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Thermal penicillinase derepression and temperature dependence of penicillinase production inducible and constitutive strains of Bacillus cereus
ARCHIVES
OF
Thermal of
BIOCHEMISTRY
AND
BIOPHYSICS
119, 50-54 (1967)
Penicillinase
Derepression
and
Penicillinase
Production
Inducible
Strains AMIRA BERNSTEIN,
KENNETH
of Bacillus
Temperature
Constitutive
cereus
W. NICKERSON
Department of Chemistry, University
and
Dependence
of Cincinnati,
AND
Cincinnati,
RICHARD
A. DAY’12
Ohio .J52Sl
Received August 15, 1966 The basal level of penicillinase production in strain 569 reaches a maximum at 42”. The induction ratio (induced/basal) reaches a maximum (42.6) at approximately 30” and falls to approximately 1.4 at 42”. Culturing Bacillus cereua 569 (inducible) at 42” and lowering the temperature leads to an increase in penicillinase production (thermal derepression) equal to the level induced by cephalothin (CT). As reported earlier, induction in strain 569 at 42” does not occur when the cells are treated with a penicillin, here the analogue CT, at 42”; however, treatment with CT at 37” followed by incubation at 42” gave the induced response. Constitutive mutants 569/H and 5/B showed a twofold change in the level of penicillinase upon going from 20” to 46”, with specific activity increasing steadily to 46”. A model is presented to explain these and other related data in the B. cereus penicillinase system.
Re-examination of the reported failure of “adaptive formation” of penicillinase in Badus cereus 569 at 42’ (1) led to an investigation of the temperature dependence of penicillinase production in certain strains of B. cereus.The results reported here show that induction in B. cereus 569 can indeed be effected under appropriate conditions at 42”. During the course of this work it was reported that penicillinase production in Staphylococcus aureus could be thermally derepressed, and the characteristics of this process were described (2). This was not unlike the thermal derepression of P-gala&osidase described earlier for Escherichia coli (3). It appears that the temperature de1 This work was supported in part by the American Cancer Society Grant P343A and by the National Institutes of Health Grant AI-06375. We thank Eli Lilly & Co. for generously supplying us with samples of cephalosporin C and cephalothin. Mr. Graf’s technical assistance was invaluable. 2 To whom correspondence concerning this paper should be addressed. 50
pendence of penicillinase production provides supportive evidence for a two-point metabolic control of penicillinase production (4, 5), and in this context a model of control is proposed. PROCEDURE Materials. Penicillin G and hydrolyzed casein were obtained from Nutritional Biochemical Corp. Cephalosporin C and cephalothin were generously donated by Eli Lilly and Co. Growth of cultures. Cultures were grown in a modified CH/C medium (6). In addition to the components of CH/C specified by Pollock (6), the medium was made 0.1% in gelatin. All of the constituents were autoclaved together. Cultures were grown to an optical density of 0.30 f 0.03 in a water bath at the appropriate temperature. The temperature was controlled to f0.1”. Penicillinase assay. Essentially the method of Perret (7) was used throughout. Dry weight of cells. The cells were dried in vacua to a constant weight after washing with distilled water. The relationship of optical density to dry weight of cells was determined in order to report the “specific activity” as units of penicillinase per milligram of dry weight of the cells (Fig. 1). The
PENICILLINASE
51
DEREPRESSION
optical density to dry weight relationship was the same for the entire temperature range examined except where clumping of the cells occurred. Clumping was sometimes observed to a variable degree at the extremes of culturing temperature. RESULTS
Modification of the culturing medium of Pollock (6) by combining all of the components recommended and adding gelatin (0.1%) caused no measurable difference in growth or penicillinase production. Addition of gelatin was essential to prevent adsorption of relatively large amounts of penicillinase to the walls of the culture flask; culturing in polypropylene also obviated the need for gelatin but was less convenient for following the growth visually. Induction of penicillinase activity in B. cereus 569 (NRRL 569), an inducible strain, by the “standard growth” method (3, 4) was somewhat higher when cephalothin (CT) was compared with penicillin G or cephalosporin C (Table I). Penicillinase production in the macroconstitutive strain of B. cereus 5/B (NCTC 9946) was found to be moderately dependent on temperat’ure. The specific 1650 1550 1450 1350 -
