENS STABLE mRNA
A 6
4
2
+
10
30
20
GEL
SLICE
40-
NO.
FIG. 5. Radioactive gel profile from cell extracts of 5-h sporulating cells. (A) culture labeled for 10 min with 3H-amino acid mixture (1 CXiml); (B) culture exposed to rifampin (40 pg/ml) for 60 min before addition oflabel; and (C) culture exposed to rifampin for 60 min before addition oflabel and chloramphenicol (300 pg/ml).
of five precipitin bands obtained by gel immunodiffusion of 5-h cell extracts against antisera prepared using spore coat protein extracted from whole spores (data not shown). Thus the major peak of Fig. 5A and B probably represents coat protein and may indicate a role for stable mRNA in general coat protein synthesis. Chloramphenicol added together with labeled amino acids partially inhibited synthesis of the major protein peak of radioactivity and eliminated the minor peak (Fig. 50). Older sporulating cells of some Bacillus species are known to become increasingly resistant to chloramphenicol (2), which could account for continued synthesis of some protein in the presone
of the antibiotic. This may also be the case in C. perfringens.
ence
DISCUSSION Results reported here strongly suggest that stable mRNA is involved in synthesizing enterotoxin and spore coat protein(s). Rifampin was effective in preventing RNA synthesis and pre-
847
vented prolonged (greater than 10 to 15 min) protein synthesis by 3-h but not by 5-h sporulating cells. If one assumes that continued protein synthesis was due to residual mRNA, then long-lived messenger(s) must be functioning during the late stages of sporulation by C. perfringens. Apparently this is not the case with 3h cells. The differences between 3-h and 5-h cells can perhaps be explained if one considers the major biosynthetic events occurring at these two times. Three-hour cells (primarily stage II and III) are probably synthesizing a variety of proteins, only a few of which are sporulation specific. By 5 h (primarily stage IV and V), it is likely that the sporulating cell is synthesizing spore products, e.g., enzymes involved in dipicolinic acid and perhaps cortex synthesis. A major spore-specific protein at this time would also be coat protein. Frieben and Duncan (11) found that 20 to 30% of the total spore protein of C. perfringens could be solubilized by methods known to remove spore coat. From the results reported here, it appears that biosynthesis of coat protein is coded for by stable mRNA. Whether synthesis of all spore coat proteins involves stable mRNA is not clear at this time. Since C. perfringens enterotoxin has been shown to be a structural component of spore coat (11), our finding that it is probably coded for by a long-lived messenger is not unusual and might be expected. B. thuringiensis is similar to C. perfringens in that it is a spore former that produces a parasporal crystalline protein structurally similar to its coat protein. However, the exact relationship between the paracrystalline inclusions produced by C. perfringens (9) and coat protein has yet to be determined. The mRNA coding for B. thuringiensis crystal formation is long lived, with a half-life of 10 min. We found that enterotoxin mRNA had a minimum half-life of 58 min. Dipicolinic acid synthetase mRNA of B. subtilis was reported to have a half-life of 15 min (4). The previous longest estimated mRNA half-life reported in bacterial systems is 40 to 80 min from E. coli minicells (23). Considering that the normal half-life of mRNA is about 2 min, it is obvious that degradation of messenger is effected by different mechanisms in these various systems. Aronson showed that stable mRNA in sporulating B. cereus was tightly bound to cytoplasmic membrane and that membrane-bound polysomes from sporulating cells were more resistant to ribonuclease than those from young cells (1). Similarly, Petit-Glatron and Rapoport reported that the stable mRNA coding for B. thuringiensis crystalline parasporal protein produced, in the presence of rifampin, crystal
