INFECTION AND IMMUNry, May 1977, p. 610-616 Copyright C) 1977 American Society for Microbiology

Vol. 16, No. 2 Printed in U.S.A.

Regulation of Staphylococcal Enterotoxin B1 JOHN J. IANDOLO* AND WILLIAM M. SHAFER Division of Biology, Microbiology Group, Kansas State University Manhattan, Kansas 66506 Received for publication 3 January 1977

The effect of glucose and the glucose analogues 2-deoxyglucose and a-methylglucoside on the synthesis and regulation of staphylococcal enterotoxin B was examined. The attenuating effect of glucose on staphylococcal enterotoxin B synthesis was observed. However, when this effect was examined with analogues of glucose, contradictory responses were seen. a-Methylglucoside had a slight stimulatory effect on enterotoxin production and other extracellular proteins, whereas 2-deoxyglucose markedly inhibited enterotoxin production. f3hemolysin and staphylococcal nuclease were also inhibited by 2-deoxyglucose, but the synthesis of nuclease could be rescued by the addition of glucose to 2deoxyglucose-containing cultures. Enterotoxin and f-hemolysin synthesis were not subject to glucose rescue. The cells used in this study were permeable to cyclic 3',5'-adenosine monophosphate, but the addition of this compound did not reverse glucose repression or 2-deoxyglucose inhibition of enterotoxin B synthesis. We conclude from these data that the regulation of enterotoxin is not under catabolite control as previously reported. Inconsistencies and controversy surround the regulation of the synthesis of enterotoxin B (SEB). They range from the common notion of SEB as an unregulated secondary metabolite whose function is unknown (2) to the contention that SEB is subject to coordinated control mechanisms (5). As a result of such reports, little definitive information is available describing the events controlling the synthesis of this important food-poisoning toxin. Recently, a series of papers by Morse and coworkers described a glucose-mediated regulatory mechanism for SEB which strongly suggests that toxin production is under catabolite control (10-13). Their data showed that SEB synthesis was retarded when glucose was present in the growth medium. Furthermore, the addition of pyruvate, a glucose catabolite, to a growing culture severely and immediately decreased the differential rate of toxin synthesis. In this instance, repression was dependent upon reactions associated with pyruvate decarboxylation. Anaerobic shock, which inhibited oxidative decarboxylation of pyruvate, could relieve repression, whereas the addition of a surrogate electron acceptor (NO3-) prevented anaerobic relief by allowing decarboxylation to

been shown to reduce toxin production, and cyclic 3',5'-adenosine monophosphate (cAMP) has not been shown to reverse toxin inhibition (although present in the cell [1], it was not known whether Staphylococcus aureus was permeable to this compound). The purpose of this paper is to present more information regarding this phenomenon and to provide data suggesting that toxin regulation by glucose and its catabolites is more complex than the classical considerations implicit in the idea of catabolite control.

MATERIALS AND METHODS Organism and cultural conditions. S. aureus S-6 was obtained from M. S. Bergdoll (Food Research Institute, University of Wisconsin). It was routinely cultured at 37°C in a medium (3% NAK-PHP) consisting of 3% N-Z Amine-NAK (Kraftco Corp., Oneonta, N.Y.), 3% protein hydrolysate powderPHP (Mead Johnson International, Evansville, Ind.), and 0.075% minimal essential medium vitamin mixture (GIBCO). Stock slants were maintained on the same medium containing 2% agar. Inocula for experiments consisted of 1 to 50 dilutions of overnight cultures in fresh medium. The cultures were grown to early stationary phase (ca. 500 Klett units) and divided into as many portions as necessary for the experiment. Additions of glucose, occur. In spite of these similarities, the regulation glucose analogues (2-deoxyglucose [2-DOG] and a[AMG]) were made from 25% stock of SEB by catabolite repression is equivocal. methylglucoside to minimize volume changes. The final Acid production due to glucose metabolism has solutions concentration of the adducts was 0.25%. cAMP was added to final concentrations of 1 x 10-3 M and 5 x 1 Contribution no. 1300 j, Kansas Agricultural Experi10-3 (17). To control pH, K2HPO4 was added to all ment Station, Manhattan, Kan. 610