TABLE
I
INDUCTION OF PENICILLINASE ACTIVITY BY PENICILLIN G (PG), CEPHBLOTHIB (CT), AND CEPHALOSPORIN C (CC) BY THE ST.LNU~RD METHOD Inducer molarity -
10-4 10-s 10-C
NOIXS
Penicillinax
5.6 -
activity,
units/ml
induced”
PG
CT
cc
58.8 56.4 59.4
60.0 58.0 65.4
60.8 60.0 28.6
n One unit of penicillinase is that amount of enzyme producing sufficient penicilloic acid in one hour at 30”, pH 6.5, to consume 9 peq of iodine by the procedure of Perret (4). TABLE
II
SPECIFIC ACTIVITIES OF THE ENZYME PRODUCTION (BASIL) IN Bacillus cereus STR~IX 5/B >\T DIFPERENT TEMPERATURES Temperature
18 22 25 30 35 38 40 42 44 4G
Total medium assayed (units/mg)
608 700 668 958 1201 1056 1218 1531 1306 853
f rt f f f rt f f f f
18.5 23 210 76 283 71 52 132 113 39
exe-Penicillinase (units/mg)
582 581 630 935 1063 889 1068 1401 1116 266
rt i rt rt + i rt z!z + f
33 134 193 10 167 87 191 12 114 53
1250 .I150 1050 .09502
OS50
F .0750 065U 0550-045U 0350.0250 0150 0050111lIllll111llllIll111
025
100
175
250 OPTICAL
FIG.
weight
325
400 DENSITY
475 (6501~1
550
1. Relationship of optical density to dry of cultures of Bacillus cereu.s strains.
activity of the penicillinase increased by a factor of 2.5 upon going from a culturing temperature of 18” to one of 42’ (Table II). On the other hand, the basal production of penicillinase in strain 569 was more strongly dependent on temperature (Table III). The specific activity increased by a factor of approximately nine upon increasing the culturing temperature from 18” to 42’. The inductive ratio is apparently even more temperature dependent (Table IV). The induction ratio rises from -6 at 20” to -40 at 30”, and then declines to <2 at 42”. It was not possible to measure levels of penicillinase production over as wide a temperature range in B. cereus strain 5 (ATCC 10702) since this organism produces immeasurably low levels of the enzyme as
52
BERNSTEIN,
NICKERSON
culturing temperatures of near 20’ are approached. The amounts cell-bound and exo-penicillinase was determined for the strains described (Tables II and III). Strains 569/H, a constitutive strain arising by spontaneous mutation from strain 569, and strain 5 were found to give similar ratios of ezo-penicillinase to total penicillinase over all culturing temperatures examined. In a previous report from this laboratory (5) it had been reported that strain 5 produced very little ezo-penicillinase, but by culturing the organism in the presence of gelatin the exoenzyme was found to be produced in the proportion typical of other strains of B. cereus. Thermal derepression of penicillinase production in strain 569 was effected by culturing at 42’, then transferring the cells to a 37” bath (Expt. 1, Table V). Exposure of the culture to CT prior to transfer from the 42-37” bath led to little increase in TABLE
II1
SPECIFIC ACTIVITIES OF THE PENICILLINASE PRODUCTION (BASAL)IN Bacilluscereus STR.~IN 569 AT DIFFERENT TEMPERATURES Teltlp erature 18" 22O 25” 30” 35” 38” 40” 42” 44” 36’
Total medium assayed (units/mg.) 13.2 14.9 43.3 37.4 43.0 49.0 99.5 122.9 15.6 20.1
era-units/mg medium assayed
f. 1.4 f 0.2 f 8.0 zk 3.5 f 7.0 xt 8.6 f 9.5 f 13.3 f 3.0
9.8 12.0 38.9 29.3 31.2 36.6 61.5 112 11.2 16.1
f f f f f f f f f
1.2 1.3 5.6 8.0 1.5 1.2 3.7 8.0 1.5
TABLE IV DEPENDENCEOFINDUCTIONONTEMPERATURE Temperature
Control (units/ml)
Cephalothin treatment (units/ml assayed after 3 hrs)
Induction ratio
20 30 38 40 42
3.8 6.6 25.8 54.4 56.8
20.4 280 326 316 82
5.4 42.6 12.6 5.8 1.4
AND
DAY
TABLE V PENICILLINASE PRODUCTION IN Bacillus cereus 569 AS A FUNCTION OF TEMPERATURE AND TREATMENT WITH CEPIL~L~THIN Expt. 1
2 3
Culturing Cephalothin temperature treatment at “CD (“w 37 37 42 42 38 38 42 42
37d 42 38 42
Regrown at “Cb
Activities (units/ml)c
42 42 37 37 38 38 42 42
10 180 592 632 36 326 57 82
a Grown in Pollock’s CH/C medium in a rotary shaker maintained within f 0.1”. b Cephalothin was added to culture to give a concentration of 10-6~. Cephalothin gave better induct.ion than cephalosporin C or penicillin G under the conditions of these experiments. c All experiments were run with complete sets of duplicates or triplicates. The optical density (A& was 0.30 f 0.03 at the beginning of the experiment; thus the absolute activities are approximately proportional to specific activities (=t 10%). d The cells remained at 37” for approximately 2 minutes after addition of CT and prior to transfer at the 42” bath.
