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A second regulatory region involved in penicillinase synthesis in Staphylococcus aureus
J. Mol. Biol. (1967) 26, 357-360
A Second Regulatory Region involved in Penicillinase Synthesis in Staphylococcus aweus Previous experiments (Richmond, 1965a,b) have shown that there is a class of GOTstitutive mutants in the penicillinase system of Xtaphylococcus aweus which are analogous to the i- mutants in the ,&galactosidase system (Jacob & Monod, 1961) in that constitutive synthesis of penicillinase is repressed by an i + allele acting tram in a plasmid diploid. Phenotypically, all mutants in this class in S. aureu.s have an uninduced rate of enzyme synthesis greater than the wild type. Some synthesize enzyme at a level characteristic of the fully induced wild type and are not further inducible, whereas in others the rate of enzyme synthesis can be increased by inducer to the fully induced level. Treatment of the fully inducible wild-type strain 8325(cr.i+pi)-for nomenclature, see Xovick & Richmond (1965)- with EMS?, however, also gives rise to a second class of regulatory mutants. These are strains with an uninduced level similar to that of the wild type but which are either uninducible (baso-constitutive) or are capable of induction to a lower level than the wild type (Table 1). Although basoTABLE 1 characters of certain EMS-induced
Phenotypic
regdatory
mutants of
Staphylococcus aureus straim 8325(a.i+pi). Specific enzyme activity units/mg dry wt bacteria. uniuduced induced
Phenotype
Parent
Mutant Mutant Mutant
Induction
ratio
All cultures John, 1964).
1 2 3
=
fully inducible base-constitutive semi-oonstitutive semi-constitutive units/mg units/mg
dry wt bacteria dry wt bacteria
induced where neoessary with
9.4 8.7 6.9 6.3
312 8.1 47 61
Induction ratio
33 0.9 6.7 9-6
induced uninduced’ 0.5 pg methicillin/ml.
(Richmond,
Parker,
Jevons &
constitutive mutants of this class are phenotypically similar to the is mutants in the Eat system (Willson, Perrin, Cohn, Jacob & Monod, 1964), they behave completely differently when their behaviour is studied in diploids. In a diploid with an i- mutation in the tram position (e.g. in the diploid 147 (u.baso- wn.stitutive.pi//3.i-ps)), the expression of the immunological C-type penicillinase (Richmond, 196%) (which is characteristic of the /3-plasmid) was repressed in the absence of inducer; and this suggests that the a-plasmid, which carries the baso-constitutive mutation and codes for the immunological A.-type of enzyme, carries an intact i+ gene (Table 2). However, induction of this diploid led to an 7 Abbreviation
used: EMS, ethyl-methane
sulphonate. 357
147(/3.i-p:
.ems)
tAbout
i .ems)
half the enzyme in the uninduced
147(/3Z2,; .ew@) 147(or.baso-eonstitutive.emR)
2 .ema/fi.i-p
Segregants
8325(a.6aso-constitutive.p~.emR)
147(cr.baso-constitutive.p
(recipient) (donor)
Diploid
Parent Parent
Genotype
Phenotypic
culture
enzyme.
9.3
6.6
A-type
_-217
-__J
135
8.1
-
11t
-
--
241
8.7
242
Specific enzyme activity (nnits/mg. dry wt bacteria) Uninduced enzyme Induced A-type A-type c-type
was immunological
c A
A+C
C A
Enzyme type
characters of a S. aureus diploid constructed by transducing strain 147(/3.i -p s .em”) with phage obtained from the erythromycin-resistant version of mutant 1 (Table 1)
TABLE 2
231 -
107
216 -_
enzyme C-type
LETTERS
TO THE
359
EDITOR
increased rate of synthesis of both the A- and C-type penicillinases, despite the fact that the A-type enzyme of the baso-constitutive mutant is uninducible in the haploid state. Thus in this diploid complementation occurred: the i- mutation OII the @plasmid was phenotypically repaired by the i+ allele on u acting tram; and the baso-constitutive mutation on u was phenotypically repaired by a product of p, but this property was only manifest in the presence of inducer. In order to dispose of the possibility that phenotypic repair of the baso-constitutive mutation might specifically be due to the i- mutant used in the test, or to an unsuspected second mutation in this strain, a diploid was constructed between the baso-constitutive mutant and the wild-type ,&plasmid, namely, 147 (a.baso-constiIt was clear from the behaviour of this diploid that the unmutated tutive.p$j/3.iCp7t;). ,&plasmid was as capable of repairing the baso-constitutive lesion of plasmid CLas the