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Catabolite repression and the Pasteur effect in Escherichia coli
ARCHIVES
OF
BIOCHEMISTRY
Catabolite
AND
Repression
RICHARD Department
BIOPHYSICS
and the Pasteur
T. OKINAKA
of Microbiology,
451-453 (1967)
120,
North
Effect in Escherichia
AND WALTER
Carolina State University,
co/i’
J. DOBROGOSZ Raleigh,
North
Carolina 27607
Received December 12, 1966 Previous studies from this laboratory indicated that certain reactions at the level of pyruvate metabolism play a key role in glucose repression of the /3-galactosidase system in .Eschetihia coli (Dobrogosz, 1966; Okinaka and Dobrogosz, 1966). Further information along this line is described in this paper. In addition, studies are presented that suggest that the Pasteur effect and catabolite repression are metabolically related phenomena. MATERIALS
AND METHODS
Escherichia coli ML30 (i+z+y+) was used throughout this study. All growth was conducted at 37” in a mineral salts medium supplemented with 0.25%) casein hydrolyzate, as previously described l(Dobrogosz, 1965, 1966). Conditions concerning aerobic and anaerobic growth, induction of ,%galactosidase formation with 2.5 X 10m3 M isopropyl-@-n-thiogalactoside (IPTG) and assay of @-galactosidase activity by use of o-nitrophenylp-n-galactoside (ONPG) as substrate, and toluenetreated cells as enzyme source have been described (Dobrogosz, 1965). A unit of fl-galactosidase activity was defined as the amount of enzyme that hydrolyzes 1 rmole of ONPG per hour at 30” in the presence of 2 X 1OV M ONPG, 1.24 X lo+ M reduced glutathione, and 0.05 M sodium phosphate buffer, pH 7.5. Rates of enzyme formation were determined by plotting units of enzyme activity per millilit,er of culture against growth of the culture, calculated as micrograms dry weight of cells per milliliter of culture.
1 This work was supported by grants from the National Science Foundation (GB-831) and by the National Institute of Allergy and Infectious Diseases (AI-06072) of the Public Health Service. Published with the approval of the Director of the N.C. Agricultural Experiment Station as Paper No. 2298 of the Journal Series.
Glucose was measured with a commercial enzyme-chromogen preparation, Blood Sugar (Biochemica-Boehringer, Calbiochem, Los Angeles, California). Pyruvate w&s determined in the culture medium after stopping growth with cold 5% trichloroacetic acid and removing the cells by centrifugation. Pyruvate was assayed by the indirect phynylhydrazone procedure of Friedemann and Haugen (1943). Cultures growing anaerobically in the presence of nitrate accumulated nitrite in the medium. This was found to interfere with the pyruvate assay. To circumvent this problem, a 0.50-ml sample in 5yo trichloroacetic acid was treated with 2 ml of 0.05 M potassium iodide prior to reaction with the phenylhydrazine. RESULTS
AND DISCUSSION
It is known that glucose repression in E. coli can be switched on or off by appropriate alterations of the atmosphere or by including nitrate in the medium (Cohn and Horibata, 1959; Dobrogosz, 1965, 1966; Okinaka and Dobrogosz, 1966). The data presented in Figs. 1 and 2 show that, under cert,ain conditions, these changes are associated with accumulation of pyruvate in the medium. As seen in Fig. 1, an aerobic culture forms P-galactosidase at a nonrepressed rate after the transition to an anaerobic state. On the other hand, if an anaerobic culture forming enzyme at a nonrepressed rate is subjected to an aerobic shock, p-galactosidase formation is almost completely repressed for the following generation of growth. This period of severe transient repression due to aerobic shock was found to occur concomitantly with a rapid accumulation of pyruvat’e in the medium. The pyruvate was eventually reutilized and aerobic glucose repression returned to a level characteristic of aerobic
451
452
OKINAKA
ilND
DOBROGOSZ
5loo-A I/. > . .$60-
80
.