848
LABBE AND DUNCAN
proteins that appeared to be attached to membrane fragments (27). The complexing of sporulation mRNA with membrane-associated polysomes may confer varying degrees of stability to the messenger. It must be noted that this same membrane-polysome-mRNA association may result in a compartmentalization effect, which could allow continued turnover of uridine into an unstable mRNA fraction that is refractory to rifampin action. The question of involvement of stable mRNA in sporulation has been controversial. It is highly probable that stable messenger codes for both enterotoxin, which is a defined spore coat structural protein, and for other spore coat protein(s) in C. perfringens. This may imply that such coat proteins are similarly coded for by stable messenger in other sporulating organisms. The recent evidence (18) against stable mRNA during sporulation in B. subtilis appears convincing for the gene products that were examined. However, in that study latestage sporulation gene products were not examined. Instead, only extracellular proteolytic activity and alkaline phosphatase activity were tested, and these were found to require continual synthesis of mRNA. If continued synthesis of coat proteins in the presence of rifampin were looked for; stable mRNA could conceivably be found. The development of spore refractility was also found to require continual synthesis of mRNA in B. subtilis (18). This may not be surprising, since several gene products may be necessary for development of refractility, not all of which would involve stable mRNA. From our results, it would appear that functional stable mRNA must be considered to achieve an understanding of developmental regulation in C. perfringens and possibly other spore-forming bacteria. ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, by research grant FD-00203-06 from the Food and Drug Administration, by Public Health Service research grant AI-11865-06 from the National Institute of Allergy and Infectious Diseases, and by contributions to the Food Research Institute by member industries. C.L.D. is the recipient of Public Health Service Research Career Development Award AI-70721-03 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Aronson, A. 1965. Membrane bound messenger RNA and polysomes in sporulating bacteria. J. Mol. Biol.
13:92-104. 2. Aronson, A., and M. del Vallee. 1964. RNA and protein synthesis required for bacterial spore formation. Biochim. Biophys. Acta 87:267-276. 3. Balassa, G. 1966. Synthese et fonction des ARN messa-
J. BACTERIOL. gers au cours de la sporulation de Bacillus subtilis. Ann. Inst. Pasteur (Paris) 110:175-191. 4. Chasin, L., and J. Szulmajster. 1969. Enzymes of dipicolinic acid biosynthesis in Bacillus subtilis, p. 133167. In L. Campbell (ed.), Spores IV. American Society for Microbiology, Bethesda, Md. 5. Chrambach, A., R. Reisfeld, M. Wyckoff, and J. Zaccan. 1967. A procedure for rapid and sensitive staining of protein fractionated by polyacrylamide gel electrophoresis. Anal. Biochem. 20:150-154. 6. Davis, B. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121:404-427. 7. del Vallee, M., and A. Aronson. 1962. Evidence for the synthesis of stable informational RNA required for bacterial spore formation. Biochem. Biophys. Res. Commun. 9:421-425. 8. Duncan, C. 1973. Time of enterotoxin formation and release during sporulation ofClostridium perfringens type A. J. Bacteriol. 113:932-936. 9. Duncan, C., G. King, and W. Frieben. 1973. A paracrystalline inclusion formed during sporulation of enterotoxin-producing strains of Clostridium perfringens type A. J. Bacteriol. 114:845-859. 10. Duncan, C., and D. Strong. 1968. Improved medium for sporulation of Clostridium perfringens. Appl. Microbiol. 16:82-89. 11. Frieben, W., and C. Duncan. 1973. Homology between enterotoxin protein and spore structural protein in Clostridium perfringens type A. Eur. J. Biochem. 39:393-401. 12. Frieben, W., and C. Duncan. 