induction above that of cells transferred from 42” to 37” without exposure to CT. Culturing at 3742” gave a slightly lower level, if any significant change, in contrast to the thermal derepression seen with the converse transfer. However, if CT was added to the 37” culture prior to transfer to 42”, induction was effected. Experiments 2 and 3 (Table V) demonstrate that CT can bring about induction at 38” but not at 42”. Induction levels were essentially the same at 37” and 38”. It was observed that strain 569/H, which has been observed to be somewhat unstable genetically, loses its penicillinase-constitutive character when cultured at 44-46” at an extremely high rate. DISCUSSION
The following conclusions can be drawn: (a) Derepression can be effected by appropriate temperature changes or by induc-
PENICILLTNASE
tion with a penicillin. (b) Penicillinase production at 42” is unimpaired in both constitutive and inducible strains. (c) InitiaGon of expression of the state of derepression is blocked at 42’ in 569, but not in 569,/H or 5/B. Thus the constitutive mutant’s apparently arising from parent st#rains by a single mutation display two phenotypic differences from t,he parental type in this experiment: constitutive character of the penicillinase production, and temperature insensitivity to expression of the penicillinase genotype. It may be useful to keep in mind the fact that derepression of penicillinase in 8. cereus falls into three categories: (I) genetic: inducible --$ constitutive; (2) chemical induction: no penicillin -+ effective load of penicillin ; and (3) thermal: higher temperat#ure --f lower temperature. According to current thinking the first case may represent mutation involving cessation of product,ion of repressor by an i locus mutant or an @ mutation; the second, the blocking of the action of the repressor as long as an inducer is present which is “permanent” in the inducible strain 569 (8) and in the constitutive strains (5). The t’hird effect described for p-galactosidase production 3, 9), for penicillinase production in S. aureus (2) and here for B. cereus, could conceivably arise from a variety of factors. The two phenotypic changes arising from a single mutation probably cannot be explained only in terms of there being no cytoplasmic repressor in 569/H and 5/B as established for the lac i- in E. coli (10). Even though treatment at 42” effectively produces a latent derepression, whether it be due to absence of repressor or dissociation from the operator locus, the prot#ein synthesis cannot start until the temperature is lowered to 37” for strain 569. No less complicated is an alternate explanation based on the assumption that, an endogenous inducer may be formed at 42”, which, however, is not functional at 42”, but is at 37”. As a step toward explaining the effects we suggest a model basically similar to that of Jacob and Monod (II), but containing additional elements. In the modification, the repressor is a section of RNA corresponding
53
DEREPREHSIOS
to the repressor locus and associated with the operator locus. The dissociation of the operator RNA from the operator locus represents derepression or induction. The release can be controlled by the inducer and/ or a product of t,he i-gene, a cytoplasmic repressor, the repressor RNA. The operator RNA when bound to the operator locus blocks release of an RNA. It is postulated that a part of the binding forces involves a protein identical in p,rimary structure to one of the enzymes which is a product of one of the structural genes in the operon. This protein is presumed to be allosteric. Its interaction with the metabolic inducer or repressor allows release of the messenger RNA which contains the transcribed cistrons and operator sequences as well as the associated allosteric enzyme. If the latter is at a position corresponding to one end of a structural gene which is adjacent to the operator, a control at the level of translation is possible. These ideas are depicted in Fig. 2. Con&itutive organisms, demonstrated to lack a cytoplasmic repressor in a bacterial system in at least one case (lo), would produce a t’ranscriptional system without repressor RKA. In these cases, control could still be exet#red at t’he level of translation. Control of protein synthesis at the level of (I)
Transcriptional
control
mRNA-penicillinase
system
polypeptide-operon
ammo
4
(2)
Translotlonal polysome
control
acid-tRNA
system
- penicillinase polypeptide amino acid tRNA 1 penicillinase
FIG. 2. Modified model for the control of the biosynthesis of penicillinase in Bacillus CWBUS. Abbreviations used are tRNA, t,ransfer ribonucleic acid, and mRNA, messenger ribonucleic acid. Points 1 and 2 are proposed sites of action of the inducer. See text for discussion of the modification.