; 600 0 52 40;
60 40 20 0
r
1.2
100 200 300 4Cil 500 600 700 100 200 300
Dry Weight (fig/ml)
400
500
600
Dry Weight U.Lg/ml)
FIG. 1. Accumulation of pyruvate during aerobic shock. A culture growing exponentially on glucose under aerobic conditions was divided into four portions and used as an inoculum at a concentration of 75 Mg dry weight cell/ml culture. Aerobic growth was continued with two cultures (A and A), and the two additional cultures were subjected to anaerobic shock (0 and 0). At the indicated points an anaerobic culture was subjected to aerobic growth (0) and an aerobic culture was subjected to anaerobic growth (A). All cultures contained 0.02 M glucose at the start of the experiment. All measurements were plotted against the respective pg dry weight/ml of culture. Graph A: &galactosidase formation; graph B: pyruvate accumulation.
growth on glucose. If a high concentration of nitrate was included in the medium (Fig. 2), glucose repression occurred in spite of anaerobic shock. Under these conditions of nitrate-induced repression, pyruvate accumulation was again found to occur during growth on glucose. The specific relationship between these transient alterations in glucose and pyruvate metabolism and repression of the /3-galactosidase system is unclear. During the course of these studies, however, it became apparent that the conditions used to induce these changes in both glucose metabolism and glucose repression were comparable with conditions known to influence regulation of glucose dissimilation via the wellknown Pasteur effect. This was readily demonstrated to be the case under our con-
FIG. 2. Pyruvate accumulation in the presence of nitrate. Two cultures, growing aerobically in medium containing 0.02 M glucose, were made anaerobic when they reached a cell mass of 95 rg dry weight/ml culture. One culture served as an anaerobic control (@) while 16 pmoles/ml of KN03 was added to the other culture (0). Graph A: ,&galactosidase formation; graph B: pyruvate accumulation. 18-
; 5
IOa0
' 100 200
300
400
I I 500 600
1 700
Dry Weight (,Ug/ml)
FIG. 3. Differential rates of glucose dissimilation under varied conditions. Glucose concentrations were measured as described elsewhere (Materials and Methods) in cultures growing aerobically (O), anaerobically (0), anaerobically with 2 rmoles/ml KNO, added (A), and anaerobically with 16 pmoles/ml KNOI added (A).
ditions (Fig. 3). Rates of glucose dissimilation-a key index in determining operation of the Pasteur effect-were measured under conditions that alter catabolite repression. Rapid glucose dissimilation was observed
CATABOLITE
REPRESSION
24r zo= .E 16h g IZ3 22 8: 2 4-
0
too
200
300
400
500
600
I 700
, 000
Dry Weight @g/ml)
FIG. 4. Pyruvate utilization during aerobic and anaerobic growth. Pyruvate concentrations were measured as described elsewhere (Materials and Methods) in cultures growing (I) aerobically in medium containing 0.02 M pyruvate (A), (2) aerobically in medium containing 0.02 M pyruvate and 0.02 151 glucose (a), (8) anaerobically in medium containing 0.02 M pyruvate (a), and (4) anaerobically in medium containing 0.02 M pyruvate and 0.02 M glucose (0 ) .
