Molec. gen. Genet. 147, 23-27 (1976) © by Springer-Verlag 1976

Characterization of Mutations in the Penicillinase Operon of Staphylococcus aureus John Imsande and Jerry L. Liileholm Department of Genetics, Iowa State Umversity,Ames, Iowa 50011

Summary. Mutant penicillinase plasmids, in which penicillinase synthesis is not inducible by penicillin or a penicillin analogue, were examined by biochemical and genetic analyses. In five of the six mutants tested, penicillinase synthesis could be induced by growth in the presence of 5-rnethyltryptophan. It is known that the tryptophan analogue 5-methyltryptophan is readily incorporated in~;o protein by S. aureus and that staphylococcal penicillinase lacks tryptophan. 5-methyltryptophan seems to induce penicillinase synthesis in wild-type plasmids by becoming incorporated into the repressor and thereby inactivating the operator binding function of the penicillinase repressor. Therefore, induction of penicillinase synthesis in the mutant plasmids by 5-methyltryptophan strongly suggests that the noninducible phenotype of these five plasmids is due to a mutation that inactivates the effector binding site of the penicillinase repressor (i.e., the five mutant plasmids carry an is genotype for the penicillinase repressor). This conclusion was supported by heterodiploid analysis. The mutant plasmid that did not respond to 5-methyltryptophan either produces an exceedingly low basal level of penicillinase or does not produce active enzyme. This plasmid seems to carry a murat:ion in the penicillinase structural gene or in the promoter for the structural gene. Thus, a genetic characterization of many mutations in the penicillinase operon can be accomplished easily and rapidly by biochemical analysis.

Introduction The gene that specifies the amino acid sequence of staphylococcal penicillinase normally is carried on a plasmid (Novick and Richmond, 1965). Also located * Journal Paper No. J-7994 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2029

on the plasmid, and tightly linked to the penicillinase structural gene (penZ) is a regulatory gene (penl) that codes for the penicillinase repressor (Richmond, 1965; Lindberg and Novick, 1973). Mutants of S. aureus that produce a low level of penicillinase in the absence of inducer and do not produce appreciably more penicillinase in the presence of inducer have been isolated in a number of laboratories (Novick, 1963; Richmond, 1966; Richmond, 1967; Cohen, Gibson, and Sweeney, 1972). A mutation responsible for the "microconstitutive" phenotype (Novick, 1963) might reside in the regulatory gene that specifies the repressor and thus cause the production of a repressor with an inactive effector binding site (i.e., an i~-type repressor, Willson et al., 1964), or the mutation might be located in some other penicillinasecontrolling genetic element (e.g., promoter, operator, or a positive control gene). Because these various regions are, or may be, closely linked on the plasmid, it can be very difficult to determine the exact nature of the mutational site by standard genetic mapping. On the other hand, one should be able to distinguish between these possible mutational sites very simply by biochemical analysis. This statement is based on the following observations and reasoning: 1, staphylococcal penicillinase lacks tryptophan (Ambler and Meadway, 1969) while the penicillinase repressor seems to contain tryptophan (Imsande, 1973); 2, 5-methyltryptophan can be readily incorporated into protein by Staphylococcus aureus (Imsande, 1973); 3, growth of S. aureus bearing the wild-type penicillinase plasmid in the presence of 5-methyltryptophan causes the induction of penicillinase (Imsande, Zyskind, Mile, 1972; Imsande, 1973). Thus, growth in the presence of 5-methyltryptophan seems to cause the formation of a faulty penicillinase repressor and thereby promotes the synthesis of penicillinase. Now, if the mutation responsible for the microconstitutive phenotype resides in the regulatory

24 gene t h a t specifies the repressor, then g r o w t h in the presence o f 5 - m e t h y l t r y p t o p h a n s h o u l d cause the newly synthesized i s r e p r e s s o r to b e c o m e c o m p l e t e l y inactive; hence, penicillinase synthesis s h o u l d result. Ifl on the o t h e r h a n d , the m i c r o c o n s t i t u t i v e m u t a t i o n resides in the p r o m o t e r for the p e n Z gene or in a positive c o n t r o l gene, then g r o w t h in the presence o f 5 - m e t h y l t r y p t o p h a n s h o u l d n o t cause an increased synthesis o f active penicillinase. Hence, the p a r t i c u l a r genetic cause o f the m i c r o c o n s t i t u t i v e p h e n o t y p e m a y be r e a d i l y e s t a b l i s h e d with the a i d o f 5 - m e t h y l t r y p t o phan. D a t a p r e s e n t e d in this c o m m u n i c a t i o n show t h a t 5 - m e t h y l t r y p t o p h a n c a n be used to c h a r a c t e r i z e s o m e o f the r e g u l a t o r y m u t a t i o n s in the s t a p h y l o c o c c a l penicillinase system. I n d u c t i o n by 5 - m e t h y l t r y p t o p h a n also shows t h a t s t a p h y l o c o c c a l penicillinase synttiesis is r e g u l a t e d by negative c o n t r o l a n d n o t by a positive c o n t r o l or a c t i v a t o r protein.