j!.;- of the previous experiment. As the phenotypic effect of the mutation in the baso-constitmive mutant is to abolishinducibility completely, it seemed that the two partially inducible strains (Table 1) might be “partial”mutants in the same region. To test this point, diploids were set up exactly as used to test the baso-constitutive mutant, and their behaviour shows that : (1) they both appear to carry an intact if gene; (2) they both can be repaired phenotypically by a product of the p-plasmidwhether ii or i--aoting tram in a diploid. Both these mutants, therefore, have similar genetic properties to the mutation causing the baso-constitutive phenotype. These results suggest, therefore, that there are two regulatory regions which cooperate in penicillinase production in S. aureus. On one hand there is a region, analogous to the i-region in the lac system (Jacob & Monad, 1961), which seems to exert a broadly repressive effect in unmutated cultures, since mutation in this region leads to cultures with a raised basal level. On the other hand, the second regulatory area seems to have an inductive role, since on mutation the ability of penicillin to induce enzyme synthesis is impaired. One possible function of the second regulatory region is to make a product which modifies the penicillin molecule to a form in which it exerts its inductive effect-an example of “effector synthesis” (Brenner, 1965). However, the inability to “force” a higher level of induction in mutants 2 and 3 (Table 1) by increasing inducer concentration (Richmond, unpublished experiments) rather argues against this possibility. Furthermore, this mechanism would depend upon there being two cistrons involved and so far there is no evidence on this point. An alternative, and rather more plausible possibility, which is unaffected by the number of cistrons involved, is one in which penicillmase production is regulated by the products of the two regulatory regions associating to form a dimer. Department of Molecular Biology University of Edinburgh King’s Buildings, West Mains Road Edinburgh, 9, Scotland
M. H. RICHMOND
Received 9 March 196’7 REFERENCES Brenner, S. (1965). Brit. Med. BUZZ. 21, 244. Jacob, F. & Monod, J. (1961). Cold Spring Harb. Symp. Quant. Btil. Noviok, R. P. & Richmond, M. H. (1966). J. Bact. 90, 467. 24
26, 193.
360 Richmond, M. H. Richmond, M. H. Richmond, M. H. Richmond, M. H., Willson, C., Perrin,
M. H. RICHMOND (1965a). J. Bact. 90, 370. (19653). Brit. Med. Bull. 21, 360. (1965~). Biochem. J. 94, 584. Parker, M. T., Jevons, M. P. Rs John, M. (1964). Lancet, i, 293. D., Cohn, M., Jacob, F. & Monod, J. (1964). J. Mol. BioE. 8, 582.
A Second Regulatory Region involved in Penicillinase Synthesis in Staphylococcus aweus Previous experiments (Richmond, 1965a,b) have shown that there is a class of GOTstitutive mutants in the penicillinase system of Xtaphylococcus aweus which are analogous to the i- mutants in the ,&galactosidase system (Jacob & Monod, 1961) in that constitutive synthesis of penicillinase is repressed by an i + allele acting tram in a plasmid diploid. Phenotypically, all mutants in this class in S. aureu.s have an uninduced rate of enzyme synthesis greater than the wild type. Some synthesize enzyme at a level characteristic of the fully induced wild type and are not further inducible, whereas in others the rate of enzyme synthesis can be increased by inducer to the fully induced level. Treatment of the fully inducible wild-type strain 8325(cr.i+pi)-for nomenclature, see Xovick & Richmond (1965)- with EMS?, however, also gives rise to a second class of regulatory mutants. These are strains with an uninduced level similar to that of the wild type but which are either uninducible (baso-constitutive) or are capable of induction to a lower level than the wild type (Table 1). Although basoTABLE 1 characters of certain EMS-induced
Phenotypic
regdatory
mutants of
Staphylococcus aureus straim 8325(a.i+pi). Specific enzyme activity units/mg dry wt bacteria. uniuduced induced
Phenotype
Parent
Mutant Mutant Mutant
Induction
ratio
All cultures John, 1964).
1 2 3
=
fully inducible base-constitutive semi-oonstitutive semi-constitutive units/mg units/mg
dry wt bacteria dry wt bacteria
induced where neoessary with
9.4 8.7 6.9 6.3
312 8.1 47 61
Induction ratio
33 0.9 6.7 9-6
induced uninduced’ 0.5 pg methicillin/ml.