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cultures to a final concentration of 1%. During the experiment, the pH, even in the glucose-containing cultures, never dropped below pH 6.8. Samples were withdrawn from the experimental cultures at intervals and placed in tubes containing thimerosol (final concentration, 0.3%). The turbidity of the samples was monitored with a Klett-Summerson colorimeter (blue filter). The cells were then removed from the medium by centrifugation, and the supernatant solutions were stored for subsequent analyses. Enterotoxin assay. SEB assays were carried out by a modification of the immunoelectrophoretic technique of Laurell (6). The buffer system used was 0.1 M tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.3 (, = 0.1), and resulted in the cathodic migration of the antigen. The antiserum (prepared in rabbits and kindly supplied by Reginald Bennett, Food and Drug Administration, Washington) was used at a dilution of 1 to 200, and at this level afforded a sensitivity of 1 t.g/ml with a 10-,ul sample in the well. The electrophoretic run lasted about 7 h, and the gel was then rinsed in running tap water for 2 h to remove unreacted antiserum. The cones rcntained in the gel were enhanced by cross-absorbing with rabbit antiglobulin prepared in goats. After removal of the unreacted goat antirabbit serum, the gels were dried and further enhanced by staining with Coomassie blue B. Extracellular protein measurement. Total extracellular protein was measured as a function of hot trichloracetic acid-precipitable protein present in the growth medium. After centrifugation of the medium to remove the cells, 1 ml of the supernatant solution was made 20% with trichloroacetic acid, allowed to stand on ice for 30 min, and then heated at 90°C for 15 min. The resulting precipitate was collected by centrifugation and washed twice with cold 5% trichloroacetic acid (protein does not precipitate from sterile growth medium). The final pellet was dissolved in 0.2 ml of 1 M NaOH and assayed by the Folin-Lowry method (7). Permeability of cAMP. The permeability of cAMP was tested in cells grown to 500 Klett units in 3% NAK-PHP medium. [8-3H]cAMP was added to 5 ml of culture at concentrations of 10-3 M (specific activity, 0.5 ,uCi/,umol) and 5 x 10-3 M (specific activity, 0.5 ,uCi/,umol). The culture was incubated at 37°C, and 0.2-ml samples were withdrawn at 1-min intervals. The cells were filtered onto 2.5-cm membrane filters (0.45-,im pore size) and washed four times with 5-ml portions of 3% NAK-PHP medium tempered to 370C. The filters were dried and counted as previously reported (8). In addition, cells were extracted with 5% trichloroacetic acid, and the filtrates were analyzed chromatographically for cAMP (solvent system: n-butanol, 60; acetic acid, 15; and water, 25). Staphylococcal nuclease was measured by the technique of Erickson and Diebel (4) and f3-hemolysin was measured according to Duncan and Cho (3).

shown in Fig. 1. Throughout the experiment (180 min), the increase in the rate of toxin synthesis in the unsupplemented medium (control) was linear. The total amount of toxin produced was 125 ,ug/ml. The addition of 0.1% sodium pyruvate or 0.25% glucose delayed the increase in the rate of synthesis for about 60 min and appeared to mimic the severe transient repression seen in similar studies on f8galactosidase synthesis in Escherichia coli (14, 18). When the rate of SEB synthesis began to increase (after 60 min), it was found that the rate of toxin production in the pyruvate culture roughly paralleled the control, whereas in this and subsequent experiments, the glucose-containing culture synthesized SEB more slowly. Morse and Baldwin (11) reported similar alterations in the differential rate of SEB synthesis in media supplemented with glucose and pyruvate. Furthermore, they found the influence of increasing pH (acidic to alkaline conditions) had an amplifying effect on the rate differences. Although we do not present primary data to explain the rate differences reported here, the pH of the media may have influenced the final

RESULTS The rate of SEB synthesis by cells of S. aureus S-6 growing in 3% NAK-PHP medium is

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FIG. 1. Rate of production of SEB by S. aureus S6 in 3% NAK-PHP medium. Symbols: (0) Control, 3% NAK-PHP medium; (0) 3% NAK-PHP medium plus 0.1% sodium pyruvate; (A) 3% NAK-PHP medium plus 0.25% glucose; (A) 3% NAK-PHP medium plus 0.25% 2-DOG. The culture was grown at 37°C to approximately 500 Klett units and split into portions to which appropriate metabolites were added. The cultures were then incubated for an additional 180 min and monitored for toxin production. The final density achieved was ca. 1,200 Klett units.