Materials and Methods Strains and Media. The six defective penicillinase plasmids, generously provided by Dr. Sidney Cohen, were carried by S. aureus

strain 8325. For each experiment, cells were transferred from a fresh (18-h) slant fortified with Brain Heart Infusion medium to nutrient broth (0.8%) supplemented with glucose (0.2%) and 0.01 M phosphate, pH 7.0. When exponential growth had been achieved, the cells were collected on a membrane filter and quickly washed and suspended in warmed casamino acid medium (Imsande et al., 1972). Cells were grown at 37°C in a gyrotory shaker, and growth was followed with a Zeiss speetrophotometer at 535 nm.

J. Imsande and J.L. Lilleholm: Mutations in the Penicillinase Operon i

,

i

I

0.8

~0.6

"~ 0 . 4 ©

2 ~0.2

0

o

I

0.3

i

I

i

0.4 Turbidity

I

i

0.5 at

535nm

Fig. 1. Penicillinase induction in strain 8325 (PI258 penI353) by 5-methyltryptophan. A culture growing exponentially on casamino acid (CAA) medium was divided into three equal 65 ml fractions. To one culture was added 10 ml of CAA medium supplemented with 7.5 mg of 5-methyltryptophan ( A - - zx);to the second culture was added 10 ml of CAA medium supplemented with 54.5 gM CBAP (o - - o); to the third culture was added 10 ml of unsupplemented CAA medium (o - - o). All cultures were grown at 37° C. Three-ml samples for penicillinase assay were transferred from each culture at 0-time, and at 15-minute intervals thereafter, to iced tubes that contained 1 mg of chloramphenicol in 1 ml of 0.5 M phosphate, pH 6.0. Samples for cell growth determination were also taken at 15-minute intervals

Penicillinase Assay. Penicillinase activity was assayed by the stan-

dard Perret (1954) procedure. A unit of penicillinase is defined as the amount of enzyme activity required to hydrolyze 1 gmole of benzylpenicillin per h at pH6.4 and 30°C. Specific activity is defined as units of penicillinase per mg (dry weight) of cells. One ml of a culture with a turbidity of 1.0 at 535 nm contains 0.28 mg (dry weight) of cells. Penicillinase phenotypes of colonies grown on 0.3% casamino acids-yeast extract (CY)-glycerol plates with or without 7.25 gM CBAP 1 were tested qualitatively by the starch (0.3%)-iodine plate assay method of Sherratt and Collins (1973). Transduction. Transductions were performed with phage 80c~ by using the media and methods described by Novick (1963). Erythromycin-resistant transductants were selected on 0.3 CY-glycerol plates containing 1 lag/ml of erythromycin and were subsequently purified by streaking on plates that contained 100 gg/ml erythromycin (Peyru et al., 1969). Cadmium-resistant transductants were selected on 0.3 CY-glycerol plates supplemented with 5 x 10 -5 M cadmium nitrate (Peyru et al., 1969).

Results I n an a t t e m p t to gain f u r t h e r insight into the m e c h a n i s m o f r e g u l a t i o n o f penicillinase synthesis in S. au1 2(2'-carboxyphenol) benzoyl-6-amino-penicillanic acid (CBAP) was kindly donated by Imperial Industries, Ltd.

reus, six a p p a r e n t m i c r o c o n s t i t u t i v e penicillinase m u -

t a n t s were g r o w n in the presence o f 5 - m e t h y l t r y p t o p h a n (5MT). A l l m u t a n t p l a s m i d s were c a r r i e d by S. aureus strain 8325. A s s h o w n in F i g u r e 1, g r o w t h o f m u t a n t 353 [i.e., strain 8325 (PI258 penI353)] in the presence o f 5 - m e t h y l t r y p t o p h a n induces penicillinase synthesis a p p r o x i m a t e l y 20-fold even t h o u g h penicillinase synthesis c o u l d n o t be i n d u c e d b y C B A P 1, the classical penicillinase inducer. F u r t h e r m o r e , C B A P plus 5 - m e t h y l t r y p t o p h a n d i d n o t induce a higher level o f penicillinase synthesis t h a n d i d 5-methylt r y p t o p h a n alone. E a c h o f the six m u t a n t s was exami n e d a c c o r d i n g to the p r o c e d u r e s h o w n in F i g u r e 1, a n d the effect o f 5 - m e t h y l t r y p t o p h a n On penicillinase synthesis in each o f the m u t a n t s is p r e s e n t e d in Table 1. I n five o f the six m u t a n t s tested, penicillinase synthesis was i n d u c e d by 5 - m e t h y l t r y p t o p h a n . A g a i n , it s h o u l d be stressed t h a t penicillinase synthesis c o u l d n o t be i n d u c e d significantly b y C B A P in a n y o f the six m u t a n t s . These results suggest that, while an i st y p e m u t a t i o n seems r e s p o n s i b l e for the m i c r o c o n s t i tutive p h e n o t y p e o f five o f the m u t a n t s , the sixth