1975. Heterogeneity of enterotoxin-like protein extracted from spores of Clostridium perfringens type A. Eur. J. Biochem. 55:455-463. 13. Harris, H., and L. Sabath. 1964. Induced enzyme synthesis in the absence of concomitant ribonucleic acid synthesis. Nature (London) 202:1078-1080. 14. Horn, D., A. Aronson, and S. Golub. 1974. Development of a quantitative immunological assay for the study of spore coat synthesis and morphogenesis. J. Bacteriol. 113:313-321. 15. Kafatos, F., and R. Gelinas. 1974. mRNA stability and the control of specific protein synthesis in highly differentiated cells, p. 223. In J. Paul (ed.), Biochemistry of cell differentiation, vol. 9. Butterworths, London. 16. Labbe, R., and C. Duncan. 1974. Sporulation and enterotoxin production by Clostridium perfringens type A under conditions of controlled pH and temperature. Can. J. Microbiol. 20:1493-1501. 17. Labbe, R., and C. Duncan. 1976. Synthesis of deoxyribonucleic acid, ribonucleic acid, and protein during sporulation of Clostridium perfringens. J. Bacteriol. 125:444 452. 18. Leighton, T. 1974. Further studies on the stability of sporulation messenger ribonucleic acid in Bacillus subtilis. J. Biol. Chem. 249.7808-7812. 19. Leighton, T., and R. Doi. 1971. The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis. J. Biol. Chem. 246:3189-3195. 20. Leive, L. 1965. RNA degradation and the assembly of ribosomes in actinomycin treated Ewcherichia coli. J. Mol. Biol. 13:862-875. 21. Lester, W. 1972. Rifampin: a semisynthetic derivative of rifamycin-a prototype for the future. Annu. Rev. Microbiol. 26:85-102. 22. Levinthal, C., A. Keynan, and A. Higa. 1962. Messenger RNA turnover and protein synthesis in Bacillus subtilis inhibited by actinomycin D. Proc. Natl. Acad. Sci. U.S.A. 48:1631-1638. 23. Levy, S. 1975. Very stable procaryotic messenger RNA in chromosomeless Escherichia coli minicells. Proc.
VOL. 129, 1977 Natl. Acad. Sci. U.S.A. 72:2900-2904. 24. Lowry, O., N. Rosebrough, A. Farr, and R. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 25. Martinez, R. 1966. The formation of bacterial flagella. II. The relative stability of messenger RNA for flagellin 1iosynthesis. J. Mol. Biol. 17:10-17. 26. Pearce, S., and P. Fitz-James. 1971. Spore refractility in variants of Bacillus cereus treated with actinomycin D. J. Bacteriol. 107:337-344. 27. Petit-Glatron, M., and G. Rapoport. 1975. In vivo and in vitro evidence for the existence df stable messenger ribonucleic acids in sporulating cells ofBacillus thuringiensis, p. 255-264. In P. Gerhardt, R. N. Costilow, and H. L. Sadoff (ed.), Spores VI. American Society for Microbiology, Washington, D.C. 28. Pollock, M. 1963. The differential effect of actinomycin D on the biosynthesis of enzymes in Bacillus subtilis andBacillus cereus. Biochim. Biophys. Acta 76:80-93. 29. Schaechter, M., E. Previc, and M. Gillespie. 1965. Messenger RNA and polyribosomes in Bacillus megaterium. J. Mol. Biol. 12:119-129.
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30. Scott, V., and C. Duncan. 1975. Affinity chromatography purification of Clostridium perfringens enterotoxin. Infect. Immun. 12:536-543. 31. Siddhiko, C., J. Erbstoeszer, and B. Weisblum. 1969. Mode of action of streptolydigin. J. Bacteriol. 99:151155. 32. Stark, R., and C. Duncan. 1971. Biological characteristics of Clostridium perfringens type A enterotoxin. Infect. Immun. 4:89-96. 33. Sterlini, J., and J. Mandelstam. 1969. Commitment to sporulation in Bacillus subtilis and its relationship to development of actinomycin resistance. Biochem. J. 113:29-37. 34. Szulmajster, J., R. Canfield, and J. Blicharska. 1963. Action de l'actinomycine D sur la sporulation de Bacillus subtilis. C. R. Acad. Sci. 256:2057-2060. 35. Tipper, D., and I. Pratt. 1970. Cell wall polymers of Bacillus sphaericus 9602. II. Synthesis of the first enzyme unique to cortex synthesis during sporulation. J. Bacteriol. 103:305-317. 36. Wehrli, W., and M. Staehelin. 1971. Actions of the rifamycins. Bacteriol. Rev. 35:290-309.