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rates of toxin synthesis attained. Examination of the pH of the cultures indicated that the pyruvate and control cultures were roughly equivalent during the experiment (pH 7.2 to 7.8), whereas the pH of the glucose culture remained relatively constant (pH 6.9 to 7.2) at values not highly supportive of vigorous toxin synthesis (10). To avoid the interactions and problems created by low pH in the presence of glucose and its catabolites on the elaboration of SEB, we tested the effect ofthe non-metabolizable glucose analogue, 2-DOG on SEB production. Previous experiments (data not presented) showed that 2-DOG is readily transported into these cells (Km = 0.92 ,uM). This compound has been shown by various workers (16-18) to severely, but transiently, repress the synthesis of f3-galactosidase in E. coli. However, with regard to SEB synthesis by S. aureus (Fig. 1), total inhibition of toxin synthesis was observed. Although the differential rate of SEB synthesis appeared unchanged, this can be attributed to an artifact of presentation, because, as in the E. coli systems, 2-DOG did not support growth of S. aureus S-6. To determine whether 2-DOG inhibition of SEB synthesis was permanent and irreversible, the following experiment was performed. A portion of an actively growing and toxin-producing culture was diluted 1 to 50 in fresh medium containing 0.25% 2-DOG (Fig. 2) and maintained for '120 min while samples were removed for SEB analysis; 2-DOG was then removed by centrifugation, and the cells were resuspended in fresh medium. The culture was incubated for an additional 90 min and assayed for SEB. It can be seen in Fig. 2 that the addition of 2-DOG (point A) immediately halted SEB synthesis while the control culture continued to produce toxin. After 2-DOG was removed (point B), toxin synthesis resumed and approximated the rate of the control culture. Growth of the culture was also monitored and was found to parallel the toxin response. We therefore concluded that SEB synthesis and growth were reversibly inhibited in a nonlethal manner by the glucose analogue 2-DOG. The specificity of inhibition was examined by determining the effect of 2-DOG on other extracellular proteins of S. aureus S-6. These data are presented in Table 1. As already described, SEB synthesis was severely inhibited by 2DOG. An average of only 0.3 ,g/108 cells (5 ,ug/ ml) was produced compared with 2.2 utg/108 cells (72 gg/ml) by the control. This small amount of toxin (5 ,ug/ml) was most likely due to the release of preformed toxin (9) since similar increases in SEB are observed (unpublished data) when chloramphenicol is present at levels

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FIG. 2. Reversibility of 2-DOG inhibition of growth and toxin production. An actively growing culture in 3% NAK-PHP was split into two portions, and 0.25% 2-DOG was added to half (point A) and incubated for 2 h. At that time (point B), the culture was centrifuged and suspended in fresh 3% NAKPHP. Symbols: Cell density-solid line; toxin (a) control, (A) culture containing 2-DOG, (A) culture after 2-DOG removal.

TABLE

1. Effect of 2-DOG on extracellular protein production by S. aureus S-6a Amt/105 cells Glucose

Protein

Control Glucose 2-DOG

SEBb

,8-Hemolysinc Staphylococcal nucleased

2.2

1.6

2.2 717

2.3 823

0.3 0.2 293

+ 2DOG 0.8

0.1 891

19.9 19.5 6.9 13.8 Total proteine a Cultures were incubated for 180 min; results are averages of three experiments. b Laurell immunoelectrophoresis (micrograms). c Hemolytic units -sheep erythrocyte lytic activity. d Units of deoxyribonuclease activity. e Micrograms of hot trichloroacetic acid-precipitable protein.

(8) that totally suppress labeled amino acid incorporation into protein. Toxin synthesis was partially rescued by the addition of glucose to 2DOG cultures, but the rate of toxin synthesis was markedly lower than in the presence of glucose alone (0.8 ug/105 cells [19 ,ug/ml] versus 1.6 ttg/108 cells [57 ,ug/ml] produced in 180 min). Moreover, the glucose and control cultures continued to produce toxin for long periods beyond the duration of the experiment, but the glucose + 2-DOG cultures ceased production at about 180 min. Therefore, although partial reversal is