during anaerobic growth, whereas repression of glucose dissimilation was noted during aerobic growth or during anaerobic growth with nitrate present as an oxidant. The data presented in Fig. 4 record rates of pyruvate dissimilation under similar conditions. As with glucose utilization, pyruvate degradation was stimulated by anaerobiosis and repressed by aerobiosis. This was shown to be the case both when pyruvate was the major carbon source for growth and when it was used in combination with glucose. These data, as well as studies described earlier (Dobrogosz, 1966; Okinaka and Dobrogosz, 1966), imply that the initial reactions involved in glucose repression of @-galactosidase formation may be similar or linked in some manner to those reactions used in regulation of glucose metabolism by the Pasteur phenomenon (Lynen et al, 1959;
AND
PASTEUR
EFFECT
453
Uyeda and Racker, 1965; Williamson, 1965). Both of these processes are enhanced by respiratory reactions and relaxed by fermentative activities. Both are considered to involve competive relationships between formation and utilization of dissimilatory intermediate compounds as well as adenosine nucleotides and inorganic phosphate. Furthermore, both phenomena provide for cellular economy in carbohydrate metabolism. The Pasteur effect insures conservation of substrate under conditions in which metabolic energy is abundantly available. This same abundance prevents, via catabolite repression, formation of catabolic enzyme systems whose activities would only serve to augment the already large metabolic pools (Neidhardt and Magasanik, 1956; Magasanik, 1961). At the present time the relationship between these two regulatory processes is based on indirect evidence and parallelisms. The evidence is sufficient, nevertheless, to provide a working model for future studies along this line. REFERENCES COHN, M., AND HORIBATA, K., J. Bacterial. 76, 624 (1959). DOBROGOSZ, W. J., Biochem, Biophys. Acta 100, 553 (1965). DOBROGOSZ, W. J., J. Bacterial. 91, 2263 (1966). FRIEDEMANN, T.E., AND HAUGEN,G. E., J.BioZ. Chem. 147, 415 (1943). LYNEN, F., Hartmann, G., NETTER, K. F., AND SCHEUGRAF, A., Ciba Found. Symp. Regulation of Cell Metab. 1969, pp. 256-273. Churchill, London (1959). MAG.~SANIK, B., Cold Spring Harbor Sump. Quant. Biol. 26, 249 (1961). NEIDHARDT, F. C., AND MAGASANIK, B., Nature 178, 801 (1956). OKINAKA, R.T., AND DOBROGOSZ, W.J., Bacterial. Proc. p. 96 (1966). UYED~, K. AND RACKER, E., J. Biol. Chem. 240, 4689 (1965). WILLIAMSON, 6. R., J. Biol. Chem. 240,230s (1965).
OF
BIOCHEMISTRY
Catabolite
AND
Repression
RICHARD Department
BIOPHYSICS
and the Pasteur
T. OKINAKA
of Microbiology,
451-453 (1967)
120,
North
Effect in Escherichia
AND WALTER
Carolina State University,
co/i’
J. DOBROGOSZ Raleigh,
North
Carolina 27607
Received December 12, 1966 Previous studies from this laboratory indicated that certain reactions at the level of pyruvate metabolism play a key role in glucose repression of the /3-galactosidase system in .Eschetihia coli (Dobrogosz, 1966; Okinaka and Dobrogosz, 1966). Further information along this line is described in this paper. In addition, studies are presented that suggest that the Pasteur effect and catabolite repression are metabolically related phenomena. MATERIALS
AND METHODS
Escherichia coli ML30 (i+z+y+) was used throughout this study. All growth was conducted at 37” in a mineral salts medium supplemented with 0.25%) casein hydrolyzate, as previously described l(Dobrogosz, 1965, 1966). Conditions concerning aerobic and anaerobic growth, induction of ,%galactosidase formation with 2.5 X 10m3 M isopropyl-@-n-thiogalactoside (IPTG) and assay of @-galactosidase activity by use of o-nitrophenylp-n-galactoside (ONPG) as substrate, and toluenetreated cells as enzyme source have been described (Dobrogosz, 1965). A unit of fl-galactosidase activity was defined as the amount of enzyme that hydrolyzes 1 rmole of ONPG per hour at 30” in the presence of 2 X 1OV M ONPG, 1.24 X lo+ M reduced glutathione, and 0.05 M sodium phosphate buffer, pH 7.5. Rates of enzyme formation were determined by plotting units of enzyme activity per millilit,er of culture against growth of the culture, calculated as micrograms dry weight of cells per milliliter of culture.