54
BERNSTEIN,
NICKERSON
translation has been established for an RNA-virus (12) and indicated for penicillinase production in B. cereua 5 (5). That is, there would be the mRNA-allosteric enzyme-operon complex as shown in Fig. 2, with a control as depicted at point 2. Metabolic control has been observed in constitutive penicillinase producers (5, 13) even when not accorded any significance (cf. ref. 14). In addition it accomodates the recognized separate existence of a regulator gene which produces a cytoplasmic factor, functionally the repressor. The derepression caused by exposure to raised temperatures may fall into the category of simply increased dissociation since increased temperature in general favors dissociation. However, the effect of the dissociation on penicillinase production can only be expressed by lowering the temperature. The dissociation of polynucleotide chains exhibits a high temperature coefficient and in vitro may be reversible or irreversible depending on treatment. The constitutive mutants of B. cereua exhibit Q10 < 2 for penicillinase production whereas the Q1o> 2 for basal production in the inducible strain 569 (cf. Tables II, III, and IV). These data provide additional support for a concept implicit in the model (Fig. 2), viz., the &lo for penicillinase production in the three cases, inducible basal, inducible induced, and constitutive, may have different rate-limiting steps, and the existence, per se, of different values of &lo for each of these cases clearly supports this. In particular, the absence of a repressor RNA would eliminate a highly temperature dependent step seen in strain 569 (point 1) and one sees
AND
DAY
instead a modulation of the rate at a step (point 2) having a lower temperature dependence. One can readily calculate an apparent activation energy for each process by using these data and rate of growth which is quite similar among all strains investigated. From the above and other data in references cited (4, 5), it is likely that a greater temperature dependence is manifested at the transcriptional level (point 1, Fig. 2) and a lower temperature dependence at point 2. REFERENCES 1. KNOX, R., AND COLLARD, P., J. Gen. Microbial. 6, 369 (1952). 2. COHEN, S., SWEENEY, H., AND LEITNER, F., Science 149, 877 (1965). 3. SADLER, J. R., AND NOVICK, A., J. Mol. Biol. 12, 305 (1965). 4. DAY, R. A., YIP, L. C., AND HEAZLETT, R. A., presented at Sixth International Congress of Biochemistry, N.Y., 1964. Abstracts, Vol. III, p. 13. 5. YIP, L. C., SHAH, R., AND DAY, R. A., J. Bacteriol. 33, 297 (1964). 6. POLLOCK, M. R., J. Pharm. Pharmacol. 9, 609 (1957). 7. PERRET, C. J., Mature 174, 1012 (1954). 8. POLLOCK, M. R., Brit. J. Exptl. Pathol. 31, 739 (1950). 9. NOVICK, A., MCCOY, J. M., AND SADLER, J. R., J. Mol. Biol. 12, 328 (1965). 10. PARDEE, A. B., JACOB, F., AND MONOD, J., J. Mol. Biol. 1, 165 (1959). 11. JACOB, F., AND MONOD, J., J. Mol. Biol. 3, 318 (1961). 12. OHTAKA,~., AND SPIEGELMAN,S.,Science 142, 493 (1963). 13. SHAH, R., AND DAY, R. A., Xcience 138, 1108 (1962) . AND POLLOCK,M.R., J.Gen. 14. DUBNAU, D.A., Microbial. 41, 7 (1965).