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achieved by the addition of glucose, the synthesis of SEB appears to be significantly altered in the presence of 2-DOG. 2-DOG was also found to be a potent inhibitor of /8-hemolysin synthesis and staphylococcal nuclease synthesis. 8-hemolysin was completely (100%) inhibited, and the amount of staphylococcal nuclease was reduced by 82%. However, unlike SEB and 8-hemolysin, inhibition of staphylococcal nuclease was totally reversible by glucose rescue. The rate and total units of nuclease were at the level of the control when glucose + 2-DOG were present together. The amount of total extracellular protein synthesized reflected this effect in a twofold increase. A second glucose analogue, AMG, was also tested to determine its effect on SEB synthesis. AMG was added to actively growing cultures of S. aureus S-6. Samples were assayed for growth, SEB, total protein, and /8-hemolysin. These data are presented in Fig. 3 and Table 2. The kinetics of SEB production in the presence of AMG are presented in Fig. 3. In Fig. 3A, toxin accumulation is shown. Although the differences are not great, in comparison to the control, the glucose-containing culture produced SEB at a somewhat reduced rate. Quite unexpectedly, and completely opposite to the results obtained with 2-DOG, AMG did not inhibit SEB synthesis, but, in fact, SEB accumulated slightly faster in the AMG culture thaii in the control. Furthermore, the AMG and glucose mixture was equivalent in SEB synthesis to the glucose culture. The rate differences among these respective treatments are enhanced substantially in the differential plots presented in the remaining

Fig. 3B and C. Fig. 3B represents the synthesis of SEB as a function of cellular growth. AMG slowed cell division but did not reduce the rate of extracellular protein production. The result of this effect was translated into an increase in specific productivity of the cultures. The specific productivity of the AMG culture (micrograms of SEB per cell) was significantly greater than the control, and that of the glucose culture was considerably less. This contention is strengthened by the fact that the growth rate of the AMG culture was 1.6 times less than the control, and growth rates of the glucose and glucose-AMG cultures were 1.2-fold greater; the AMG culture produced more toxin with fewer cells than were contained in the glucosecontaining cultures. As suggested in Fig. 3A, the productivity of the glucose-AMG and glucose cultures (Fig. 3B)

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TABLE 2. Effect of AMG on extracellular protein production by S. aureus S-6a

Amt/10" cells Protein

SEBb

,8-Hemoly-

Glucose

Control

Glucose

AMG

1.7 2.2

1.2 2.3

2.0 2.2

1.7 2.0

12.3

18.4

17.6

16.9

+

AMG

sinC

Total

pro-

teind

a Cultures were incubated for 180 min; results are averages of two experiments. b Laurell immunoelectrophoresis (micrograms). c Hemolytic units -sheep erythrocyte lytic activity. d Micrograms of hot trichloroacetic acid-precipitable protein.

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FIG. 3. Effect ofAMG on toxin production by S. aureus S-6. An actively growing culture in 3% NAK-PHP was split into four portions at a density of approximately 460 Klott units. One portion was kept as a control (a), another was made 0.25% in AMG (A), another was made 0..&% in glucose (0), and the last was made 0.25% in both AMG and glucose (A). Samples were taken at intervals and assayed for toxin, growth, and extracellular protein. The data are presented as toxin accumulation (A), differential rate of toxin production as a function ofgrowth (B), and differential rate as a function of total extracellular protein (C).

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were equivalent; however, it is interesting to note that the addition of glucose to AMG cultures reduced productivity in AMG to that of glucose alone. The reason for this effect is not clear at this time, although it may also be related to pH. When toxin production was compared as a fraction of the total extracellular protein (Fig. 3C), it could be seen that AMG quantitatively changed the rate of extracellular appearance of SEB. The AMG culture initially produced toxin more rapidly than the control, and the AMGglucose culture produced more than the glucose culture. A similar pattern was seen (Table 2) regarding total extracellular protein. The glucose- and/or AMG-containing cultures produced more total protein than the control. SEB was repressed in the glucose culture, but not in the AMG culture, and /3-hemolysin synthesis was unaffected in any of the media. The startlingly different modes of action of these common glucose analogues led us to further question the relationship of the glucose effect to catabolite repression and cyclic AMP. Cyclic AMP has been shown in E. coli to play a central regulatory role in the positive expression of several genes (15). It is this compound whose intracellular level is drastically lowered by glucose, resulting in a transient inhibition of synthesis. Moreover, in E. coli, the addition of exogenous cAMP to transiently glucose-repressed cultures has resulted in reversal of repression and resumption of the normal rate of ,/-galactosidase synthesis (16). In S. aureus, however, Morse and Mah (13) preliminarily reported that exogenously added cAMP did not reverse glucose repression of SEB synthesis, even though cAMP is reportedly present at low concentration in S. aureus (1). Nevertheless, their data (13) were equivocal because they did not test cAMP permeability in this organism. To test the ability of cAMP to enter S. aureus S-6, actively growing washed cells were incubated in 3% NAK-PHP medium (control), the same medium plus 0.25% glucose, and the same medium containing 0.25% 2-DOG. cAMP was added at two concentrations, 1 x 10-3 M and 5 X 10-3 M and the cells were assayed for uptake. These data are presented in Fig. 4. It was found that cells in 3% NAK-PHP rapidly transported cAMP (ca. 2 nmol/mg of protein). The presence of either glucose or 2-DOG did not materially affect the rate or extent of uptake. Chromatographic examination of trichloracetic acid extracts from these cells did not show that cAMP was intracellularly converted to either 3' or 5' AMP. Since S. aureus S-6 was able to transport cAMP, we tested the ability of this compound to