1 This work was supported by grants from the National Science Foundation (GB-831) and by the National Institute of Allergy and Infectious Diseases (AI-06072) of the Public Health Service. Published with the approval of the Director of the N.C. Agricultural Experiment Station as Paper No. 2298 of the Journal Series.
Glucose was measured with a commercial enzyme-chromogen preparation, Blood Sugar (Biochemica-Boehringer, Calbiochem, Los Angeles, California). Pyruvate w&s determined in the culture medium after stopping growth with cold 5% trichloroacetic acid and removing the cells by centrifugation. Pyruvate was assayed by the indirect phynylhydrazone procedure of Friedemann and Haugen (1943). Cultures growing anaerobically in the presence of nitrate accumulated nitrite in the medium. This was found to interfere with the pyruvate assay. To circumvent this problem, a 0.50-ml sample in 5yo trichloroacetic acid was treated with 2 ml of 0.05 M potassium iodide prior to reaction with the phenylhydrazine. RESULTS
AND DISCUSSION
It is known that glucose repression in E. coli can be switched on or off by appropriate alterations of the atmosphere or by including nitrate in the medium (Cohn and Horibata, 1959; Dobrogosz, 1965, 1966; Okinaka and Dobrogosz, 1966). The data presented in Figs. 1 and 2 show that, under cert,ain conditions, these changes are associated with accumulation of pyruvate in the medium. As seen in Fig. 1, an aerobic culture forms P-galactosidase at a nonrepressed rate after the transition to an anaerobic state. On the other hand, if an anaerobic culture forming enzyme at a nonrepressed rate is subjected to an aerobic shock, p-galactosidase formation is almost completely repressed for the following generation of growth. This period of severe transient repression due to aerobic shock was found to occur concomitantly with a rapid accumulation of pyruvat’e in the medium. The pyruvate was eventually reutilized and aerobic glucose repression returned to a level characteristic of aerobic
451
452
OKINAKA
ilND
DOBROGOSZ
5loo-A I/. > . .$60-
80
.
; 600 0 52 40;
60 40 20 0
r
1.2
100 200 300 4Cil 500 600 700 100 200 300
Dry Weight (fig/ml)
400
500
600
Dry Weight U.Lg/ml)
FIG. 1. Accumulation of pyruvate during aerobic shock. A culture growing exponentially on glucose under aerobic conditions was divided into four portions and used as an inoculum at a concentration of 75 Mg dry weight cell/ml culture. Aerobic growth was continued with two cultures (A and A), and the two additional cultures were subjected to anaerobic shock (0 and 0). At the indicated points an anaerobic culture was subjected to aerobic growth (0) and an aerobic culture was subjected to anaerobic growth (A). All cultures contained 0.02 M glucose at the start of the experiment. All measurements were plotted against the respective pg dry weight/ml of culture. Graph A: &galactosidase formation; graph B: pyruvate accumulation.
growth on glucose. If a high concentration of nitrate was included in the medium (Fig. 2), glucose repression occurred in spite of anaerobic shock. Under these conditions of nitrate-induced repression, pyruvate accumulation was again found to occur during growth on glucose. The specific relationship between these transient alterations in glucose and pyruvate metabolism and repression of the /3-galactosidase system is unclear. During the course of these studies, however, it became apparent that the conditions used to induce these changes in both glucose metabolism and glucose repression were comparable with conditions known to influence regulation of glucose dissimilation via the wellknown Pasteur effect. This was readily demonstrated to be the case under our con-
FIG. 2. Pyruvate accumulation in the presence of nitrate. Two cultures, growing aerobically in medium containing 0.02 M glucose, were made anaerobic when they reached a cell mass of 95 rg dry weight/ml culture. One culture served as an anaerobic control (@) while 16 pmoles/ml of KN03 was added to the other culture (0). Graph A: ,&galactosidase formation; graph B: pyruvate accumulation. 18-
; 5
IOa0
' 100 200
300
400
I I 500 600
1 700
Dry Weight (,Ug/ml)
FIG. 3. Differential rates of glucose dissimilation under varied conditions. Glucose concentrations were measured as described elsewhere (Materials and Methods) in cultures growing aerobically (O), anaerobically (0), anaerobically with 2 rmoles/ml KNO, added (A), and anaerobically with 16 pmoles/ml KNOI added (A).