OF
Thermal of
BIOCHEMISTRY
AND
BIOPHYSICS
119, 50-54 (1967)
Penicillinase
Derepression
and
Penicillinase
Production
Inducible
Strains AMIRA BERNSTEIN,
KENNETH
of Bacillus
Temperature
Constitutive
cereus
W. NICKERSON
Department of Chemistry, University
and
Dependence
of Cincinnati,
AND
Cincinnati,
RICHARD
A. DAY’12
Ohio .J52Sl
Received August 15, 1966 The basal level of penicillinase production in strain 569 reaches a maximum at 42”. The induction ratio (induced/basal) reaches a maximum (42.6) at approximately 30” and falls to approximately 1.4 at 42”. Culturing Bacillus cereua 569 (inducible) at 42” and lowering the temperature leads to an increase in penicillinase production (thermal derepression) equal to the level induced by cephalothin (CT). As reported earlier, induction in strain 569 at 42” does not occur when the cells are treated with a penicillin, here the analogue CT, at 42”; however, treatment with CT at 37” followed by incubation at 42” gave the induced response. Constitutive mutants 569/H and 5/B showed a twofold change in the level of penicillinase upon going from 20” to 46”, with specific activity increasing steadily to 46”. A model is presented to explain these and other related data in the B. cereus penicillinase system.
Re-examination of the reported failure of “adaptive formation” of penicillinase in Badus cereus 569 at 42’ (1) led to an investigation of the temperature dependence of penicillinase production in certain strains of B. cereus.The results reported here show that induction in B. cereus 569 can indeed be effected under appropriate conditions at 42”. During the course of this work it was reported that penicillinase production in Staphylococcus aureus could be thermally derepressed, and the characteristics of this process were described (2). This was not unlike the thermal derepression of P-gala&osidase described earlier for Escherichia coli (3). It appears that the temperature de1 This work was supported in part by the American Cancer Society Grant P343A and by the National Institutes of Health Grant AI-06375. We thank Eli Lilly & Co. for generously supplying us with samples of cephalosporin C and cephalothin. Mr. Graf’s technical assistance was invaluable. 2 To whom correspondence concerning this paper should be addressed. 50
pendence of penicillinase production provides supportive evidence for a two-point metabolic control of penicillinase production (4, 5), and in this context a model of control is proposed. PROCEDURE Materials. Penicillin G and hydrolyzed casein were obtained from Nutritional Biochemical Corp. Cephalosporin C and cephalothin were generously donated by Eli Lilly and Co. Growth of cultures. Cultures were grown in a modified CH/C medium (6). In addition to the components of CH/C specified by Pollock (6), the medium was made 0.1% in gelatin. All of the constituents were autoclaved together. Cultures were grown to an optical density of 0.30 f 0.03 in a water bath at the appropriate temperature. The temperature was controlled to f0.1”. Penicillinase assay. Essentially the method of Perret (7) was used throughout. Dry weight of cells. The cells were dried in vacua to a constant weight after washing with distilled water. The relationship of optical density to dry weight of cells was determined in order to report the “specific activity” as units of penicillinase per milligram of dry weight of the cells (Fig. 1). The
PENICILLINASE
51
DEREPRESSION
optical density to dry weight relationship was the same for the entire temperature range examined except where clumping of the cells occurred. Clumping was sometimes observed to a variable degree at the extremes of culturing temperature. RESULTS
Modification of the culturing medium of Pollock (6) by combining all of the components recommended and adding gelatin (0.1%) caused no measurable difference in growth or penicillinase production. Addition of gelatin was essential to prevent adsorption of relatively large amounts of penicillinase to the walls of the culture flask; culturing in polypropylene also obviated the need for gelatin but was less convenient for following the growth visually. Induction of penicillinase activity in B. cereus 569 (NRRL 569), an inducible strain, by the “standard growth” method (3, 4) was somewhat higher when cephalothin (CT) was compared with penicillin G or cephalosporin C (Table I). Penicillinase production in the macroconstitutive strain of B. cereus 5/B (NCTC 9946) was found to be moderately dependent on temperat’ure. The specific 1650 1550 1450 1350 -