(Richmond,
Parker,
Jevons &
constitutive mutants of this class are phenotypically similar to the is mutants in the Eat system (Willson, Perrin, Cohn, Jacob & Monod, 1964), they behave completely differently when their behaviour is studied in diploids. In a diploid with an i- mutation in the tram position (e.g. in the diploid 147 (u.baso- wn.stitutive.pi//3.i-ps)), the expression of the immunological C-type penicillinase (Richmond, 196%) (which is characteristic of the /3-plasmid) was repressed in the absence of inducer; and this suggests that the a-plasmid, which carries the baso-constitutive mutation and codes for the immunological A.-type of enzyme, carries an intact i+ gene (Table 2). However, induction of this diploid led to an 7 Abbreviation
used: EMS, ethyl-methane
sulphonate. 357
147(/3.i-p:
.ems)
tAbout
i .ems)
half the enzyme in the uninduced
147(/3Z2,; .ew@) 147(or.baso-eonstitutive.emR)
2 .ema/fi.i-p
Segregants
8325(a.6aso-constitutive.p~.emR)
147(cr.baso-constitutive.p
(recipient) (donor)
Diploid
Parent Parent
Genotype
Phenotypic
culture
enzyme.
9.3
6.6
A-type
_-217
-__J
135
8.1
-
11t
-
--
241
8.7
242
Specific enzyme activity (nnits/mg. dry wt bacteria) Uninduced enzyme Induced A-type A-type c-type
was immunological
c A
A+C
C A
Enzyme type
characters of a S. aureus diploid constructed by transducing strain 147(/3.i -p s .em”) with phage obtained from the erythromycin-resistant version of mutant 1 (Table 1)
TABLE 2
231 -
107
216 -_
enzyme C-type
LETTERS
TO THE
359
EDITOR
increased rate of synthesis of both the A- and C-type penicillinases, despite the fact that the A-type enzyme of the baso-constitutive mutant is uninducible in the haploid state. Thus in this diploid complementation occurred: the i- mutation OII the @plasmid was phenotypically repaired by the i+ allele on u acting tram; and the baso-constitutive mutation on u was phenotypically repaired by a product of p, but this property was only manifest in the presence of inducer. In order to dispose of the possibility that phenotypic repair of the baso-constitutive mutation might specifically be due to the i- mutant used in the test, or to an unsuspected second mutation in this strain, a diploid was constructed between the baso-constitutive mutant and the wild-type ,&plasmid, namely, 147 (a.baso-constiIt was clear from the behaviour of this diploid that the unmutated tutive.p$j/3.iCp7t;). ,&plasmid was as capable of repairing the baso-constitutive lesion of plasmid CLas the j!.;- of the previous experiment. As the phenotypic effect of the mutation in the baso-constitmive mutant is to abolishinducibility completely, it seemed that the two partially inducible strains (Table 1) might be “partial”mutants in the same region. To test this point, diploids were set up exactly as used to test the baso-constitutive mutant, and their behaviour shows that : (1) they both appear to carry an intact if gene; (2) they both can be repaired phenotypically by a product of the p-plasmidwhether ii or i--aoting tram in a diploid. Both these mutants, therefore, have similar genetic properties to the mutation causing the baso-constitutive phenotype. These results suggest, therefore, that there are two regulatory regions which cooperate in penicillinase production in S. aureus. On one hand there is a region, analogous to the i-region in the lac system (Jacob & Monad, 1961), which seems to exert a broadly repressive effect in unmutated cultures, since mutation in this region leads to cultures with a raised basal level. On the other hand, the second regulatory area seems to have an inductive role, since on mutation the ability of penicillin to induce enzyme synthesis is impaired. One possible function of the second regulatory region is to make a product which modifies the penicillin molecule to a form in which it exerts its inductive effect-an example of “effector synthesis” (Brenner, 1965). However, the inability to “force” a higher level of induction in mutants 2 and 3 (Table 1) by increasing inducer concentration (Richmond, unpublished experiments) rather argues against this possibility. Furthermore, this mechanism would depend upon there being two cistrons involved and so far there is no evidence on this point. An alternative, and rather more plausible possibility, which is unaffected by the number of cistrons involved, is one in which penicillmase production is regulated by the products of the two regulatory regions associating to form a dimer. Department of Molecular Biology University of Edinburgh King’s Buildings, West Mains Road Edinburgh, 9, Scotland
M. H. RICHMOND
Received 9 March 196’7 REFERENCES Brenner, S. (1965). Brit. Med. BUZZ. 21, 244. Jacob, F. & Monod, J. (1961). Cold Spring Harb. Symp. Quant. Btil. Noviok, R. P. & Richmond, M. H. (1966). J. Bact. 90, 467. 24
26, 193.
360 Richmond, M. H. Richmond, M. H. Richmond, M. H. Richmond, M. H., Willson, C., Perrin,
M. H. RICHMOND (1965a). J. Bact. 90, 370. (19653). Brit. Med. Bull. 21, 360. (1965~). Biochem. J. 94, 584. Parker, M. T., Jevons, M. P. Rs John, M. (1964). Lancet, i, 293. D., Cohn, M., Jacob, F. & Monod, J. (1964). J. Mol. BioE. 8, 582.