reverse the glucose- and 2-DOG-mediated repression of SEB synthesis. An actively growing culture was divided into the following 3 treatments: (i) control -no additions; (ii) 0.25% glucose + 5 x 10-3 M cAMP; (iii) 0.25% 2-DOG + 5 x 10-3 M cAMP. Additional controls, consisting of a culture in NAK-PHP medium plus added glucose and another culture that contained cAMP and no glucose, were also performed. Growth and toxin production were identical to the (i) no addition control and the (ii) glucose + cAMP control and so were not included in Fig. 5. Incubation was continued at 37°C for 180 min, and samples were assayed for growth, SEB, and total extracellular protein. It can be seen in Fig. 5 that cAMP was ineffective in relieving either glucose or 2-DOG repression of SEB synthesis. In Fig. 5A, the expected patterns were observed: glucose slowed SEB production and 2-DOG severely inhibited its synthesis. The differential plots (Fig. 5B

and C) further substantiated the lack of cAMP relief. It should be mentioned that the 2-DOG data are not presented in the differential plots, because neither the culture density nor the extracellular protein increased significantly and hence could not be plotted. DISCUSSION

Glucose-mediated repression of SEB synthesis has led to the suggestion that the regulation of this protein toxin is under catabolite control (10-13). This observation and other similarities to the catabolite repression of /8-galactosidase in E. coli, namely, the requirement for pyruvate decarboxylase activity for active repression (14), comprise the main evidence for this mode of control of SEB expression. These data are superficially convincing; however, distinct inconsistencies have appeared which place significant doubt on the role of glucose in governing the expression of SEB. Although we have consistently observed a decrease in the synthesis of SEB after glucose addition to a culture, our data suggest that more complex interactions are taking place. The use of two common glucose analogues (AMG and 2-DOG), which have both been shown to induce severe repression of /3-galactosidase synthesis in E. coli (1618), have produced precisely opposite effects in S. aureus regarding their specific modes of action and regarding reported activities in known catabolically regulated systems. AMG appears to stimulate the synthesis of SEB and other extracellular proteins in S. aureus, whereas 2DOG severely inhibits SEB synthesis in a rather specific manner. These effects are especially puzzling because, although both ana-

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logues are taken up by the cells, neither is either substance is prohibited. Moreover, the metabolized. AMG can be processed via glycol- inhibition of SEB synthesis mediated by 2-DOG ysis to the level of fructose-6-phosphate and 2- (although reversible) appears to be permanent DOG can be catabolized to the level of glucose- rather than transient. In addition, 2-DOG 6-phosphate. However, further catabolism of repression cannot be relieved by the addition of cAMP, whereas, in E. coli, such relief does occur (18). It also appears that 2-DOG exerts a greater specificity of inhibition that might be expected, since the addition of glucose in the presence of 2-DOG does not restore SEB synthesis, but does allow staphylococcal nuclease production. All these findings differ from those normally associated with catabolite regulated enzymes. In systems under catabolite control, AMG and 2-DOG severely repress enzyme synthesis, but synthesis gradually resumes (18), and in the presence of non-metabolizable analogues at a rate equivalent to the control. Such proteins also respond to cAMP rescue. Those proteins in E. coli that are subject to glucose repression share other common features. They are virtually all inducible, their presence is not essential for cell survival and growth in glucose-supplemented media, and they are positively regulated by cAMP (15). Although SEB certainly appears to be a nonessential protein (toxin-negative cells grow as well as toxin-positive cells in all media) and fits 0 1 2 3 4 one of these criteria, the inducibility of this MINUTES FIG. 4. Uptake ofcAMP by S. aureus in 3% NAK- substance has yet to be established. Lastly, the PHP (@), 3% NAK-PHP plus 0.25% glucose (0), and addition of cAMP, in all cases, in E. coli reverses glucose repression (15), but in S. aureus 3% NAK-PHP with 0.25% 2-DOG and 0.25% glucose (A). All fractions contained 5 x 10-3 M cAMP (speS-6 we have shown that cAMP has no effect on cific activity, 0.5 uCi/mmol). the repression of SEB. We therefore conclude