TABLE
I
INDUCTION OF PENICILLINASE ACTIVITY BY PENICILLIN G (PG), CEPHBLOTHIB (CT), AND CEPHALOSPORIN C (CC) BY THE ST.LNU~RD METHOD Inducer molarity -
10-4 10-s 10-C
NOIXS
Penicillinax
5.6 -
activity,
units/ml
induced”
PG
CT
cc
58.8 56.4 59.4
60.0 58.0 65.4
60.8 60.0 28.6
n One unit of penicillinase is that amount of enzyme producing sufficient penicilloic acid in one hour at 30”, pH 6.5, to consume 9 peq of iodine by the procedure of Perret (4). TABLE
II
SPECIFIC ACTIVITIES OF THE ENZYME PRODUCTION (BASIL) IN Bacillus cereus STR~IX 5/B >\T DIFPERENT TEMPERATURES Temperature
18 22 25 30 35 38 40 42 44 4G
Total medium assayed (units/mg)
608 700 668 958 1201 1056 1218 1531 1306 853
f rt f f f rt f f f f
18.5 23 210 76 283 71 52 132 113 39
exe-Penicillinase (units/mg)
582 581 630 935 1063 889 1068 1401 1116 266
rt i rt rt + i rt z!z + f
33 134 193 10 167 87 191 12 114 53
1250 .I150 1050 .09502
OS50
F .0750 065U 0550-045U 0350.0250 0150 0050111lIllll111llllIll111
025
100
175
250 OPTICAL
FIG.
weight
325
400 DENSITY
475 (6501~1
550
1. Relationship of optical density to dry of cultures of Bacillus cereu.s strains.
activity of the penicillinase increased by a factor of 2.5 upon going from a culturing temperature of 18” to one of 42’ (Table II). On the other hand, the basal production of penicillinase in strain 569 was more strongly dependent on temperature (Table III). The specific activity increased by a factor of approximately nine upon increasing the culturing temperature from 18” to 42’. The inductive ratio is apparently even more temperature dependent (Table IV). The induction ratio rises from -6 at 20” to -40 at 30”, and then declines to <2 at 42”. It was not possible to measure levels of penicillinase production over as wide a temperature range in B. cereus strain 5 (ATCC 10702) since this organism produces immeasurably low levels of the enzyme as
52
BERNSTEIN,
NICKERSON
culturing temperatures of near 20’ are approached. The amounts cell-bound and exo-penicillinase was determined for the strains described (Tables II and III). Strains 569/H, a constitutive strain arising by spontaneous mutation from strain 569, and strain 5 were found to give similar ratios of ezo-penicillinase to total penicillinase over all culturing temperatures examined. In a previous report from this laboratory (5) it had been reported that strain 5 produced very little ezo-penicillinase, but by culturing the organism in the presence of gelatin the exoenzyme was found to be produced in the proportion typical of other strains of B. cereus. Thermal derepression of penicillinase production in strain 569 was effected by culturing at 42’, then transferring the cells to a 37” bath (Expt. 1, Table V). Exposure of the culture to CT prior to transfer from the 42-37” bath led to little increase in TABLE
II1
SPECIFIC ACTIVITIES OF THE PENICILLINASE PRODUCTION (BASAL)IN Bacilluscereus STR.~IN 569 AT DIFFERENT TEMPERATURES Teltlp erature 18" 22O 25” 30” 35” 38” 40” 42” 44” 36’
Total medium assayed (units/mg.) 13.2 14.9 43.3 37.4 43.0 49.0 99.5 122.9 15.6 20.1
era-units/mg medium assayed
f. 1.4 f 0.2 f 8.0 zk 3.5 f 7.0 xt 8.6 f 9.5 f 13.3 f 3.0
9.8 12.0 38.9 29.3 31.2 36.6 61.5 112 11.2 16.1
f f f f f f f f f
1.2 1.3 5.6 8.0 1.5 1.2 3.7 8.0 1.5
TABLE IV DEPENDENCEOFINDUCTIONONTEMPERATURE Temperature
Control (units/ml)
Cephalothin treatment (units/ml assayed after 3 hrs)
Induction ratio
20 30 38 40 42
3.8 6.6 25.8 54.4 56.8
20.4 280 326 316 82
5.4 42.6 12.6 5.8 1.4
AND
DAY
TABLE V PENICILLINASE PRODUCTION IN Bacillus cereus 569 AS A FUNCTION OF TEMPERATURE AND TREATMENT WITH CEPIL~L~THIN Expt. 1
2 3
Culturing Cephalothin temperature treatment at “CD (“w 37 37 42 42 38 38 42 42
37d 42 38 42
Regrown at “Cb
Activities (units/ml)c
42 42 37 37 38 38 42 42
10 180 592 632 36 326 57 82
a Grown in Pollock’s CH/C medium in a rotary shaker maintained within f 0.1”. b Cephalothin was added to culture to give a concentration of 10-6~. Cephalothin gave better induct.ion than cephalosporin C or penicillin G under the conditions of these experiments. c All experiments were run with complete sets of duplicates or triplicates. The optical density (A& was 0.30 f 0.03 at the beginning of the experiment; thus the absolute activities are approximately proportional to specific activities (=t 10%). d The cells remained at 37” for approximately 2 minutes after addition of CT and prior to transfer at the 42” bath.