J. Imsande and J.L. Lilleholm: Mutations in the Penicillinase Operon

mutant, subsequently identified as 8325 (PII147

penZ378), seems to carry a different defect. In order to confirm that the mutants inducible by 5MT possess an i ~genotype, heterodiploid analysis Table 1. 5-methyltryptophan-induced penicillinase synthesis in strains that do not respond to the normal penicillinase inducer a Mutant number

Plasmid

Increase in rate of penicillinase synthesis (fold)

Apparent plasmid genotype

353 356 357 358 395 378

PI258 PI258 PI258 PI258 PI524 PII147

20 10~0 20 10~0 20 0

PI258 pen is PI258 pen ? PI258 pen ? PI258 pen is PI524 pen i~ PII147 penZ-

a

See Fig. 1 for experimental details

25

was performed. A transducing lysate was prepared from each of the mutant strains and these lysates were used to transduce a strain that is partially constitutive for penicillinase production [i.e., 8325 (PII147 penI220)]. Three or more clones of each of the heterodiploids constructed [i.e., 8325 (PII147 penI220); (PI258 pen is)] were then assayed for basal penicillinase production as well as for inducibility by CBAP or 5MT (see Table 2). It can be seen that penicillinase synthesis is inducible in these heterodiploid strains by either CBAP or 5MT. These results suggest that the penicillinase repressor is composed of at least two subunits and that the heterologous repressor is inactivated by the normal induction process. Proof of the heterodiploid nature of these strains was provided by segregational analysis. To increase the frequency of plasmid loss, each diploid was treated with ethidium bromide (Cohen and Sweeney,

Table 2. Properties of heterodiploids co,reposed of a partial constitutive plasmid and an is mutant plasmid Penicillinase specific activity

Ratio

Basal

Plus CBAP

Plus 5MT

CBAP/ Basal

2.0 1.2 8.0 8.9

70 70 92 150

86 140 145 159

35 58 12 17

1.2 1.5 1.2

1.2 1.5 1.3

24 30 25

1 1

1.0

1.0

19

4.7 0.3 3.5 1.3 0.2 1.1

103 1.6 78.0 111.0 0.8 85.0

Parental-type segregants 8325 (PI258 penI353a) 8325 (PII147 penI220)

0.3-0.6

0.2~).5

8325 (PI258 penI356) 8325 (PII147 penI220)

0.5 6.0

0.6 82

8325 (PI258 penI357a) 8325 (PII147 penI220)

0.6-l. 1 4.2

0.4-1.2 160

38

8325 (PI258 penI358) 8325 (PII147 penI220)

1.0-2.0

0.9 1.2

1

Strain

Controls 8325-4 (PI258) 8325 (PII147) 8325 (PII 147 penI220) 8325 (PII147) (PI258 penI443) i s Class 8325 (PI258 8325 (PI258 8325 (PI258 8325 (PI258

penI353) penI356) penI357) penI358)

Diploid classes 8325 (PII147 penI220) 8325 (PII147 penI220) 8325 (PII147 penI220) 8325 (PII147pen1220) 8325 (PII147 penI220) 8325 (PII147 penI220)

(PI258 penI353a) (PI258 penI353b) (PI258 penI356)

(PI258penI357a) (PI258 penI357b) (PI258 penI358)

N.F, signifies that this segregant was not found.

87 2.7 147.0 200.0 0.8 144.0

1 1

22 6 22 85 4 77

1

95

Number of class assayed

5 N.F.

1

1

14

2

1

2 1

4 N.F.

26

J. Imsande and J.L. Lilleholm: Mutations in the Penicitlinase Operon

Table 3. Properties of transductants

Colony number

Penicillinase (units/rag dry weight) (Induced)

Growth on indicator plate

~378 la a

0.5 96

+ +

0 +

20

90

+

+

3d

99

+

+

1hu 2h 3h

95