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FIG. 5. Effect of cAMP on 2-DOG inhibition of SEB synthesis. The cells were prepared as given in the legend to Fig. 3. The glucose and 2-DOG cultures also contained 5 x 10-3 M cAMP. Symbols: Control (0), glucose (0), 2-DOG (A).

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that although a glucose-repressive effect on the synthesis of SEB exists in S. aureus, the mode of action is not equatable with catabolite repression and exhibits properties of a more complex phenomenon. ACKNOWLEDGMENTS We acknowledge the expert technical assistance of Martha S. Gentry. This work was supported by a research grant from the Biology Research fund, Division of Biology, Kansas State University. LITERATURE CITED 1. Blumenthal, H. J. 1972. Glucose catabolism in staphylococci. p. 111-136. In Jay 0. Cohen (ed.), The staphylococci. Wiley-Interscience, a division of John Wiley and Sons, New York. 2. Dietrich, G. G., R. J. Watson, and G. J. Silverman. 1972. Effect of shaking speed on the secretion of enterotoxin B by Staphylococcus aureus. Appl. Microbiol. 24:561-566. 3. Duncan, J. L., and G. J. Cho. 1971. Production of staphylococcal alpha toxin. I. Relationship between cell growth and toxin formation. Infect. Immun. 4:456461. 4. Erickson, A., and R. H. Diebel. 1973. Turbidimetric assay of staphylococcal nuclease. Appl. Microbiol. 25:337-341. 5. Katsuno, S., and M. Kondo. 1973. Regulation of staphylococcal enterotoxin B synthesis and its relation to other extracellular proteins. Jpn. J. Med. Sci. Biol. 26:26-29. 6. Laurell, C-B. 1966. Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal. Biochem. 15:45-52.

INFECT. IMMUN. 7. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 913:265-280. 8. Martin, S. E., and J. J. landolo. 1975. Translational control of protein sysnthesis in Staphylococcus aureus. J. Bacteriol. 122:1136-1143. 9. Miller, R. D., and D. Y. C. Fung. 1976. The occurrence of cell-associated enterotoxin B in Staphylococcus aureus. Can. J. Microbiol. 22:1215-1221. 10. Morse, S. A., R. A. Mah, and W. J. Dobrogosz. 1969. Regulation of staphylococcal enterotoxin B. J. Bacteriol. 98:4-9. 11. Morse, S. A., and J. N. Baldwin. 1971. Regulation of staphylococcal enterotoxin B: effect of thiamine starvation. Appl. Microbiol. 22:242-249. 12. Morse, S. A., and J. N. Baldwin. 1973. Factors affecting the regulation of staphylococcal enterotoxin B. Infect. Immun. 7:839-846. 13. Morse, S. A., and R. A. Mah. 1973. Regulation of staphylococcal enterotoxin B: effect of anaerobic shock. Appl. Microbiol. 25:553-557. 14. Okinaka, R. T., and W. J. Dobrogosz. 1967. Catabolite repression and pyruvate metabolism in E. coli. J. Bacteriol. 93:1644-1650. 15. Pastan, I., and S. Adhya. 1976. Cyclic adenosine 5'monophosphate in Escherichia coli. Bacteriol. Rev. 40:527-551. 16. Perlman, R. L., and I. Pastan. 1968. Regulation of f8galactosidase synthesis in Escherichia coli by cyclic adenosine 3',5'-monophosphate. J. Biol. Chem. 243:5420-5427. 17. Perlman, R. L., B. De Crombrugghe, and I. Pastan. 1969. Cyclic AMP regulates catabolite and transient repression in E. coli. Nature (London) 223:810-812. 18. Tyler, B., and B. Magasanik. 1970. Physiological basis of transient repression of catabolic enzymes in Escherichia coli. J. Bacteriol. 102:411-422.

Regulation of staphylococcal enterotoxin B.

INFECTION AND IMMUNry, May 1977, p. 610-616 Copyright C) 1977 American Society for Microbiology Vol. 16, No. 2 Printed in U.S.A. Regulation of Staph...
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