induction above that of cells transferred from 42” to 37” without exposure to CT. Culturing at 3742” gave a slightly lower level, if any significant change, in contrast to the thermal derepression seen with the converse transfer. However, if CT was added to the 37” culture prior to transfer to 42”, induction was effected. Experiments 2 and 3 (Table V) demonstrate that CT can bring about induction at 38” but not at 42”. Induction levels were essentially the same at 37” and 38”. It was observed that strain 569/H, which has been observed to be somewhat unstable genetically, loses its penicillinase-constitutive character when cultured at 44-46” at an extremely high rate. DISCUSSION
The following conclusions can be drawn: (a) Derepression can be effected by appropriate temperature changes or by induc-
PENICILLTNASE
tion with a penicillin. (b) Penicillinase production at 42” is unimpaired in both constitutive and inducible strains. (c) InitiaGon of expression of the state of derepression is blocked at 42’ in 569, but not in 569,/H or 5/B. Thus the constitutive mutant’s apparently arising from parent st#rains by a single mutation display two phenotypic differences from t,he parental type in this experiment: constitutive character of the penicillinase production, and temperature insensitivity to expression of the penicillinase genotype. It may be useful to keep in mind the fact that derepression of penicillinase in 8. cereus falls into three categories: (I) genetic: inducible --$ constitutive; (2) chemical induction: no penicillin -+ effective load of penicillin ; and (3) thermal: higher temperat#ure --f lower temperature. According to current thinking the first case may represent mutation involving cessation of product,ion of repressor by an i locus mutant or an @ mutation; the second, the blocking of the action of the repressor as long as an inducer is present which is “permanent” in the inducible strain 569 (8) and in the constitutive strains (5). The t’hird effect described for p-galactosidase production 3, 9), for penicillinase production in S. aureus (2) and here for B. cereus, could conceivably arise from a variety of factors. The two phenotypic changes arising from a single mutation probably cannot be explained only in terms of there being no cytoplasmic repressor in 569/H and 5/B as established for the lac i- in E. coli (10). Even though treatment at 42” effectively produces a latent derepression, whether it be due to absence of repressor or dissociation from the operator locus, the prot#ein synthesis cannot start until the temperature is lowered to 37” for strain 569. No less complicated is an alternate explanation based on the assumption that, an endogenous inducer may be formed at 42”, which, however, is not functional at 42”, but is at 37”. As a step toward explaining the effects we suggest a model basically similar to that of Jacob and Monod (II), but containing additional elements. In the modification, the repressor is a section of RNA corresponding
53
DEREPREHSIOS
to the repressor locus and associated with the operator locus. The dissociation of the operator RNA from the operator locus represents derepression or induction. The release can be controlled by the inducer and/ or a product of t,he i-gene, a cytoplasmic repressor, the repressor RNA. The operator RNA when bound to the operator locus blocks release of an RNA. It is postulated that a part of the binding forces involves a protein identical in p,rimary structure to one of the enzymes which is a product of one of the structural genes in the operon. This protein is presumed to be allosteric. Its interaction with the metabolic inducer or repressor allows release of the messenger RNA which contains the transcribed cistrons and operator sequences as well as the associated allosteric enzyme. If the latter is at a position corresponding to one end of a structural gene which is adjacent to the operator, a control at the level of translation is possible. These ideas are depicted in Fig. 2. Con&itutive organisms, demonstrated to lack a cytoplasmic repressor in a bacterial system in at least one case (lo), would produce a t’ranscriptional system without repressor RKA. In these cases, control could still be exet#red at t’he level of translation. Control of protein synthesis at the level of (I)
Transcriptional
control
mRNA-penicillinase
system
polypeptide-operon
ammo
4
(2)
Translotlonal polysome
control
acid-tRNA
system
- penicillinase polypeptide amino acid tRNA 1 penicillinase
FIG. 2. Modified model for the control of the biosynthesis of penicillinase in Bacillus CWBUS. Abbreviations used are tRNA, t,ransfer ribonucleic acid, and mRNA, messenger ribonucleic acid. Points 1 and 2 are proposed sites of action of the inducer. See text for discussion of the modification.