ditions (Fig. 3). Rates of glucose dissimilation-a key index in determining operation of the Pasteur effect-were measured under conditions that alter catabolite repression. Rapid glucose dissimilation was observed
CATABOLITE
REPRESSION
24r zo= .E 16h g IZ3 22 8: 2 4-
0
too
200
300
400
500
600
I 700
, 000
Dry Weight @g/ml)
FIG. 4. Pyruvate utilization during aerobic and anaerobic growth. Pyruvate concentrations were measured as described elsewhere (Materials and Methods) in cultures growing (I) aerobically in medium containing 0.02 M pyruvate (A), (2) aerobically in medium containing 0.02 M pyruvate and 0.02 151 glucose (a), (8) anaerobically in medium containing 0.02 M pyruvate (a), and (4) anaerobically in medium containing 0.02 M pyruvate and 0.02 M glucose (0 ) .
during anaerobic growth, whereas repression of glucose dissimilation was noted during aerobic growth or during anaerobic growth with nitrate present as an oxidant. The data presented in Fig. 4 record rates of pyruvate dissimilation under similar conditions. As with glucose utilization, pyruvate degradation was stimulated by anaerobiosis and repressed by aerobiosis. This was shown to be the case both when pyruvate was the major carbon source for growth and when it was used in combination with glucose. These data, as well as studies described earlier (Dobrogosz, 1966; Okinaka and Dobrogosz, 1966), imply that the initial reactions involved in glucose repression of @-galactosidase formation may be similar or linked in some manner to those reactions used in regulation of glucose metabolism by the Pasteur phenomenon (Lynen et al, 1959;
AND
PASTEUR
EFFECT
453
Uyeda and Racker, 1965; Williamson, 1965). Both of these processes are enhanced by respiratory reactions and relaxed by fermentative activities. Both are considered to involve competive relationships between formation and utilization of dissimilatory intermediate compounds as well as adenosine nucleotides and inorganic phosphate. Furthermore, both phenomena provide for cellular economy in carbohydrate metabolism. The Pasteur effect insures conservation of substrate under conditions in which metabolic energy is abundantly available. This same abundance prevents, via catabolite repression, formation of catabolic enzyme systems whose activities would only serve to augment the already large metabolic pools (Neidhardt and Magasanik, 1956; Magasanik, 1961). At the present time the relationship between these two regulatory processes is based on indirect evidence and parallelisms. The evidence is sufficient, nevertheless, to provide a working model for future studies along this line. REFERENCES COHN, M., AND HORIBATA, K., J. Bacterial. 76, 624 (1959). DOBROGOSZ, W. J., Biochem, Biophys. Acta 100, 553 (1965). DOBROGOSZ, W. J., J. Bacterial. 91, 2263 (1966). FRIEDEMANN, T.E., AND HAUGEN,G. E., J.BioZ. Chem. 147, 415 (1943). LYNEN, F., Hartmann, G., NETTER, K. F., AND SCHEUGRAF, A., Ciba Found. Symp. Regulation of Cell Metab. 1969, pp. 256-273. Churchill, London (1959). MAG.~SANIK, B., Cold Spring Harbor Sump. Quant. Biol. 26, 249 (1961). NEIDHARDT, F. C., AND MAGASANIK, B., Nature 178, 801 (1956). OKINAKA, R.T., AND DOBROGOSZ, W.J., Bacterial. Proc. p. 96 (1966). UYED~, K. AND RACKER, E., J. Biol. Chem. 240, 4689 (1965). WILLIAMSON, 6. R., J. Biol. Chem. 240,230s (1965).