54
BERNSTEIN,
NICKERSON
translation has been established for an RNA-virus (12) and indicated for penicillinase production in B. cereua 5 (5). That is, there would be the mRNA-allosteric enzyme-operon complex as shown in Fig. 2, with a control as depicted at point 2. Metabolic control has been observed in constitutive penicillinase producers (5, 13) even when not accorded any significance (cf. ref. 14). In addition it accomodates the recognized separate existence of a regulator gene which produces a cytoplasmic factor, functionally the repressor. The derepression caused by exposure to raised temperatures may fall into the category of simply increased dissociation since increased temperature in general favors dissociation. However, the effect of the dissociation on penicillinase production can only be expressed by lowering the temperature. The dissociation of polynucleotide chains exhibits a high temperature coefficient and in vitro may be reversible or irreversible depending on treatment. The constitutive mutants of B. cereua exhibit Q10 < 2 for penicillinase production whereas the Q1o> 2 for basal production in the inducible strain 569 (cf. Tables II, III, and IV). These data provide additional support for a concept implicit in the model (Fig. 2), viz., the &lo for penicillinase production in the three cases, inducible basal, inducible induced, and constitutive, may have different rate-limiting steps, and the existence, per se, of different values of &lo for each of these cases clearly supports this. In particular, the absence of a repressor RNA would eliminate a highly temperature dependent step seen in strain 569 (point 1) and one sees
AND
DAY
instead a modulation of the rate at a step (point 2) having a lower temperature dependence. One can readily calculate an apparent activation energy for each process by using these data and rate of growth which is quite similar among all strains investigated. From the above and other data in references cited (4, 5), it is likely that a greater temperature dependence is manifested at the transcriptional level (point 1, Fig. 2) and a lower temperature dependence at point 2. REFERENCES 1. KNOX, R., AND COLLARD, P., J. Gen. Microbial. 6, 369 (1952). 2. COHEN, S., SWEENEY, H., AND LEITNER, F., Science 149, 877 (1965). 3. SADLER, J. R., AND NOVICK, A., J. Mol. Biol. 12, 305 (1965). 4. DAY, R. A., YIP, L. C., AND HEAZLETT, R. A., presented at Sixth International Congress of Biochemistry, N.Y., 1964. Abstracts, Vol. III, p. 13. 5. YIP, L. C., SHAH, R., AND DAY, R. A., J. Bacteriol. 33, 297 (1964). 6. POLLOCK, M. R., J. Pharm. Pharmacol. 9, 609 (1957). 7. PERRET, C. J., Mature 174, 1012 (1954). 8. POLLOCK, M. R., Brit. J. Exptl. Pathol. 31, 739 (1950). 9. NOVICK, A., MCCOY, J. M., AND SADLER, J. R., J. Mol. Biol. 12, 328 (1965). 10. PARDEE, A. B., JACOB, F., AND MONOD, J., J. Mol. Biol. 1, 165 (1959). 11. JACOB, F., AND MONOD, J., J. Mol. Biol. 3, 318 (1961). 12. OHTAKA,~., AND SPIEGELMAN,S.,Science 142, 493 (1963). 13. SHAH, R., AND DAY, R. A., Xcience 138, 1108 (1962) . AND POLLOCK,M.R., J.Gen. 14. DUBNAU, D.A., Microbial. 41, 7 (1965).